{"paper_id":"27255851-6f82-4e01-bb1a-d174d2b60038","body_text":"Dysregulation of STS in keratinocytes promotes calcium signaling and hyperkeratinization: Insights into the mechanisms underlying X-linked ichthyosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dysregulation of STS in keratinocytes promotes calcium signaling and hyperkeratinization: Insights into the mechanisms underlying X-linked ichthyosis Tae-Uk Kwon, Yeo-Jung Kwon, Hyemin Park, Yoon-ji Kang, Young-Jin Chun This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5115056/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Steroid sulfatase (STS) is a key enzyme for the desulfation of steroid sulfates, converting them into their biologically active forms. Notably, X-linked ichthyosis (XLI), a genetic disorder characterized by hyperkeratinization, arises as a direct result of STS deficiency. Keratinocyte differentiation is essential for proper keratinization. In this study, gene ontology analysis from STS-deficient mice revealed enhanced differentiation and upregulation of calcium-related signaling. Calcium plays a key role in regulating keratinocyte differentiation, with STS-deficient cells showing a marked increase in intracellular calcium influx. Additionally, these cells significantly upregulated calcium-sensing receptors (CasR), leading to elevated tyrosine phosphorylation, increased differentiation signaling, and the upregulation of early differentiation markers, including keratin 1 and keratin 10, as seen in HaCaT cells and mouse primary keratinocytes. Furthermore, STS inhibitors enhanced the expression of E-cadherin and late differentiation markers such as involucrin and loricrin. Due to increased calcium sensitivity, STS-deficient cells treated with calcium exhibited a significant upregulation of differentiation markers and reduced sensitivity to calcium chelation. Collectively, our findings demonstrate that reduced STS expression and inhibition of its activity enhance calcium responsiveness, induce CasR expression, and amplify calcium signaling, thereby promoting keratinocyte differentiation. These findings offer valuable insights into the mechanisms underlying STS deficiency-induced hyperkeratinization. Biological sciences/Cell biology/Mechanisms of disease Health sciences/Diseases/Skin diseases Biological sciences/Cell biology/Cell signalling/Calcium signalling Biological sciences/Biochemistry/Enzyme mechanisms Biological sciences/Molecular biology/Transcriptomics STS XLI RNA-seq CasR calcium influx keratinocyte Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Steroid sulfatase (STS), an enzyme found in varying amounts across nearly all tissues, desulfates steroid hormones such as cholesterol sulfate and dehydroepiandrosterone sulfate (DHEAS), converting them into their biologically active forms 1,2 . In skin tissue, STS is predominantly localized in the epidermis 1 . Dysregulation of STS has been linked to several skin conditions. For example, elevated plasma DHEAS levels are associated with conditions such as androgenetic alopecia and hirsutism 3 . Notably, STS deficiency is linked to X-linked ichthyosis (XLI), a congenital disorder characterized by abnormal keratinization, resulting in large, dark brown scaly patches and a thickened stratum corneum 1,2 . These symptoms are thought to result from the accumulation of cholesterol sulfate 1,2 , although the precise mechanisms underlying STS deficiency-induced keratinization remain unclear. Thickening of the stratum corneum primarily occurs due to excessive keratinocyte differentiation and stratification, which weakens exfoliation by increasing cell cohesion 6–7 . Calcium is a key regulator of differentiation-related signaling pathways and plays a critical role in keratinocyte differentiation both in vivo and in vitro 8,9 . As keratinocytes move through the epidermal layers, calcium gradients drive their differentiation, forming the stratum corneum’s barrier 9–13 . Changes in extracellular calcium concentrations activate calcium-sensing receptors (CasR), triggering intracellular signaling essential for differentiation 14 . Elevated calcium levels promote intercellular contact formation 15 , and cadherins such as E-cadherin translocate to the membrane, facilitating the assembly of adherens junctions and desmosomes 16–19 . Upon calcium stimulation, E-cadherin binds to neighboring cells, while its cytoplasmic tail interacts with catenins to form the core of adherens junctions 20 . E-cadherin-mediated adhesion is essential for maintaining proper differentiation and remodeling cell-cell interactions 21,22 . Loss of E-cadherin disrupts adherens junctions and impairs terminal differentiation 23 . Tyrosine phosphorylation also positively regulates cell adhesion during keratinocyte differentiation 16 , and Src family kinases such as Src and Fyn exhibit elevated activity in calcium-differentiated keratinocytes 9,24,25 . CasR responds to extracellular calcium by triggering intracellular signaling cascades that drive differentiation 14 . As keratinocytes transition from growth to differentiation, they undergo proliferation, cell cycle arrest in the G 0 /G 1 phase, and increased expression of early differentiation markers such as keratin 1 and keratin 10 4 . These markers play critical roles in regulating gene expression during both proliferation and differentiation 26,27 . Involucrin and loricrin, markers of terminal differentiation, are also important indicators of keratinocyte maturation 28,29 , accumulating in the membranes of keratinized cells 30–32 . In our previous work, we identified impaired cell motility and increased cell death as contributing factors to keratinization induced by STS deficiency 33 . To gain further insights into the underlying mechanisms of this phenomenon, we analyzed calcium signaling and differentiation factors using RNA-seq in STS-deficient and STS-overexpressing mouse models. Additionally, we modulated STS expression in keratinocytes to assess calcium influx, CasR levels, and differentiation markers. Therefore, this study provides a deeper understanding of the pathogenesis of skin diseases, such as XLI, caused by STS deficiency. Results STS deficiency amplifies calcium signaling in mouse skin tissue RNA-seq analysis was conducted on skin tissue from STS knockout (KO) and transgenic (TG) mouse models (8-week-old males) to investigate the role of STS in the epidermis. Volcano plots representing approximately 39,000 gene expression levels were generated using a 1.5-fold cutoff for visualization (Fig. 1 A). Using the Mouse Genome Informatics (MGI) database ( https://www.informatics.jax.org/ ), we analyzed the effects of STS deficiency on keratinocyte differentiation. Gene ontology (GO) analysis revealed a significant upregulation of genes related to tyrosine phosphorylation, tyrosine kinase activity, keratinization, calcium signaling, and keratinocyte differentiation in STS KO mice compared to controls (Fig. 1 B) 16,24,25,34 . In contrast, these genes were notably downregulated in STS TG mice (Fig. 1 B). We further identified genes involved in calcium signaling and generated a heatmap that showed a significant upregulation of calcium-related genes in STS KO mice (Fig. 1 C, Table S1 ). These genes regulate calcium binding, dependence, ion regulation, and transport. Gene set enrichment analysis (GSEA) also revealed an upregulation of factors involved in calcium ion transport and regulation in STS KO mice compared to STS TG mice (Fig. 1 D). RNA-seq data indicated elevated expression of calmodulin and calcium/calmodulin-dependent protein kinases, which regulate intracellular calcium levels, in STS KO mice (Fig. 1 E) 35,36 . Furthermore, STS deficiency increased the expression of CasR and calcium-binding proteins such as calsequestrin and parvalbumin in STS KO mice (Fig. 1 F) 37 . We also observed a significant increase in filamin A expression, which stabilizes CasR (Fig. 1 G) 38 . These results suggest that STS deficiency enhances calcium signaling, leading to significant changes in keratinocyte differentiation. STS deficiency increases intracellular calcium influx and upregulates CasR expression To further validate the increase in calcium signaling observed in Fig. 1 , we assessed calcium levels in STS-deficient and overexpressing cells. Confocal microscopy revealed higher calcium content in STS-deficient cells, while STS overexpression reduced calcium influx (Fig. 2 A). Additionally, STS-deficient cells exhibited greater calcium influx when cultured in a high-calcium medium compared to controls. Treatment with the calcium chelator BAPTA-AM 39 reduced this influx, albeit to a lesser extent in STS-deficient cells (Fig. 2 B). These results suggest that inhibiting STS enhances intracellular calcium influx and may activate calcium signaling. Next, we examined CasR expression, a key regulator of calcium homeostasis. RNA-seq analysis confirmed the upregulation of CasR in STS KO mice (Fig. 1 G), a pattern also observed in STS-deficient keratinocytes (Fig. 2 C, D). Treatment with STX-64, an STS inhibitor 40 , led to a dose-dependent increase in CasR expression (Fig. 2 E, F). These findings indicate that the upregulation of CasR following STS inhibition contributes to the enhanced intracellular calcium influx. STS deficiency enhances keratinocyte differentiation in both mouse skin and keratinocytes We investigated the effects of enhanced calcium signaling on keratinocyte differentiation resulting from STS suppression. RNA-seq data revealed an upregulation of genes related to tyrosine kinase activity and tyrosine phosphorylation, which promote differentiation, in STS KO mice compared to controls, while genes related to tyrosine phosphatase activity were downregulated (Fig. 3 A, Table S2 ). Additionally, genes involved in keratinocyte differentiation and keratinization were significantly upregulated (Fig. 3 B, Table S3). Calcium-induced keratinocyte differentiation requires phosphoinositide 3-kinase (PI3K) activation 15 , and RNA-seq data confirmed increased expression of PI3K subunits, including PIK3CA and PIK3CB, in STS-deficient mice (Fig. 3 C). We then analyzed STS-deficient and STS-overexpressing cells to assess the impact of STS suppression on keratinocyte differentiation, which typically occurs during the G 0 /G 1 phase 9 . STS-deficient cells exhibited G 0 /G 1 arrest (Fig. 3 D). Moreover, mRNA levels of differentiation markers, including keratin 1, keratin 10, filaggrin 1, and filaggrin 10, were significantly upregulated in STS-deficient keratinocytes (Fig. 3 E), and protein levels of keratin 1 and keratin 10 were similarly elevated (Fig. 3 D). Treatment of primary cells with STX-64 at concentrations of 0, 0.1, 1, or 10 µM resulted in a dose-dependent increase in keratin 1 and keratin 10 expression. These findings suggest that STS reduction enhances keratinocyte differentiation, thereby promoting keratinization. Increased calcium responsiveness in STS-deficient keratinocytes elevates the expression of late differentiation markers We assessed the expression of E-cadherin and the late