SR8278 inhibits cell proliferation independent of REV-ERB

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Kemp This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6727727/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The small molecule SR8278 was initially identified as an antagonist of the REV-ERB (reverse c-ERBAa) nuclear receptor proteins, which play an important role in metabolism and circadian rhythms. Though SR8278 has been shown to have beneficial physiological effects in a variety of preclinical disease contexts, its impact on gene expression and cell proliferation in keratinocytes has not previously been examined. Methods An RNA-seq analysis was used to identify genes differentially impacted by SR8278 treatment in human HaCaT keratinocytes, which was confirmed by RT-qPCR and western blotting. Cell growth and viability assays were further used to examine cell proliferation in HaCaT and other cell lines. CRISPR/Cas9 genome editing was used to generate cells lacking REV-ERBα and β. Results RNA-seq analysis indicated genes involved in the G1/S transition of the cell cycle were significantly impacted by SR8278 treatment, which was confirmed via RT-qPCR and western blotting. Cell proliferation assays showed that SR8278 slowed cell growth but did not induce apoptosis. Finally, the knockout of the REV-ERBs did not impact the effect of SR8278 on gene expression and cell proliferation. Conclusions We conclude that the anti-proliferative effects of SR8278 are not mediated by the REV-ERB proteins, and thus care should be taken when interpreting studies involving this compound unless complementary genetic approaches are also shown. Cell proliferation small molecule circadian REV-ERB DNA synthesis nuclear receptor Figures Figure 1 Figure 2 Figure 3 Introduction The NR1D1 and NR1D2 genes (nuclear receptor subfamily 1 group D members 1 and 2) encode the REV-ERB alpha (REV-ERBα) and REV-ERB beta (REV-ERBβ) nuclear receptor proteins, which utilize heme as a natural ligand [ 1 , 2 ] and compete with retinoic acid-related orphan receptors (RORs) for binding to ROR-response elements (RORE) in the promoters of target genes [ 3 ]. The REV-ERBs have historically been thought to function as transcriptional repressors via the recruitment of the nuclear co-repressor (NCoR)/histone deacetylase 3 (HDAC2) complex. However, recent work indicates that in cancer cells, REV-ERBα becomes a transcriptional activator by interacting with BRD3/p300 to drive the expression of thousands of genes involved in tumorigenesis, including genes involved in MAPK and PI3K-Akt signaling [ 4 ]. Nonetheless, the major known functions of the REV-ERBs are in circadian rhythms and metabolism [ 5 ]. The REV-ERBs are both transcriptionally activated by the CLOCK (circadian locomotor output kaput)-BMAL1 (brain and muscle Arnt-like protein-1) complex [ 6 ], and REV-ERBα then feeds back to inhibit the transcription of both BMAL1 [ 7 ] and CLOCK [ 8 ]. Via interactions with UCP1 (uncoupling protein 1), REV-ERBα also regulates body temperature and enzymes involved in gluconeogenesis [ 9 ]. Furthermore, REV-ERBα interacts with apolipoproteins to regulate cholesterol metabolism [ 10 ]. Double-knockout of both REV-ERBs in mice leads to major disruptions in both circadian rhythms and lipid homeostasis [ 5 ]. Given the physiological processes governed by REV-ERB, there has been interest in pharmacologically targeting REV-ERB [ 11 ]. This has led to the discovery and study of both agonists and antagonists of REV-ERB. The agonist SR9009 has been demonstrated to show beneficial effects in both healthy and diseased model systems ranging from cancer to neuroinflammation to heart failure [ 12 – 17 ]. Similarly, the REV-ERB antagonist SR8278 [ 16 ] has been shown in mice to promote corneal repair [ 18 ], reduce fibrosis in dystrophic muscle [ 19 ], stimulate amyloid plaque deposition [ 20 ], prevent kidney injury [ 21 , 22 ], stabilize mood disorder [ 23 ], protect against ischemia-reperfusion lung injury [ 24 ], and slow tumor cell growth in mice [ 4 ]. Thus, these REV-ERB modulators have been reported to have favorable benefits in a variety of tissues and disease states. Though skin exhibits robust circadian rhythms [ 25 , 26 ], the function of REV-ERBs in skin and keratinocytes that comprise the major cell type of skin epidermis has not been extensively examined. However, prior studies have