differentiation markers involucrin and loricrin, which play key roles in keratinocyte differentiation 15,28,31 . Inhibiting STS activity significantly increased their expression (Fig. 4 A, B), with a similar pattern observed in STS-overexpressing cells (Fig. 4 C, D). Conversely, treatment with TNF-α, a known inducer of STS 1,41 , reduced the expression of these markers as STS levels increased (Fig. 4 E, F). This upregulation of E-cadherin and late differentiation markers was also observed in mouse primary keratinocytes (Fig. 4 G, H), indicating that reducing STS affects both early and late stages of keratinocyte differentiation. Furthermore, we analyzed the expression of E-cadherin, involucrin, and loricrin in response to calcium treatment in keratinocytes. The expression of these factors increased in a calcium concentration-dependent manner (Fig. 5 A). Calcium treatment significantly upregulated all markers in STS-deficient cells, with E-cadherin and involucrin levels increasing by 5.7- and 7.1-fold, respectively, compared to controls. These markers were less sensitive to BAPTA-AM-induced calcium suppression, showing only a 32.3% and 21.2% decrease, respectively. Co-treatment with BAPTA-AM and calcium restored their expression to levels similar to those seen in untreated STS-deficient cells (Fig. 5 B). In contrast, calcium treatment in STS-overexpressing cells increased E-cadherin, involucrin, and loricrin expression by 1.85- to 2.36-fold, but this increase was smaller compared to STS-deficient cells (Fig. 5 C). Overall, STS inhibition significantly enhanced responsiveness to calcium, leading to upregulated expression of E-cadherin, involucrin, and loricrin. Discussion Our study used RNA-seq data from STS KO and TG mouse models to investigate the impact of STS deficiency on skin diseases, particularly XLI (Fig. 1 A, B). We focused on genes and signaling pathways involved in keratinocyte differentiation, given the abnormal keratinization observed in XLI 1,2 . Our findings demonstrate that STS deficiency amplifies calcium signaling and accelerates keratinocyte differentiation, likely contributing to the abnormal keratinization seen in skin conditions such as XLI. These insights provide a deeper understanding of the role of STS deficiency in the pathogenesis of skin disorders. Calcium is a crucial factor in keratinocyte differentiation 8,9 , and patients with XLI have been shown to exhibit elevated calcium levels in the stratum corneum 42 . Our study confirmed that STS deficiency intensifies calcium signaling in mouse skin. RNA-seq analysis revealed a significant upregulation of genes related to calcium signaling, binding, and ion transport in STS-deficient mice (Fig. 1 C-G). This finding aligns with the increased intracellular calcium levels observed in STS-deficient keratinocytes (Fig. 2 A, B), further supporting the key role of calcium as a regulator of keratinocyte differentiation and skin barrier formation. The heightened calcium signaling in STS KO mice likely drives excessive keratinocyte differentiation, contributing to impaired keratinization. Additionally, STS-deficient keratinocytes showed increased expression of CasR (Fig. 2 C-F), supporting the hypothesis that STS modulates calcium homeostasis. CasR regulates extracellular calcium and triggers calcium-dependent signaling within keratinocytes, promoting calcium influx 14,43 . These findings suggest that STS deficiency upregulates CasR, resulting in increased intracellular calcium influx and enhanced calcium signaling. However, further research is needed to clarify the mechanisms by which STS regulates CasR expression. STS deficiency also enriched genes related to tyrosine kinases and phosphorylation, which are key drivers of differentiation and cell adhesion during calcium-induced keratinocyte differentiation (Fig. 3 A). Genes involved in keratinocyte differentiation and keratinization were similarly upregulated (Fig. 3 B, C), showing increased expression of E-cadherin, keratin 1, keratin 10, involucrin, and loricrin (Fig. 3 E-G, Fig. 4 ). E-cadherin plays a crucial role in mediating calcium-induced cell adhesion and promoting keratinocyte differentiation 44 . The enhanced expression of E-cadherin, alongside its role in cell-cell adhesion, suggests that STS may influence keratinocyte differentiation and adhesion through calcium signaling. Several studies have examined the interactions between CasR, filamin A, and E-cadherin 15,38,44 . Extracellular calcium induces differentiation by activating CasR-mediated signaling and E-cadherin-mediated adhesion 15 . The sequential binding of catenins such as E-cadherin to the cytoskeleton and other signaling molecules, including PI3K 21,45 . PI3K activation is essential for calcium-induced keratinocyte differentiation, and CasR has been shown to regulate the E-cadherin/PI3K pathway via Src family kinases 15 . The increased expression of PI3K observed in STS KO mice is consistent with this regulatory pathway (Fig. 3 C). Additionally, STS-deficient keratinocytes demonstrated heightened sensitivity to calcium, along with a significant increase in differentiation markers. Thus, STS deficiency not only upregulates E-cadherin expression but also strengthens E-cadherin-mediated adhesion in response to external calcium, potentially leading to dysregulation of the differentiation process. In summary, STS plays a key role in regulating calcium signaling and keratinocyte differentiation. STS deficiency increases intracellular calcium influx and signaling, driving the expression of crucial differentiation markers (Fig. 6 ). These findings offer valuable insights into the mechanisms underlying STS deficiency-related skin disorders, such as XLI, and suggest potential therapeutic strategies that target calcium signaling modulation. Materials and Methods Cell culture. HaCaT, a human keratinocyte cell line, was obtained from CLS Cell Lines Service (Germany). HaCaT, pLjm1-Control HaCaT, and pLjm1-STS HaCaT cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Cas9-control HaCaT and Cas9-STS +/− HaCaT cell lines were grown in DMEM with 20% (v/v) FBS, along with the same concentrations of penicillin and streptomycin. All cell lines were incubated at 37°C in a humidified atmosphere containing 5% CO 2 . After 24 hours of incubation, cells were treated with TNF-α or STX-64 for either 24 or 48 hours. Reagents. STX-64 and Ultra Cruz™ mounting medium (sc-24941) were purchased from Santa Cruz Biotechnology (Dallas, TX). TNF-α and MG132 were purchased from Enzo Biochem (Farmingdale, NY), and CaCl 2 , and cycloheximide from Sigma-Aldrich (Burlington, MA). DMEM was obtained from HyClone (Logan, UT), while Fluo-4 AM was acquired from Glpbio (California, CA). FBS and charcoal-stripped FBS were supplied by Tissue Culture Biologicals (Long Beach, CA). The Neon™ Transfection System, BAPTA-AM, and Hoechst 33258 were procured from Thermo Fisher Scientific (Waltham, MA), and the D-Plus™ ECL solution from Dongin LS (Seoul, Korea). Except for TNF-α (dissolved in triple-distilled water), all chemicals were prepared in dimethylsulfoxide (DMSO), stored in small aliquots at − 20°C, and diluted in cell culture media prior to use. The following antibodies were used in our experiments: anti-STS polyclonal antibody (ab62219), involucrin antibody (ab53112), and loricrin antibody (ab85679) from Abcam (Cambridge, MA); cytokeratin 1 antibody (ab93652) and cytokeratin 10 antibody (ab76318) from Proteintech (Rosemont, IL); goat anti-rabbit IgG-Texas Red antibody (sc-2780) and CaSR antibody (6D4) from Santa Cruz Biotechnology (Santa Cruz, CA); E-cadherin antibody (07-697) from Merck Millipore (Burlington, MA); and GAPDH antibody from Cusabio Technology (Houston, TX). All other chemicals were sourced from commercial suppliers. Stable transfection. The STS coding sequence was cloned into the pcDNA3.1/Zeo + vector for transient transfection experiments. For lentiviral production, the plasmid vectors pLJM1-Empty (provided by Joshua Mendell, Addgene plasmid #91980), pLKO.1 puro (provided by Bob Weinberg, Addgene plasmid #8453), pMD2.G, and psPAX2 (provided by Didier Trono, Addgene plasmids #12259 and #12260) were utilized. HEK293T cells were co-transfected with pLJM1-STS, pMD2.G, and psPAX2. After 48 hours, the lentiviral supernatant containing the STS gene was collected and used to infect HaCaT cells in the presence of polybrene (8 µg/mL) for 24 hours. HaCaT cells stably overexpressing STS were selected with puromycin (1 µg/mL). Generation of Cas9-STS +/− HaCaT cells. The plasmid vectors pLentiCas9-T2A-GFP (Addgene plasmid #78548, a gift from Roderic Guigo and Rory Johnson) and tet-pLKO-sgRNA-puro (Addgene plasmid #104321, a gift from Nathanael Gray) were obtained from Addgene. Lentiviral supernatants containing either the Cas9 gene or STS-specific sgRNA were generated using HEK293T cells. Cas9-STS +/− HaCaT cells were then isolated through single-cell cloning into 96-well plates. Quantitative reverse transcription PCR (RT-qPCR). RT-qPCR analysis was performed using the Rotor-Gene Q system (Qiagen, Netherlands) with data processed using the QIAGEN Rotor-Gene Q Series software. Each RT-qPCR reaction mixture consisted of 10 µL of Q Green 2× qPCR Master Mix, 1 µM of specific oligonucleotide primers, and 20 ng of cDNA, totaling a volume of 20 µL. The amplification protocol included an initial denaturation at 95°C for 5 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing/extension at 56°C for 45 seconds. The following primer sets were used for RT-qPCR: 18S rRNA : 5′-GTA ACC CGT TGA ACC CCA TT-3′ and 5′-CCA TCC AAT CGG TAG TAG CG-3′; Keratin 1 (mouse) : 5′- GAG CAG ATC AAG TCA CTC AAT GA -3′ and 5′- CCC ATT TGG TTT GTA GCA CCT − 3′; Keratin 10 (mouse) : 5′- GCC TCC TAC ATG GAC AAA GTC − 3′ and 5′- GCC TCC TAC ATG GAC AAA GTC − 3′; Filaggrin 1 (mouse) : 5′- ATG TCC GCT CTC CTG GAA AG -3′ and 5′- TGG ATT CTT CAA GAC TGC CTG TA -3′; and Filaggrin 2 (mouse) : 5′- CTA GAG GGC ATG AGT TAG TCA − 3′ and 5′- CAA GAC TGG ACA GTT GGC TGG − 3′. Western blotting analysis. Cells and mouse skin samples were lysed in ice-cold PE buffer containing 50 mM NaF. The protein lysates were separated by SDS-PAGE on 8%, 10%, or 15% polyacrylamide gels and then transferred onto 0.45 µm PVDF membranes via electrophoresis. Membranes were blocked for 2 hours at 4°C with 5% (w/v) bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBST). Following blocking, the membranes were incubated with primary antibodies diluted 1:1000 in TBST. After primary antibody incubation, the membranes were treated with secondary antibodies for 2 hours. Protein bands were visualized using D-Plus™ ECL solution (Dongin LS, Korea) and analyzed on a ChemiDoc XRS system (Bio-Rad, CA). Immunofluorescence. Samples were fixed in 10% neutral formalin for 30 minutes at room temperature (24°C), then blocked for 45 minutes in PBS containing 10% goat serum and 0.2% Triton X-100. After blocking, the samples were incubated overnight at 4°C with a primary antibody diluted at a 1:200 ratio. The following day, they were treated with goat anti-rabbit IgG conjugated to Alexa Fluor 594 (1:200) for overnight staining. After three washes with PBS, coverslips were mounted on glass slides using Ultra Cruz™ mounting medium. Fluorescence signals were visualized using a confocal laser scanning microscope (LSM 800, Carl Zeiss, Germany). Ethics statement. This study complies with all relevant ethical regulations. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Chung-Ang University (protocol numbers 2017-00096 and 2019-00003) and were reviewed and conducted in accordance with approved guidelines. All animal experiment reporting complies with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Experimental mice were bred and maintained in the clean animal facility of Chung-Ang University. Two to three mice were housed per cage and maintained at 20 to 24°C with a 12-hour light/dark cycle. Food and water were provided ad libitum to all mice. Room temperature was maintained at 20 to 26°C, daily temperature changes were kept below 4°C, and relative humidity was maintained at 40 to 60%. Neonatal mice were euthanized by physical means and adult mice by CO2 inhalation, both in accordance with IACUC guidelines. Mouse primary keratinocytes. Primary epidermal keratinocytes were isolated from the skin of one-day-old neonatal C57BL/6 mice. The skin was removed as a single sheet and incubated overnight at 4°C in 0.25% trypsin. The epidermis and dermis were then mechanically separated using forceps, and the epidermal cells were gently scraped off. The collected cells were centrifuged and resuspended in minimum essential medium (MEM) supplemented with 1.3 mM calcium. The cell suspension was passed through a 100 µm cell strainer, centrifuged again, and then seeded onto plates in 10% MEM medium. All animal procedures were conducted in accordance with protocols approved by the institutional animal care and use committee. Generation of STS TG and STS KO mice. To generate STS transgenic mice, a 7,088-bp fragment of the human STS gene, including the CMV promoter, was synthesized and inserted into the AmpR site of the pcDNA3.1 vector, creating the STS plasmid for microinjection. The plasmid was digested with PvuI and AvrII restriction enzymes, removing the 4.4-kb backbone fragment and isolating the desired 2.2-kb fragment via gel extraction. This STS DNA was injected into the male pronucleus of zygotes, which were transferred into the oviducts of pseudopregnant female mice. After the F0 generation was born, genotyping was performed using PCR on tail samples to confirm the presence of the STS gene, with a forward primer specific to the CMV promoter and a reverse primer for the human STS gene. For the generation of STS KO mice, a Cas9 targeting vector was constructed, incorporating guide RNAs (gRNAs) designed to induce double-strand breaks (DSBs) in the Cas9 gene, alongside a selectable marker such as the puromycin resistance gene. This vector was introduced into embryonic stem (ES) cells via PEG-mediated transfection, and the transfected cells were cultured under puromycin selection to isolate cells that incorporated the targeting vector. Successful targeting events were confirmed using PCR and Southern blot analysis, with PCR primers amplifying regions surrounding the Cas9 gene and the targeting vector, and Southern blot probes hybridizing to specific sites within the Cas9 gene. Successfully targeted ES cells were injected into blastocysts, which were transferred into pseudopregnant females to generate chimeric mice. These chimeric mice contained a mixture of cells from both the original blastocyst and the injected ES cells. All animal experiments adhered to the guidelines of the Institutional Animal Care and Use Committee at Chung-Ang University. The STS transgenic mice were maintained under pathogen-free conditions at Macrogen, Inc. (Seoul, Korea). RNA-seq. Skin tissue samples from 8-week-old male mice were used for RNA-seq. Total RNA was extracted and processed to prepare libraries using the NEB Next Ultra II Directional RNA-Seq Kit (New England Biolabs, UK). Poly(A) RNA was isolated with the Poly(A) RNA Selection Kit (Lexogen, Austria), and the resulting mRNA was used for cDNA synthesis and shearing according to the manufacturer’s instructions. Illumina indexes 1–12 were utilized for sample indexing, with PCR included for enrichment. Fragment sizes were assessed using the Agilent 2100 Bioanalyzer and the DNA High Sensitivity Kit. Library quantification was performed using the Library Quantification Kit and a Step One Real-Time PCR System (Life Technologies, CA). High-throughput paired-end 100 sequencing was carried out on a HiSeq X10 sequencer (Illumina, CA). Transcriptome analysis. Mouse skin samples were sequenced following the standard Illumina protocol (Shanghai Bingwang Biotechnology Co., Ltd.). Sequencing reads were aligned to the mouse genome (Mm9) using HISAT2 (v2.1), and gene counts were estimated with ‘HTSeq.’ Differential expression analysis was conducted using the ‘DESeq2’ R package. Genes with a fold-change > 2 and an adjusted p -value < 0.05 were considered differentially expressed genes (DEGs). GO enrichment analysis for the DEGs was performed using the ‘topGO’ R package, while GSEA was carried out according to the GSEA manual. Statistical analysis. Dunnett’s pairwise multiple comparison t -test was performed using the GraphPad Prism 7 software (GraphPad Software, CA). Differences were considered statistically significant at * p < 0.05. Abbreviations CasR Calcium-sensing receptor GO Gene Ontology GSEA Gene set enrichment analysis PI3K phosphoinositide 3-kinase RNA-seq RNA-sequencing STS Steroid sulfatase TNF-α Tumor necrosis factorα XLI X-linked ichthyosis. Declarations Compliance with ethical standards All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Chung-Ang University (protocol numbers 2017-00096 and 2019-00003) and were conducted in accordance with the approved guidelines. Corresponding author Correspondence should be addressed to Young-Jin Chun. Conflict of interest The authors declare no conflicts of interest. Data and materials availability All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Funding This research was supported by the National Research Foundation of Korea (NRF), funded by the Korean government (MSIP) (NRF-2021R1A2C201239514 and NRF-2022R1A5A600076013), and by the Ministry of Food and Drug Safety of South Korea (22183MFDS366) in 2022–2025. The funding agencies had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript. Author Contribution T. U. Kwon designed and conducted the study; T. U. Kwon, Y. J. Kwon, H. Park, and Y. J. Kang analyzed the data; and T. U. Kwon and Y. J. Chun wrote the manuscript. Data Availability Data and materials availabilityAll data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. References Reed, M. J., Purohit, A., Woo, L. W. L., Newman, S. P. & Potter, B. V. L. Steroid sulfatase: Molecular biology, regulation, and inhibition. Endocr. Rev. 26 , 171–202, https://doi.org/10.1210/er.2004-0003 (2005). Mueller, J. 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L., Chang, W., Xie, Z. & Bikle, D. D. Inactivation of the calcium sensing receptor inhibits E-cadherin-mediated cell-cell adhesion and calcium-induced differentiation in human epidermal keratinocytes. J. Biol. Chem. 283 , 3519–3528, https://doi.org/10.1074/jbc.M708318200 (2008). Calautti, E. et al. Tyrosine phosphorylation and src family kinases control keratinocyte cell-cell adhesion. J. Cell Biol. 141 , 1449–1465, https://doi.org/10.1083/jcb.141.6.1449 (1998). O'Keefe, E. J., Briggaman, R. A. & Herman, B. Calcium-induced assembly of adherens junctions in keratinocytes. J. Cell Biol. 105 , 807–817, https://doi.org/10.1083/jcb.105.2.807 (1987). Lewis, J. E., Jensen, P. J. & Wheelock, M. J. Cadherin function is required for human keratinocytes to assemble desmosomes and stratify in response to calcium. J. Invest. Dermatol. 102 , 870–877, https://doi.org/10.1111/1523-1747.ep12382690 (1994). Nam, M. W., Kim, C. W. & Choi, K. C. Epithelial-mesenchymal transition-inducing factors involved in the progression of lung cancers. Biomol. Ther. (Seoul) 30 , 213–220, https://doi.org/10.4062/biomolther.2021.178 (2022). Pokutta, S. & Weis, W. I. Structure and mechanism of cadherins and catenins in cell-cell contacts. Annu. Rev. Cell Dev. Biol. 23 , 237–261, https://doi.org/10.1146/annurev.cellbio.22.010305.104241 (2007). Perez-Moreno, M., Jamora, C. & Fuchs, E. Sticky business: Orchestrating cellular signals at adherens junctions. Cell 112 , 535–548, https://doi.org/10.1016/S0092-8674(03)00108-9 (2003). Furukawa, F. et al. Cadherins in cutaneous biology. J. Dermatol. 21 , 802–813, https://doi.org/10.1111/j.1346-8138.1994.tb03294.x (1994). Young, P. et al. E-cadherin controls adherens junctions in the epidermis and the renewal of hair follicles. EMBO J. 22 , 5723–5733, https://doi.org/10.1093/emboj/cdg560 (2003). Zhao, Y., Sudol, M., Hanafusa, H. & Krueger, J. 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Dermatol. 81 , 100s-103s, https://doi.org/10.1111/1523-1747.ep12540786 (1983). Shen, C. S. et al. Premature apoptosis of keratinocytes and the dysregulation of keratinization in porokeratosis. Br. J. Dermatol. 147 , 498–502, https://doi.org/10.1046/j.1365-2133.2002.04853.x (2002). Baek, H. S., Kwon, T. U., Shin, S., Kwon, Y. J. & Chun, Y. J. Steroid sulfatase deficiency causes cellular senescence and abnormal differentiation by inducing Yippee-like 3 expression in human keratinocytes. Sci. Rep. 11 , 20867, https://doi.org/10.1038/s41598-021-00051-w (2021). Steinert, P. M. & Marekov, L. N. The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope. J. Biol. Chem. 270 , 17702–17711, https://doi.org/10.1074/jbc.270.30.17702 (1995). Jung, M. H., Jung, S. M. & Shin, H. S. Co-stimulation of HaCaT keratinization with mechanical stress and air-exposure using a novel 3D culture device. Sci. Rep. 6 , 33889, https://doi.org/10.1038/srep33889 (2016). Kwon, T. U. et al. Unraveling the molecular mechanisms of cell migration impairment and apoptosis associated with steroid sulfatase deficiency: Implications for X-linked ichthyosis. Biochim. Biophys. Acta. Mol. Basis. Dis. , 167004, https://doi.org/10.1016/j.bbadis.2023.167004 (2024). Xie, Z., Singleton, P. A., Bourguignon, L. Y. & Bikle, D. D. Calcium-induced human keratinocyte differentiation requires src- and fyn-mediated phosphatidylinositol 3-kinase-dependent activation of phospholipase C-gamma1. Mol. Biol. Cell. 16 , 3236–3246, https://doi.org/10.1091/mbc.e05-02-0109 (2005). Zhang, M. et al. Structural basis for calmodulin as a dynamic calcium sensor. Structure 20 , 911–923, https://doi.org/10.1016/j.str.2012.03.019 (2012). Junho, C. V. C., Caio-Silva, W., Trentin-Sonoda, M. & Carneiro-Ramos, M. S. An Overview of the Role of Calcium/Calmodulin-Dependent Protein Kinase in Cardiorenal Syndrome. Front. Physiol. 11 , 735, https://doi.org/10.3389/fphys.2020.00735 (2020). Elies, J. et al. An Update to Calcium Binding Proteins. Adv. Exp. Med. Biol. 1131 , 183–213, https://doi.org/10.1007/978-3-030-12457-1_8 (2020). Tu, C. L., Chang, W. & Bikle, D. D. The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and filamin A. J. Invest. Dermatol. 