included examination of the effects of REV-ERB inhibition with SR8278 on cellular responses to UV radiation [ 27 ] and viral infection [ 28 ]. To better understand the genes that are regulated by REV-ERB and SR8278 in keratinocytes, we performed RNA-seq analysis of SR8278-treated HaCaT keratinocytes and identified genes involved in cell proliferation and DNA synthesis as a major pathway impacted by SR8278. However, although SR8278 slowed cell proliferation in HaCaT cells and other cell lines, we show here that this effect is independent of REV-ERB. Materials and Methods Cell culture : HaCaT keratinocytes and HeLa, U2OS, and A549 cells were cultured and maintained in DMEM containing 10% FBS, an additional 2 mM L-glutamine, 10,000 U/ml penicillin, and 10,000 µg/ml streptomycin. Telomerase-immortalized human neonatal foreskin keratinocytes (N-TERTs) [ 29 ] were grown in EpiLife medium with human keratinocyte growth supplement (HKGS) (Thermo Fisher Scientific) and penicillin/streptomycin. REV-ERBα and β-knockout (REV-ERBα/β-KO) HaCaT cells were generated by transfecting HaCaT cells with plasmids expressing Cas9 and guide RNAs targeting either REV-ERBα or REV-ERBβ and a homology template (Santa Cruz sc-401211, sc-401211-HDR, sc-402616 and sc-401616-HDR), selection with puromycin, and expansion of single cell clones. Double-KO (DKO) cells were generated by co-transfecting REV-ERBα-KO cells with the REV-ERBβ CRISPR plasmids along with pcDNA3 and then selection with geneticin. Cells were treated with DMSO vehicle (0.02–0.1%) (Sigma) or with the indicated concentrations of SR8278 (Sigma) diluted from a 50 mM stock in DMSO. Assays of cell survival Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assays were used to monitor cell viability/proliferation by adding the MTT reagent to cell culture medium at a final concentration of 0.25 mg/ml, incubating for 30 min, and then solubilizing the samples in DMSO for measurement of absorbance at 570 nm on a Synergy H1 spectrophotometer (Bio-Tek). Clonogenic assays were performed by treating low numbers of cells in 6-well plates with SR8278 and then staining colonies with crystal violet 10–14 days later. Increases in relative cell number were also determined by staining cells with crystal violet after various periods of time, solubilizing the dye in 1% SDS, and measuring the absorbance at 535 nm. RNA analyses Cell pellets from treated cells were placed on ice, homogenized in TriZol, extracted with phenol, and then purified using RNeasy columns (Qiagen). RNA was reverse transcribed a QuantiTect Reverse Transcription Kit (Qiagen). Library preparation and Illumina sequencing was performed by Azenta Life Sciences. PCRs were prepared using 2X TaqMan Fast Universal PCR Master Mix and TaqMan probes targeting the indicated genes (Applied Biosystems). PCRs were run on an Azure Cielo 6 real-time PCR machine using an initial 3 min melting step at 95°C followed by 40 cycles of 95°C for 10 sec and 55°C for 30 sec. The ∆∆C t method was used to determine fold-changes in gene expression using beta-2-microglobulin (B2M) as a housekeeping gene. Protein immunoblotting Cells were lysed in either 1X SDS-PAGE sample buffer or ice-cold RIPA buffer, and then soluble protein lysates were separated on Tris-Glycine SDS gels. Proteins were then transferred to a nitrocellulose membrane using a semi-dry transfer apparatus. Blots were stained with 0.5% Ponceau S (Sigma) to ensure equal loading. The blots were blocked in 5% non-fat milk in TBST (Tris-buffered saline containing 0.1% Tween-20) and then probed overnight with primary antibodies from Cell Signaling Technology recognizing E2F1 (#3742), RRM2 (#65939), Cyclin E2 (#4132), PARP (#9542), or REV-ERBα (#13418) or antibodies from Santa Cruz Biotechnology recognizing PCNA (sc-56) or REV-ERBβ (sc-398252). After washing with TBST, blots were probed with HRP-coupled anti-rabbit IgG (Invitrogen) secondary antibodies for one hour at room temperature. Chemiluminescence was visualized with Clarity Western ECL substrate (Bio-Rad) using an Azure 600 western blot imager. Signals in the linear range of detection were quantified by densitometry using Image Lab (Bio-Rad) and normalized to the Ponceau S-stained membranes. Statistical analyses GraphPad Prism version 10 was used for all data analyses. ANOVAs and paired t-tests were used to compare treatment groups. Results SR8278 impacts the expression of genes