131 , 1119–1128, https://doi.org/10.1038/jid.2010.414 (2011). Tsien, R. Y. A Non-disruptive technique for loading calcium buffers and indicators into cells. Nature 290 , 527–528, https://doi.org/10.1038/290527a0 (1981). Nussbaumer, P. & Billich, A. Steroid sulfatase inhibitors. Med. Res. Rev. 24 , 529–576, https://doi.org/10.1002/med.20008 (2004). Suh, B. Y. et al. Induction of steroid sulfatase expression by tumor necrosis factor-α through phosphatidylinositol 3-kinase/Akt signaling pathway in PC-3 human prostate cancer cells. Exp. Mol. Med. 43 , 646–652, https://doi.org/10.3858/emm.2011.43.11.073 (2011). Elias, P. M., Williams, M. L., Choi, E. H. & Feingold, K. R. Role of cholesterol sulfate in epidermal structure and function: lessons from X-linked ichthyosis. Biochim. Biophys. Acta. 1841 , 353–361, https://doi.org/10.1016/j.bbalip.2013.11.009 (2014). Tu, C. L., Oda, Y., Komuves, L. & Bikle, D. D. The role of the calcium-sensing receptor in epidermal differentiation. Cell Calcium 35 , 265–273, https://doi.org/10.1016/j.ceca.2003.10.019 (2004). Tu, C. L. & You, M. Obligatory roles of filamin A in E-cadherin-mediated cell-cell adhesion in epidermal keratinocytes. J. Dermatol. Sci. 73 , 142–151, https://doi.org/10.1016/j.jdermsci.2013.09.007 (2014). Wheelock, M. J. & Johnson, K. R. Cadherin-mediated cellular signaling. Curr. Opin. Cell Biol. 15 , 509–514, https://doi.org/10.1016/s0955-0674(03)00101-7 (2003). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-5115056\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":372142892,\"identity\":\"cf21bba5-a12e-4e2e-b76e-29917c1a4462\",\"order_by\":0,\"name\":\"Tae-Uk Kwon\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chung-Ang University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Tae-Uk\",\"middleName\":\"\",\"lastName\":\"Kwon\",\"suffix\":\"\"},{\"id\":372142896,\"identity\":\"1a0e9a59-f597-4c7c-aff3-516295f2d771\",\"order_by\":1,\"name\":\"Yeo-Jung Kwon\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chung-Ang University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yeo-Jung\",\"middleName\":\"\",\"lastName\":\"Kwon\",\"suffix\":\"\"},{\"id\":372142898,\"identity\":\"b9e168ed-76f4-4198-b81c-2195f84f0685\",\"order_by\":2,\"name\":\"Hyemin Park\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chung-Ang University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hyemin\",\"middleName\":\"\",\"lastName\":\"Park\",\"suffix\":\"\"},{\"id\":372142899,\"identity\":\"9fba675b-061b-4dfb-ba3f-f7b8395f338e\",\"order_by\":3,\"name\":\"Yoon-ji Kang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Chung-Ang University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yoon-ji\",\"middleName\":\"\",\"lastName\":\"Kang\",\"suffix\":\"\"},{\"id\":372142901,\"identity\":\"13d46446-af0d-4ab3-b19d-21ff25fedc48\",\"order_by\":4,\"name\":\"Young-Jin Chun\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYFCCA2xAwgbGSyBaSxpJWhhAWg6ToMXg4OFnj3l3nJfnn5HA+OEHQ1o+YS0Hjpkb8565bTjjRgKzZA9DjmUDYS1n2KR5224nMNxIYJBmYKgwIMIWsJZzCfJAW36TouVAgsGNBDagLTmEtUgeOGYmObct2XDjmYdtlj0GaYS18N04/EzibZudvNzx5MM3flQkE9aicOMAjMnYAHQnQQ0MDPL9DUSoGgWjYBSMgpENAP4DO631TKsaAAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"Chung-Ang University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Young-Jin\",\"middleName\":\"\",\"lastName\":\"Chun\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-09-19 08:01:36\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-5115056/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-5115056/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1038/s41598-024-84701-9\",\"type\":\"published\",\"date\":\"2025-03-21T15:56:49+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":71234174,\"identity\":\"c830941d-06d9-41d3-bd7b-1bbc0cb9559e\",\"added_by\":\"auto\",\"created_at\":\"2024-12-12 11:26:16\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":7479506,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSTS deficiency enhances calcium signaling in mouse skin.\\u003c/strong\\u003e Volcano plots, heat maps, and GSEA of RNA-seq data from STS KO, STS TG, and normal mouse skin. \\u003cstrong\\u003e(A)\\u003c/strong\\u003e Volcano plots showing DEGs in STS KO vs. normal, STS TG vs. normal, and STS TG vs. STS KO mouse skin. Upregulated genes are shown in red, downregulated genes in green. The fold-change threshold is set at 1.5, with a significance level of \\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt;0.05. \\u003cstrong\\u003e(B) \\u003c/strong\\u003eGene ontology analysis of upregulated (red) and downregulated (green) gene clusters in STS KO and STS TG mouse skin. The numbers above the bars indicate the number of genes in each category. \\u003cstrong\\u003e(C) \\u003c/strong\\u003eHeatmaps depicting expression levels of genes related to calcium signaling, binding, dependency, ion regulation, and transport in STS KO, STS TG, and normal mouse skin. Expression levels are adjusted and normalized on a log\\u003csub\\u003e2\\u003c/sub\\u003e scale. Upregulation and downregulation are indicated in red and green, respectively. \\u003cstrong\\u003e(D) \\u003c/strong\\u003eGSEA results revealing significant enrichment of calcium ion transport, calcium ion-regulated, and calcium channel complex genes in STS KO and STS TG mouse skin. \\u003cstrong\\u003e(E) \\u003c/strong\\u003eRNA-seq analysis of gene expression in calmodulins and calcium/calmodulin-dependent protein kinases. Data are presented as mean ± SD (n = 3). Statistical significance is indicated by *, \\u003csup\\u003e#\\u003c/sup\\u003e\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05. \\u003cstrong\\u003e(F) \\u003c/strong\\u003eRNA-seq analysis of gene expression in calcium-sensing receptors and calcium-binding proteins. Data are presented as mean ± SD (n = 3). Statistical significance is indicated by *, \\u003csup\\u003e#\\u003c/sup\\u003e\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05. \\u003cstrong\\u003e(G) \\u003c/strong\\u003eRNA-seq analysis of Filamin A gene expression. Data are presented as mean ± SD (n = 3). Statistical significance is indicated by *, \\u003csup\\u003e#\\u003c/sup\\u003e\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5115056/v1/70dbe98bc93ac95042cf17c1.png\"},{\"id\":71234177,\"identity\":\"24838db6-20fc-4ca4-aae1-d1b295f168e0\",\"added_by\":\"auto\",\"created_at\":\"2024-12-12 11:26:16\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":6476670,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eReduced STS expression in keratinocytes increases intracellular calcium influx and upregulates CasR (A) \\u003c/strong\\u003eRepresentative confocal microscopy images of Cas9- and pLJM1-HaCaT cells. Intracellular calcium was stained with Fluo-4 AM (green), and nuclei were stained with Hoechst-33258 (blue). Scale bar = 20 μm. \\u003cstrong\\u003e(B) \\u003c/strong\\u003eRepresentative confocal microscopy images of Cas9-HaCaT cells treated with calcium (140 nM) and BAPTA-AM (5 μM). Intracellular calcium was stained with Fluo-4 AM (green), and nuclei were stained with Hoechst-33258 (blue). Scale bar = 20 μm. \\u003cstrong\\u003e(C)\\u003c/strong\\u003e Western blot analysis of total cellular protein (20 μg) from Cas9- and pLJM1-HaCaT cells, with GAPDH as a loading control. The blot shows STS and CasR protein levels. Data are expressed as mean ± SEM of three experiments. *\\u003cem\\u003ep \\u003c/em\\u003e\\u0026lt; 0.05 compared to the control. \\u003cstrong\\u003e(D, F)\\u003c/strong\\u003e Immunofluorescence staining of CasR. Cells were fixed, incubated with CasR antibodies, and stained with Alexa Fluor 594-labeled secondary antibody. Images were captured using fluorescence microscopy. Blue = DAPI; scale bar = 20 μm. \\u003cstrong\\u003e(D)\\u003c/strong\\u003e Representative immunofluorescence images showing CasR expression in Cas9- or pLJM1-HaCaT cells. \\u003cstrong\\u003e(E, F)\\u003c/strong\\u003e HaCaT cells treated with STX-64 (0, 0.1, 1, or 10 μM) for 48 hours. \\u003cstrong\\u003e(E)\\u003c/strong\\u003e Western blot analysis of total cellular protein (20 μg) showing CasR protein levels in HaCaT cells, with GAPDH as a loading control. \\u003cstrong\\u003e(F) \\u003c/strong\\u003eRepresentative immunofluorescence images of CasR expression in HaCaT cells.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5115056/v1/0483b4338ee3bfeed1610a5c.png\"},{\"id\":71234173,\"identity\":\"ed503837-0a99-4512-b936-9871a43b85cd\",\"added_by\":\"auto\",\"created_at\":\"2024-12-12 11:26:16\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3487849,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSuppression of STS expression promotes differentiation of keratinocytes. (A) \\u003c/strong\\u003eHeatmaps showing the expression of tyrosine kinase, tyrosine phosphorylation, and tyrosine phosphatase-related genes in STS KO, STS TG, and normal mouse skin. \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Heatmaps of keratinocyte differentiation and keratinization genes in STS KO, STS TG, and normal mouse skin. \\u003cstrong\\u003e(C)\\u003c/strong\\u003e RNA-seq analysis of gene expression in PI3K subunits. Data are expressed as mean ± SD (n = 3). *, \\u003csup\\u003e#\\u003c/sup\\u003e\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05. \\u003cstrong\\u003e(D)\\u003c/strong\\u003e Cell cycle distribution was measured using a Muse cell cycle kit. Data are presented as mean ± SD (n = 3). \\u003cstrong\\u003e(E)\\u003c/strong\\u003e Real-time qPCR analysis of mRNA expression levels for keratin 1, keratin 10, filaggrin 1, and filaggrin 2 in STS KO and STS TG mouse skin tissues. Data are expressed as mean ± SEM of three experiments. *\\u003cem\\u003ep \\u003c/em\\u003e\\u0026lt; 0.05 compared to the control. \\u003cstrong\\u003e(F, G)\\u003c/strong\\u003e Western blot analysis of total cellular protein (20 μg) with GAPDH as a loading control. Data are presented as mean ± SEM of three experiments. *\\u003cem\\u003ep \\u003c/em\\u003e\\u0026lt; 0.05 compared to the control. \\u003cstrong\\u003e(F)\\u003c/strong\\u003e Representative western blot image showing STS, keratin 1, and keratin 10 protein levels in Cas9-STS\\u003csup\\u003e+/-\\u003c/sup\\u003e and pLJM1-STS HaCaT cells. \\u003cstrong\\u003e(G)\\u003c/strong\\u003e Primary mouse keratinocytes treated with STX-64 (0, 0.1, 1, or 10 μM) for 48 hours. Representative western blot image displaying keratin 1 and keratin 10 protein levels.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5115056/v1/8a7a125f2d0bd9dd67184cdd.png\"},{\"id\":71235351,\"identity\":\"f3724b95-e0a7-48bc-9874-1fffe0e1e72e\",\"added_by\":\"auto\",\"created_at\":\"2024-12-12 11:42:16\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":11415233,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eInhibition of STS activity upregulates the expression of E-cadherin, involucrin, and loricrinin keratinocytes.\\u003c/strong\\u003e \\u003cstrong\\u003e(A, C, E-G)\\u003c/strong\\u003e Western blot analysis of total cellular protein (20 μg), with GAPDH as a loading control. Data are presented as mean ± SEM of three experiments. *\\u003cem\\u003ep \\u003c/em\\u003e\\u0026lt; 0.05 compared to the control. \\u003cstrong\\u003e(A, C)\\u003c/strong\\u003eRepresentative western blot images showing E-cadherin, involucrin, and loricrinprotein levels. \\u003cstrong\\u003e(A, B)\\u003c/strong\\u003e HaCaT cells were treated with STX-64 (0, 0.1, 1, or 10 μM) for 48 hours. \\u003cstrong\\u003e(B, D)\\u003c/strong\\u003e Immunofluorescence staining was conducted. Cells were fixed, incubated with antibodies against E-cadherin, involucrin, and loricrin, and stained with Alexa Fluor 594-labeled secondary antibody. Images were captured using fluorescence microscopy. Blue = DAPI; scale bar = 20 μm. \\u003cstrong\\u003e(C, D)\\u003c/strong\\u003e pLJM1-HaCaT cells were treated with STX-64 (0 or 10 μM) for 48 hours. \\u003cstrong\\u003e(D)\\u003c/strong\\u003e Representative immunofluorescence images showing E-cadherin, involucrin, and loricrin expression in Cas9 or pLJM1-HaCaT cells. \\u003cstrong\\u003e(E, F)\\u003c/strong\\u003e Representative western blot images showing STS, E-cadherin, involucrin, and loricrin protein levels. \\u003cstrong\\u003e(E)\\u003c/strong\\u003e HaCaT cells treated with TNF-α (0, 25, 50, or 100 ng/ml) for 48 hours. \\u003cstrong\\u003e(F)\\u003c/strong\\u003eCas9-HaCaT cells treated with TNF-α (0 or 100 ng/ml) for 48 hours.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5115056/v1/e9edcbc5b1b5b8f701340cba.png\"},{\"id\":71234181,\"identity\":\"d6eb8d83-9b3c-4d3d-9af7-0f7ec07aaa1d\",\"added_by\":\"auto\",\"created_at\":\"2024-12-12 11:26:18\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1358355,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCalcium significantly increases differentiation marker expression in STS-deficient keratinocytes. (A-C) \\u003c/strong\\u003eRepresentative immunofluorescence images of E-cadherin, involucrin, and loricrin expressions in (A) HaCaT cells treated with Ca\\u003csup\\u003e2+\\u003c/sup\\u003e (0, 35, 70, or 140 nM) for 48 hours, \\u003cstrong\\u003e(B) \\u003c/strong\\u003eCas9-HaCaT cells treated with Ca\\u003csup\\u003e2+\\u003c/sup\\u003e (0 or 140 nM), or BAPT-AM (0 or 5μM) for 48 hours, and \\u003cstrong\\u003e(C) \\u003c/strong\\u003epLjm1-HaCaT cells treated with Ca\\u003csup\\u003e2+\\u003c/sup\\u003e (0 or 140 nM) for 48 h. Western blot analysis was performed on total cellular protein (20 μg), with GAPDH serving as a loading control. Data are shown as mean ± SEM of three experiments. *\\u003cem\\u003ep \\u003c/em\\u003e\\u0026lt; 0.05 compared to the control.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5115056/v1/52d22ab759b1212c4f129285.png\"},{\"id\":71234298,\"identity\":\"8d3f1b9b-6ae0-4d9f-a497-4a21e7e6e0ec\",\"added_by\":\"auto\",\"created_at\":\"2024-12-12 11:34:16\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3805389,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eDysregulation of STS contributes to skin disorders such as XLI, characterized by abnormal keratinization. This study used STS-deficient mouse and cell models to show that STS inhibition enhances calcium signaling and keratinocyte differentiation. These findings offer insights into the mechanisms behind hyperkeratinization associated with STS deficiency.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig.6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5115056/v1/e170d1adf2c666509e84a065.png\"},{\"id\":79120361,\"identity\":\"f7cf71cd-2bb8-498d-bde8-6379b20235e8\",\"added_by\":\"auto\",\"created_at\":\"2025-03-24 15:59:35\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":33231138,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5115056/v1/98a1c12f-edc3-4ece-af18-af01a1630a53.pdf\"},{\"id\":71234178,\"identity\":\"8e0dce08-a363-4391-a921-e1fcbc9d3b34\",\"added_by\":\"auto\",\"created_at\":\"2024-12-12 11:26:16\",\"extension\":\"pdf\",\"order_by\":8,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":335101,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Sourcedatawesternblot.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5115056/v1/e0d02af552d4919faf09bbf1.pdf\"},{\"id\":71234179,\"identity\":\"0c1d1c88-d609-4951-b7b3-fb57621e4e22\",\"added_by\":\"auto\",\"created_at\":\"2024-12-12 11:26:16\",\"extension\":\"xlsx\",\"order_by\":9,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":695172,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Sourcedata.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5115056/v1/b4766952b2e54e4521767b9f.xlsx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Dysregulation of STS in keratinocytes promotes calcium signaling and hyperkeratinization: Insights into the mechanisms underlying X-linked ichthyosis\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eSteroid sulfatase (STS), an enzyme found in varying amounts across nearly all tissues, desulfates steroid hormones such as cholesterol sulfate and dehydroepiandrosterone sulfate (DHEAS), converting them into their biologically active forms\\u003csup\\u003e1,2\\u003c/sup\\u003e. In skin tissue, STS is predominantly localized in the epidermis\\u003csup\\u003e1\\u003c/sup\\u003e. Dysregulation of STS has been linked to several skin conditions. For example, elevated plasma DHEAS levels are associated with conditions such as androgenetic alopecia and hirsutism\\u003csup\\u003e3\\u003c/sup\\u003e. Notably, STS deficiency is linked to X-linked ichthyosis (XLI), a congenital disorder characterized by abnormal keratinization, resulting in large, dark brown scaly patches and a thickened stratum corneum\\u003csup\\u003e1,2\\u003c/sup\\u003e. These symptoms are thought to result from the accumulation of cholesterol sulfate\\u003csup\\u003e1,2\\u003c/sup\\u003e, although the precise mechanisms underlying STS deficiency-induced keratinization remain unclear.\\u003c/p\\u003e \\u003cp\\u003eThickening of the stratum corneum primarily occurs due to excessive keratinocyte differentiation and stratification, which weakens exfoliation by increasing cell cohesion\\u003csup\\u003e6\\u0026ndash;7\\u003c/sup\\u003e. Calcium is a key regulator of differentiation-related signaling pathways and plays a critical role in keratinocyte differentiation both \\u003cem\\u003ein vivo\\u003c/em\\u003e and \\u003cem\\u003ein vitro\\u003c/em\\u003e\\u003csup\\u003e8,9\\u003c/sup\\u003e. As keratinocytes move through the epidermal layers, calcium gradients drive their differentiation, forming the stratum corneum\\u0026rsquo;s barrier\\u003csup\\u003e9\\u0026ndash;13\\u003c/sup\\u003e. Changes in extracellular calcium concentrations activate calcium-sensing receptors (CasR), triggering intracellular signaling essential for differentiation\\u003csup\\u003e14\\u003c/sup\\u003e. Elevated calcium levels promote intercellular contact formation\\u003csup\\u003e15\\u003c/sup\\u003e, and cadherins such as E-cadherin translocate to the membrane, facilitating the assembly of adherens junctions and desmosomes\\u003csup\\u003e16\\u0026ndash;19\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eUpon calcium stimulation, E-cadherin binds to neighboring cells, while its cytoplasmic tail interacts with catenins to form the core of adherens junctions\\u003csup\\u003e20\\u003c/sup\\u003e. E-cadherin-mediated adhesion is essential for maintaining proper differentiation and remodeling cell-cell interactions\\u003csup\\u003e21,22\\u003c/sup\\u003e. Loss of E-cadherin disrupts adherens junctions and impairs terminal differentiation\\u003csup\\u003e23\\u003c/sup\\u003e. Tyrosine phosphorylation also positively regulates cell adhesion during keratinocyte differentiation\\u003csup\\u003e16\\u003c/sup\\u003e, and Src family kinases such as Src and Fyn exhibit elevated activity in calcium-differentiated keratinocytes\\u003csup\\u003e9,24,25\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eCasR responds to extracellular calcium by triggering intracellular signaling cascades that drive differentiation\\u003csup\\u003e14\\u003c/sup\\u003e. As keratinocytes transition from growth to differentiation, they undergo proliferation, cell cycle arrest in the G\\u003csub\\u003e0\\u003c/sub\\u003e/G\\u003csub\\u003e1\\u003c/sub\\u003e phase, and increased expression of early differentiation markers such as keratin 1 and keratin 10\\u003csup\\u003e4\\u003c/sup\\u003e. These markers play critical roles in regulating gene expression during both proliferation and differentiation\\u003csup\\u003e26,27\\u003c/sup\\u003e. Involucrin and loricrin, markers of terminal differentiation, are also important indicators of keratinocyte maturation\\u003csup\\u003e28,29\\u003c/sup\\u003e, accumulating in the membranes of keratinized cells\\u003csup\\u003e30\\u0026ndash;32\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003eIn our previous work, we identified impaired cell motility and increased cell death as contributing factors to keratinization induced by STS deficiency \\u003csup\\u003e33\\u003c/sup\\u003e. To gain further insights into the underlying mechanisms of this phenomenon, we analyzed calcium signaling and differentiation factors using RNA-seq in STS-deficient and STS-overexpressing mouse models. Additionally, we modulated STS expression in keratinocytes to assess calcium influx, CasR levels, and differentiation markers. Therefore, this study provides a deeper understanding of the pathogenesis of skin diseases, such as XLI, caused by STS deficiency.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSTS deficiency amplifies calcium signaling in mouse skin tissue\\u003c/h2\\u003e \\u003cp\\u003eRNA-seq analysis was conducted on skin tissue from STS knockout (KO) and transgenic (TG) mouse models (8-week-old males) to investigate the role of STS in the epidermis. Volcano plots representing approximately 39,000 gene expression levels were generated using a 1.5-fold cutoff for visualization (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). Using the Mouse Genome Informatics (MGI) database (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.informatics.jax.org/\\u003c/span\\u003e\\u003cspan address=\\\"https://www.informatics.jax.org/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e), we analyzed the effects of STS deficiency on keratinocyte differentiation. Gene ontology (GO) analysis revealed a significant upregulation of genes related to tyrosine phosphorylation, tyrosine kinase activity, keratinization, calcium signaling, and keratinocyte differentiation in STS KO mice compared to controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB)\\u003csup\\u003e16,24,25,34\\u003c/sup\\u003e. In contrast, these genes were notably downregulated in STS TG mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eWe further identified genes involved in calcium signaling and generated a heatmap that showed a significant upregulation of calcium-related genes in STS KO mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC, Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e). These genes regulate calcium binding, dependence, ion regulation, and transport. Gene set enrichment analysis (GSEA) also revealed an upregulation of factors involved in calcium ion transport and regulation in STS KO mice compared to STS TG mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD). RNA-seq data indicated elevated expression of calmodulin and calcium/calmodulin-dependent protein kinases, which regulate intracellular calcium levels, in STS KO mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE)\\u003csup\\u003e35,36\\u003c/sup\\u003e. Furthermore, STS deficiency increased the expression of CasR and calcium-binding proteins such as calsequestrin and parvalbumin in STS KO mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eF)\\u003csup\\u003e37\\u003c/sup\\u003e. We also observed a significant increase in filamin A expression, which stabilizes CasR (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eG)\\u003csup\\u003e38\\u003c/sup\\u003e. These results suggest that STS deficiency enhances calcium signaling, leading to significant changes in keratinocyte differentiation.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eSTS deficiency increases intracellular calcium influx and upregulates CasR expression\\u003c/h3\\u003e\\n\\u003cp\\u003eTo further validate the increase in calcium signaling observed in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e, we assessed calcium levels in STS-deficient and overexpressing cells. Confocal microscopy revealed higher calcium content in STS-deficient cells, while STS overexpression reduced calcium influx (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). Additionally, STS-deficient cells exhibited greater calcium influx when cultured in a high-calcium medium compared to controls. Treatment with the calcium chelator BAPTA-AM\\u003csup\\u003e39\\u003c/sup\\u003e reduced this influx, albeit to a lesser extent in STS-deficient cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). These results suggest that inhibiting STS enhances intracellular calcium influx and may activate calcium signaling.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eNext, we examined CasR expression, a key regulator of calcium homeostasis. RNA-seq analysis confirmed the upregulation of CasR in STS KO mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eG), a pattern also observed in STS-deficient keratinocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC, D). Treatment with STX-64, an STS inhibitor\\u003csup\\u003e40\\u003c/sup\\u003e, led to a dose-dependent increase in CasR expression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE, F). These findings indicate that the upregulation of CasR following STS inhibition contributes to the enhanced intracellular calcium influx.\\u003c/p\\u003e\\n\\u003ch3\\u003eSTS deficiency enhances keratinocyte differentiation in both mouse skin and keratinocytes\\u003c/h3\\u003e\\n\\u003cp\\u003eWe investigated the effects of enhanced calcium signaling on keratinocyte differentiation resulting from STS suppression. RNA-seq data revealed an upregulation of genes related to tyrosine kinase activity and tyrosine phosphorylation, which promote differentiation, in STS KO mice compared to controls, while genes related to tyrosine phosphatase activity were downregulated (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA, Table \\u003cspan refid=\\\"MOESM2\\\" class=\\\"InternalRef\\\"\\u003eS2\\u003c/span\\u003e). Additionally, genes involved in keratinocyte differentiation and keratinization were significantly upregulated (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB, Table S3). Calcium-induced keratinocyte differentiation requires phosphoinositide 3-kinase (PI3K) activation\\u003csup\\u003e15\\u003c/sup\\u003e, and RNA-seq data confirmed increased expression of PI3K subunits, including PIK3CA and PIK3CB, in STS-deficient mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eWe then analyzed STS-deficient and STS-overexpressing cells to assess the impact of STS suppression on keratinocyte differentiation, which typically occurs during the G\\u003csub\\u003e0\\u003c/sub\\u003e/G\\u003csub\\u003e1\\u003c/sub\\u003e phase\\u003csup\\u003e9\\u003c/sup\\u003e. STS-deficient cells exhibited G\\u003csub\\u003e0\\u003c/sub\\u003e/G\\u003csub\\u003e1\\u003c/sub\\u003e arrest (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD). Moreover, mRNA levels of differentiation markers, including keratin 1, keratin 10, filaggrin 1, and filaggrin 10, were significantly upregulated in STS-deficient keratinocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE), and protein levels of keratin 1 and keratin 10 were similarly elevated (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD). Treatment of primary cells with STX-64 at concentrations of 0, 0.1, 1, or 10 \\u0026micro;M resulted in a dose-dependent increase in keratin 1 and keratin 10 expression. These findings suggest that STS reduction enhances keratinocyte differentiation, thereby promoting keratinization.\\u003c/p\\u003e\\n\\u003ch3\\u003eIncreased calcium responsiveness in STS-deficient keratinocytes elevates the expression of late differentiation markers\\u003c/h3\\u003e\\n\\u003cp\\u003eWe assessed the expression of E-cadherin and the late differentiation markers involucrin and loricrin, which play key roles in keratinocyte differentiation\\u003csup\\u003e15,28,31\\u003c/sup\\u003e. Inhibiting STS activity significantly increased their expression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA, B), with a similar pattern observed in STS-overexpressing cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC, D). Conversely, treatment with TNF-α, a known inducer of STS\\u003csup\\u003e1,41\\u003c/sup\\u003e, reduced the expression of these markers as STS levels increased (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eE, F). This upregulation of E-cadherin and late differentiation markers was also observed in mouse primary keratinocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eG, H), indicating that reducing STS affects both early and late stages of keratinocyte differentiation.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eFurthermore, we analyzed the expression of E-cadherin, involucrin, and loricrin in response to calcium treatment in keratinocytes. The expression of these factors increased in a calcium concentration-dependent manner (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA). Calcium treatment significantly upregulated all markers in STS-deficient cells, with E-cadherin and involucrin levels increasing by 5.7- and 7.1-fold, respectively, compared to controls. These markers were less sensitive to BAPTA-AM-induced calcium suppression, showing only a 32.3% and 21.2% decrease, respectively. Co-treatment with BAPTA-AM and calcium restored their expression to levels similar to those seen in untreated STS-deficient cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB). In contrast, calcium treatment in STS-overexpressing cells increased E-cadherin, involucrin, and loricrin expression by 1.85- to 2.36-fold, but this increase was smaller compared to STS-deficient cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC). Overall, STS inhibition significantly enhanced responsiveness to calcium, leading to upregulated expression of E-cadherin, involucrin, and loricrin.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eOur study used RNA-seq data from STS KO and TG mouse models to investigate the impact of STS deficiency on skin diseases, particularly XLI (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA, B). We focused on genes and signaling pathways involved in keratinocyte differentiation, given the abnormal keratinization observed in XLI\\u003csup\\u003e1,2\\u003c/sup\\u003e. Our findings demonstrate that STS deficiency amplifies calcium signaling and accelerates keratinocyte differentiation, likely contributing to the abnormal keratinization seen in skin conditions such as XLI. These insights provide a deeper understanding of the role of STS deficiency in the pathogenesis of skin disorders.\\u003c/p\\u003e \\u003cp\\u003eCalcium is a crucial factor in keratinocyte differentiation\\u003csup\\u003e8,9\\u003c/sup\\u003e, and patients with XLI have been shown to exhibit elevated calcium levels in the stratum corneum\\u003csup\\u003e42\\u003c/sup\\u003e. Our study confirmed that STS deficiency intensifies calcium signaling in mouse skin. RNA-seq analysis revealed a significant upregulation of genes related to calcium signaling, binding, and ion transport in STS-deficient mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC-G). This finding aligns with the increased intracellular calcium levels observed in STS-deficient keratinocytes (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, B), further supporting the key role of calcium as a regulator of keratinocyte differentiation and skin barrier formation. The heightened calcium signaling in STS KO mice likely drives excessive keratinocyte differentiation, contributing to impaired keratinization.\\u003c/p\\u003e \\u003cp\\u003eAdditionally, STS-deficient keratinocytes showed increased expression of CasR (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC-F), supporting the hypothesis that STS modulates calcium homeostasis. CasR regulates extracellular calcium and triggers calcium-dependent signaling within keratinocytes, promoting calcium influx\\u003csup\\u003e14,43\\u003c/sup\\u003e. These findings suggest that STS deficiency upregulates CasR, resulting in increased intracellular calcium influx and enhanced calcium signaling. However, further research is needed to clarify the mechanisms by which STS regulates CasR expression.\\u003c/p\\u003e \\u003cp\\u003eSTS deficiency also enriched genes related to tyrosine kinases and phosphorylation, which are key drivers of differentiation and cell adhesion during calcium-induced keratinocyte differentiation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). Genes involved in keratinocyte differentiation and keratinization were similarly upregulated (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB, C), showing increased expression of E-cadherin, keratin 1, keratin 10, involucrin, and loricrin (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE-G, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). E-cadherin plays a crucial role in mediating calcium-induced cell adhesion and promoting keratinocyte differentiation\\u003csup\\u003e44\\u003c/sup\\u003e. The enhanced expression of E-cadherin, alongside its role in cell-cell adhesion, suggests that STS may influence keratinocyte differentiation and adhesion through calcium signaling.\\u003c/p\\u003e \\u003cp\\u003eSeveral studies have examined the interactions between CasR, filamin A, and E-cadherin\\u003csup\\u003e15,38,44\\u003c/sup\\u003e. Extracellular calcium induces differentiation by activating CasR-mediated signaling and E-cadherin-mediated adhesion\\u003csup\\u003e15\\u003c/sup\\u003e. The sequential binding of catenins such as E-cadherin to the cytoskeleton and other signaling molecules, including PI3K\\u003csup\\u003e21,45\\u003c/sup\\u003e. PI3K activation is essential for calcium-induced keratinocyte differentiation, and CasR has been shown to regulate the E-cadherin/PI3K pathway via Src family kinases\\u003csup\\u003e15\\u003c/sup\\u003e. The increased expression of PI3K observed in STS KO mice is consistent with this regulatory pathway (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC). Additionally, STS-deficient keratinocytes demonstrated heightened sensitivity to calcium, along with a significant increase in differentiation markers. Thus, STS deficiency not only upregulates E-cadherin expression but also strengthens E-cadherin-mediated adhesion in response to external calcium, potentially leading to dysregulation of the differentiation process.\\u003c/p\\u003e \\u003cp\\u003eIn summary, STS plays a key role in regulating calcium signaling and keratinocyte differentiation. STS deficiency increases intracellular calcium influx and signaling, driving the expression of crucial differentiation markers (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e). These findings offer valuable insights into the mechanisms underlying STS deficiency-related skin disorders, such as XLI, and suggest potential therapeutic strategies that target calcium signaling modulation.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cp\\u003e \\u003cb\\u003eCell culture.\\u003c/b\\u003e HaCaT, a human keratinocyte cell line, was obtained from CLS Cell Lines Service (Germany). HaCaT, pLjm1-Control HaCaT, and pLjm1-STS HaCaT cells were cultured in Dulbecco\\u0026rsquo;s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 \\u0026micro;g/mL streptomycin. Cas9-control HaCaT and Cas9-STS\\u003csup\\u003e+/\\u0026minus;\\u003c/sup\\u003e HaCaT cell lines were grown in DMEM with 20% (v/v) FBS, along with the same concentrations of penicillin and streptomycin. All cell lines were incubated at 37\\u0026deg;C in a humidified atmosphere containing 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. After 24 hours of incubation, cells were treated with TNF-α or STX-64 for either 24 or 48 hours.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eReagents.\\u003c/b\\u003e STX-64 and Ultra Cruz\\u0026trade; mounting medium (sc-24941) were purchased from Santa Cruz Biotechnology (Dallas, TX). TNF-α and MG132 were purchased from Enzo Biochem (Farmingdale, NY), and CaCl\\u003csub\\u003e2\\u003c/sub\\u003e, and cycloheximide from Sigma-Aldrich (Burlington, MA). DMEM was obtained from HyClone (Logan, UT), while Fluo-4 AM was acquired from Glpbio (California, CA). FBS and charcoal-stripped FBS were supplied by Tissue Culture Biologicals (Long Beach, CA). The Neon\\u0026trade; Transfection System, BAPTA-AM, and Hoechst 33258 were procured from Thermo Fisher Scientific (Waltham, MA), and the D-Plus\\u0026trade; ECL solution from Dongin LS (Seoul, Korea). Except for TNF-α (dissolved in triple-distilled water), all chemicals were prepared in dimethylsulfoxide (DMSO), stored in small aliquots at \\u0026minus;\\u0026thinsp;20\\u0026deg;C, and diluted in cell culture media prior to use. The following antibodies were used in our experiments: anti-STS polyclonal antibody (ab62219), involucrin antibody (ab53112), and loricrin antibody (ab85679) from Abcam (Cambridge, MA); cytokeratin 1 antibody (ab93652) and cytokeratin 10 antibody (ab76318) from Proteintech (Rosemont, IL); goat anti-rabbit IgG-Texas Red antibody (sc-2780) and CaSR antibody (6D4) from Santa Cruz Biotechnology (Santa Cruz, CA); E-cadherin antibody (07-697) from Merck Millipore (Burlington, MA); and GAPDH antibody from Cusabio Technology (Houston, TX). All other chemicals were sourced from commercial suppliers.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eStable transfection.\\u003c/b\\u003e The STS coding sequence was cloned into the pcDNA3.1/Zeo\\u0026thinsp;+\\u0026thinsp;vector for transient transfection experiments. For lentiviral production, the plasmid vectors pLJM1-Empty (provided by Joshua Mendell, Addgene plasmid #91980), pLKO.1 puro (provided by Bob Weinberg, Addgene plasmid #8453), pMD2.G, and psPAX2 (provided by Didier Trono, Addgene plasmids #12259 and #12260) were utilized. HEK293T cells were co-transfected with pLJM1-STS, pMD2.G, and psPAX2. After 48 hours, the lentiviral supernatant containing the STS gene was collected and used to infect HaCaT cells in the presence of polybrene (8 \\u0026micro;g/mL) for 24 hours. HaCaT cells stably overexpressing STS were selected with puromycin (1 \\u0026micro;g/mL).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eGeneration of Cas9-STS\\u003c/b\\u003e \\u003csup\\u003e \\u003cb\\u003e+/\\u0026minus;\\u003c/b\\u003e \\u003c/sup\\u003e \\u003cb\\u003eHaCaT cells.\\u003c/b\\u003e The plasmid vectors pLentiCas9-T2A-GFP (Addgene plasmid #78548, a gift from Roderic Guigo and Rory Johnson) and tet-pLKO-sgRNA-puro (Addgene plasmid #104321, a gift from Nathanael Gray) were obtained from Addgene. Lentiviral supernatants containing either the Cas9 gene or STS-specific sgRNA were generated using HEK293T cells. Cas9-STS\\u003csup\\u003e+/\\u0026minus;\\u003c/sup\\u003e HaCaT cells were then isolated through single-cell cloning into 96-well plates.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eQuantitative reverse transcription PCR (RT-qPCR).\\u003c/b\\u003e RT-qPCR analysis was performed using the Rotor-Gene Q system (Qiagen, Netherlands) with data processed using the QIAGEN Rotor-Gene Q Series software. Each RT-qPCR reaction mixture consisted of 10 \\u0026micro;L of Q Green 2\\u0026times; qPCR Master Mix, 1 \\u0026micro;M of specific oligonucleotide primers, and 20 ng of cDNA, totaling a volume of 20 \\u0026micro;L. The amplification protocol included an initial denaturation at 95\\u0026deg;C for 5 minutes, followed by 40 cycles of denaturation at 95\\u0026deg;C for 15 seconds and annealing/extension at 56\\u0026deg;C for 45 seconds. The following primer sets were used for RT-qPCR: \\u003cb\\u003e18S rRNA\\u003c/b\\u003e: 5\\u0026prime;-GTA ACC CGT TGA ACC CCA TT-3\\u0026prime; and 5\\u0026prime;-CCA TCC AAT CGG TAG TAG CG-3\\u0026prime;; \\u003cb\\u003eKeratin 1 (mouse)\\u003c/b\\u003e: 5\\u0026prime;- GAG CAG ATC AAG TCA CTC AAT GA -3\\u0026prime; and 5\\u0026prime;- CCC ATT TGG TTT GTA GCA CCT \\u0026minus;\\u0026thinsp;3\\u0026prime;; \\u003cb\\u003eKeratin 10 (mouse)\\u003c/b\\u003e: 5\\u0026prime;- GCC TCC TAC ATG GAC AAA GTC \\u0026minus;\\u0026thinsp;3\\u0026prime; and 5\\u0026prime;- GCC TCC TAC ATG GAC AAA GTC \\u0026minus;\\u0026thinsp;3\\u0026prime;; \\u003cb\\u003eFilaggrin 1 (mouse)\\u003c/b\\u003e: 5\\u0026prime;- ATG TCC GCT CTC CTG GAA AG -3\\u0026prime; and 5\\u0026prime;- TGG ATT CTT CAA GAC TGC CTG TA -3\\u0026prime;; and \\u003cb\\u003eFilaggrin 2 (mouse)\\u003c/b\\u003e: 5\\u0026prime;- CTA GAG GGC ATG AGT TAG TCA \\u0026minus;\\u0026thinsp;3\\u0026prime; and 5\\u0026prime;- CAA GAC TGG ACA GTT GGC TGG \\u0026minus;\\u0026thinsp;3\\u0026prime;.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eWestern blotting analysis.\\u003c/b\\u003e Cells and mouse skin samples were lysed in ice-cold PE buffer containing 50 mM NaF. The protein lysates were separated by SDS-PAGE on 8%, 10%, or 15% polyacrylamide gels and then transferred onto 0.45 \\u0026micro;m PVDF membranes via electrophoresis. Membranes were blocked for 2 hours at 4\\u0026deg;C with 5% (w/v) bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBST). Following blocking, the membranes were incubated with primary antibodies diluted 1:1000 in TBST. After primary antibody incubation, the membranes were treated with secondary antibodies for 2 hours. Protein bands were visualized using D-Plus\\u0026trade; ECL solution (Dongin LS, Korea) and analyzed on a ChemiDoc XRS system (Bio-Rad, CA).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eImmunofluorescence.\\u003c/b\\u003e Samples were fixed in 10% neutral formalin for 30 minutes at room temperature (24\\u0026deg;C), then blocked for 45 minutes in PBS containing 10% goat serum and 0.2% Triton X-100. After blocking, the samples were incubated overnight at 4\\u0026deg;C with a primary antibody diluted at a 1:200 ratio. The following day, they were treated with goat anti-rabbit IgG conjugated to Alexa Fluor 594 (1:200) for overnight staining. After three washes with PBS, coverslips were mounted on glass slides using Ultra Cruz\\u0026trade; mounting medium. Fluorescence signals were visualized using a confocal laser scanning microscope (LSM 800, Carl Zeiss, Germany).\\u003c/p\\u003e \\u003cp\\u003e\\u003cb\\u003eEthics statement.\\u003c/b\\u003e This study complies with all relevant ethical regulations. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Chung-Ang University (protocol numbers 2017-00096 and 2019-00003) and were reviewed and conducted in accordance with approved guidelines. All animal experiment reporting complies with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Experimental mice were bred and maintained in the clean animal facility of Chung-Ang University. Two to three mice were housed per cage and maintained at 20 to 24\\u0026deg;C with a 12-hour light/dark cycle. Food and water were provided ad libitum to all mice. Room temperature was maintained at 20 to 26\\u0026deg;C, daily temperature changes were kept below 4\\u0026deg;C, and relative humidity was maintained at 40 to 60%. Neonatal mice were euthanized by physical means and adult mice by CO2 inhalation, both in accordance with IACUC guidelines.\\u003c/p\\u003e \\u003cp\\u003e\\u003cb\\u003eMouse primary keratinocytes.\\u003c/b\\u003e Primary epidermal keratinocytes were isolated from the skin of one-day-old neonatal C57BL/6 mice. The skin was removed as a single sheet and incubated overnight at 4\\u0026deg;C in 0.25% trypsin. The epidermis and dermis were then mechanically separated using forceps, and the epidermal cells were gently scraped off. The collected cells were centrifuged and resuspended in minimum essential medium (MEM) supplemented with 1.3 mM calcium. The cell suspension was passed through a 100 \\u0026micro;m cell strainer, centrifuged again, and then seeded onto plates in 10% MEM medium. All animal procedures were conducted in accordance with protocols approved by the institutional animal care and use committee.\\u003c/p\\u003e \\u003cp\\u003e\\u003cb\\u003eGeneration of STS TG and STS KO mice.\\u003c/b\\u003e To generate STS transgenic mice, a 7,088-bp fragment of the human STS gene, including the CMV promoter, was synthesized and inserted into the AmpR site of the pcDNA3.1 vector, creating the STS plasmid for microinjection. The plasmid was digested with PvuI and AvrII restriction enzymes, removing the 4.4-kb backbone fragment and isolating the desired 2.2-kb fragment via gel extraction. This STS DNA was injected into the male pronucleus of zygotes, which were transferred into the oviducts of pseudopregnant female mice. After the F0 generation was born, genotyping was performed using PCR on tail samples to confirm the presence of the STS gene, with a forward primer specific to the CMV promoter and a reverse primer for the human STS gene. For the generation of STS KO mice, a Cas9 targeting vector was constructed, incorporating guide RNAs (gRNAs) designed to induce double-strand breaks (DSBs) in the Cas9 gene, alongside a selectable marker such as the puromycin resistance gene. This vector was introduced into embryonic stem (ES) cells via PEG-mediated transfection, and the transfected cells were cultured under puromycin selection to isolate cells that incorporated the targeting vector. Successful targeting events were confirmed using PCR and Southern blot analysis, with PCR primers amplifying regions surrounding the Cas9 gene and the targeting vector, and Southern blot probes hybridizing to specific sites within the Cas9 gene. Successfully targeted ES cells were injected into blastocysts, which were transferred into pseudopregnant females to generate chimeric mice. These chimeric mice contained a mixture of cells from both the original blastocyst and the injected ES cells. All animal experiments adhered to the guidelines of the Institutional Animal Care and Use Committee at Chung-Ang University. The STS transgenic mice were maintained under pathogen-free conditions at Macrogen, Inc. (Seoul, Korea).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eRNA-seq.\\u003c/b\\u003e\\u0026nbsp;Skin tissue samples from 8-week-old male mice were used for RNA-seq.\\u0026nbsp;Total RNA was extracted and processed to prepare libraries using the NEB Next Ultra II Directional RNA-Seq Kit (New England Biolabs, UK). Poly(A) RNA was isolated with the Poly(A) RNA Selection Kit (Lexogen, Austria), and the resulting mRNA was used for cDNA synthesis and shearing according to the manufacturer\\u0026rsquo;s instructions. Illumina indexes 1\\u0026ndash;12 were utilized for sample indexing, with PCR included for enrichment. Fragment sizes were assessed using the Agilent 2100 Bioanalyzer and the DNA High Sensitivity Kit. Library quantification was performed using the Library Quantification Kit and a Step One Real-Time PCR System (Life Technologies, CA). High-throughput paired-end 100 sequencing was carried out on a HiSeq X10 sequencer (Illumina, CA).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eTranscriptome analysis.\\u003c/b\\u003e Mouse skin samples were sequenced following the standard Illumina protocol (Shanghai Bingwang Biotechnology Co., Ltd.). Sequencing reads were aligned to the mouse genome (Mm9) using HISAT2 (v2.1), and gene counts were estimated with \\u0026lsquo;HTSeq.\\u0026rsquo; Differential expression analysis was conducted using the \\u0026lsquo;DESeq2\\u0026rsquo; R package. Genes with a fold-change\\u0026thinsp;\\u0026gt;\\u0026thinsp;2 and an adjusted \\u003cem\\u003ep\\u003c/em\\u003e-value\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05 were considered differentially expressed genes (DEGs). GO enrichment analysis for the DEGs was performed using the \\u0026lsquo;topGO\\u0026rsquo; R package, while GSEA was carried out according to the GSEA manual.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eStatistical analysis.\\u003c/b\\u003e Dunnett\\u0026rsquo;s pairwise multiple comparison \\u003cem\\u003et\\u003c/em\\u003e-test was performed using the GraphPad Prism 7 software (GraphPad Software, CA). Differences were considered statistically significant at *\\u003cem\\u003ep\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cdiv class=\\\"DefinitionList\\\"\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eCasR\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eCalcium-sensing receptor\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eGO\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eGene Ontology\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eGSEA\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eGene set enrichment analysis\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003ePI3K\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003ephosphoinositide 3-kinase\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eRNA-seq\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eRNA-sequencing\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eSTS\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eSteroid sulfatase\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eTNF-α\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eTumor necrosis factorα\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv class=\\\"DefinitionListEntry\\\"\\u003e \\u003cdiv class=\\\"Term\\\"\\u003eXLI\\u003c/div\\u003e \\u003cdiv class=\\\"Description\\\"\\u003e \\u003cp\\u003eX-linked ichthyosis.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e \\u003ch2\\u003eCompliance with ethical standards\\u003c/h2\\u003e \\u003cp\\u003e All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Chung-Ang University (protocol numbers 2017-00096 and 2019-00003) and were conducted in accordance with the approved guidelines.\\u003c/p\\u003e \\u003c/p\\u003e\\u003cp\\u003e \\u003ch2\\u003eCorresponding author\\u003c/h2\\u003e \\u003cp\\u003eCorrespondence should be addressed to Young-Jin Chun.\\u003c/p\\u003e \\u003c/p\\u003e\\u003cp\\u003e \\u003ch2\\u003eConflict of interest\\u003c/h2\\u003e \\u003cp\\u003eThe authors declare no conflicts of interest.\\u003c/p\\u003e \\u003c/p\\u003e\\u003cp\\u003e \\u003ch2\\u003eData and materials availability\\u003c/h2\\u003e \\u003cp\\u003eAll data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eFunding\\u003c/h2\\u003e \\u003cp\\u003eThis research was supported by the National Research Foundation of Korea (NRF), funded by the Korean government (MSIP) (NRF-2021R1A2C201239514 and NRF-2022R1A5A600076013), and by the Ministry of Food and Drug Safety of South Korea (22183MFDS366) in 2022\\u0026ndash;2025. The funding agencies had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript.\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eT. U. Kwon designed and conducted the study; T. U. Kwon, Y. J. Kwon, H. Park, and Y. J. Kang analyzed the data; and T. U. Kwon and Y. J. Chun wrote the manuscript.\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eData and materials availabilityAll data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eReed, M. J., Purohit, A., Woo, L. W. L., Newman, S. P. \\u0026amp; Potter, B. V. L. Steroid sulfatase: Molecular biology, regulation, and inhibition. \\u003cem\\u003eEndocr. 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Cell Biol.\\u003c/em\\u003e\\u003cstrong\\u003e15\\u003c/strong\\u003e, 509\\u0026ndash;514, https://doi.org/10.1016/s0955-0674(03)00101-7 (2003).\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"STS, XLI, RNA-seq, CasR, calcium influx, keratinocyte\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5115056/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5115056/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eSteroid sulfatase (STS) is a key enzyme for the desulfation of steroid sulfates, converting them into their biologically active forms. Notably, X-linked ichthyosis (XLI), a genetic disorder characterized by hyperkeratinization, arises as a direct result of STS deficiency. Keratinocyte differentiation is essential for proper keratinization. In this study, gene ontology analysis from STS-deficient mice revealed enhanced differentiation and upregulation of calcium-related signaling. Calcium plays a key role in regulating keratinocyte differentiation, with STS-deficient cells showing a marked increase in intracellular calcium influx. Additionally, these cells significantly upregulated calcium-sensing receptors (CasR), leading to elevated tyrosine phosphorylation, increased differentiation signaling, and the upregulation of early differentiation markers, including keratin 1 and keratin 10, as seen in HaCaT cells and mouse primary keratinocytes. Furthermore, STS inhibitors enhanced the expression of E-cadherin and late differentiation markers such as involucrin and loricrin. Due to increased calcium sensitivity, STS-deficient cells treated with calcium exhibited a significant upregulation of differentiation markers and reduced sensitivity to calcium chelation. Collectively, our findings demonstrate that reduced STS expression and inhibition of its activity enhance calcium responsiveness, induce CasR expression, and amplify calcium signaling, thereby promoting keratinocyte differentiation. These findings offer valuable insights into the mechanisms underlying STS deficiency-induced hyperkeratinization.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Dysregulation of STS in keratinocytes promotes calcium signaling and hyperkeratinization: Insights into the mechanisms underlying X-linked ichthyosis\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-12-12 11:26:11\",\"doi\":\"10.21203/rs.3.rs-5115056/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2024-10-30T09:13:01+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-10-25T09:02:59+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-10-18T08:35:41+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2024-10-13T03:09:40+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"209911736654919531819857636604834579494\",\"date\":\"2024-10-09T07:03:28+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"131428812737432664858230031441690053065\",\"date\":\"2024-10-08T13:28:43+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"278675188043105036929594707724053383414\",\"date\":\"2024-10-07T06:20:24+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2024-10-07T01:31:05+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2024-10-07T01:19:42+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2024-10-06T16:49:02+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2024-10-03T12:39:52+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2024-09-19T07:59:51+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"b69cef61-3441-4704-a031-bc390ad82df5\",\"owner\":[],\"postedDate\":\"December 12th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":39605105,\"name\":\"Biological sciences/Cell biology/Mechanisms of disease\"},{\"id\":39605106,\"name\":\"Health sciences/Diseases/Skin diseases\"},{\"id\":39605107,\"name\":\"Biological sciences/Cell biology/Cell signalling/Calcium signalling\"},{\"id\":39605108,\"name\":\"Biological sciences/Biochemistry/Enzyme mechanisms\"},{\"id\":39605109,\"name\":\"Biological sciences/Molecular biology/Transcriptomics\"}],\"tags\":[],\"updatedAt\":\"2025-03-24T15:58:34+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-5115056\",\"link\":\"https://doi.org/10.1038/s41598-024-84701-9\",\"journal\":{\"identity\":\"scientific-reports\",\"isVorOnly\":false,\"title\":\"Scientific Reports\"},\"publishedOn\":\"2025-03-21 15:56:49\",\"publishedOnDateReadable\":\"March 21st, 2025\"},\"versionCreatedAt\":\"2024-12-12 11:26:11\",\"video\":\"\",\"vorDoi\":\"10.1038/s41598-024-84701-9\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41598-024-84701-9\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5115056\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5115056\",\"identity\":\"rs-5115056\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}