involved in cell proliferation Though the small molecule SR8278 is reported to be an inhibitor of the REV-ERB transcription factors [ 30 ], the genes impacted by SR8278 treatment in human keratinocytes have not been examined. Because of our prior work with the compound in human keratinocytes in vitro [ 27 ], we treated HaCaT keratinocytes with either vehicle (0.1% DMSO) or 50 µM SR8278 for 24 hr and total RNA was subjected to RNA-seq analysis to identify genes differentially affected by SR8278 treatment. A total of 2,686 genes met the threshold criteria to be classified as significantly altered by SR8278 treatment, and a subset of the most differentially affected genes is shown in Fig. 1 A. Gene ontology enrichment analysis indicated that the genes could be classified into two major biological processes, including the regulation of cholesterol biosynthesis (Fig. 1 B) and of the G1/S phase transition of the mitotic cell cycle (Fig. 1 C). The most significantly altered genes in each of these two biological pathways are shown in Fig. 1 D-E). Because of our interest in DNA metabolism, we decided to use Taqman-based RT-qPCR to confirm a subset of the genes identified with RNA-seq. As shown in Fig. 1 F, SR8278 induced a significant decrease in the expression of several genes involved in the G1/S phase transition and cell proliferation, including the pro-S phase transcription factor E2F1, the dNTP synthesizing gene RRM2, the cyclin-dependent kinase regulator Cyclin E2 (CCNE2), and the DNA synthesis factor PCNA. Western blot analysis further confirmed that SR8278 treatment for 24 or 48 hr led to reduced expression of these genes at the protein level (Fig. 1 G). SR8278 slows cell proliferation in multiple cell lines To determine whether the reduced expression of these gene products by SR8278 treatment is correlated with slower cell growth, we treated HaCaT keratinocytes with SR8278 and then visualized cell growth by staining cells with crystal violet. As shown in Fig. 2 A and quantified in Fig. 2 B, SR8278 significantly inhibited cell growth and proliferation. Though somewhat dose-dependent, only a high concentration of SR8278 (50 µM) caused a statistically significant decrease in cell proliferation after 3 days of treatment as measured by MTT assay (Fig. 2 C). To confirm these results with other cell lines, we treated telomerase-immortalized N-TERT keratinocytes, A549 lung carcinoma, U2OS osteosarcoma, and HeLa cervical cancer cells with either vehicle or DMSO and performed MTT assays to assess cell proliferation. SR8278 treatment resulted in lower cell proliferation in all tested cell lines (Fig. 2 D). Similarly, western blot analysis showed that SR8278 induced significant reductions in the expression of both RRM2 and Cyclin E in both USOS and HeLa cells (Fig. 2 E). Finally, to investigate whether the reduced cell proliferation is associated with increased apoptosis, we treated HaCaT cells with either DMSO, SR8278, or the anti-cancer drug cisplatin. Only cisplatin induced a significant cleavage of the caspase substrate protein PARP (Fig. 2 F). Thus, the anti-proliferative effect of SR8278 does not appear to be due to the induction of apoptosis. SR8278 slows cell proliferation independent of REV-ERB To provide genetic evidence that SR8278 acts via either REV-ERBα or REV-ERBβ, we used CRISPR/Cas9 genome editing to create HaCaT cell lines lacking expression of one or both REV-ERB proteins. As shown in Fig. 3 A, western blot analysis showed that single knockout was associated with a corresponding increase in the expression of the other REV-ERB protein. Furthermore, we were also able to generate REV-ERBα/β double-knockout (DKO) cell lines (Fig. 3 A). Though the purported REV-ERB antagonist SR8278 slowed cell growth (Fig. 2 ), we noted no difference in cell growth rate between single- or double-knockout cell lines (Fig. 3 B), indicating that genetic loss of REV-ERB does not impact growth rate of HaCaT cells. Moreover, when we treated the single- and double-knockout cells with different concentrations of SR8278, we observed similar inhibition of cell proliferation (Fig. 3 C). Consistent with these results, SR8278 caused a similar decrease in E2F1 protein expression in all the cell lines (Fig. 3 D). We conclude that the effect of SR8278 on cell proliferation is independent of the REV-ERB proteins. Discussion Related to our work here on the effects of SR8278 on slowing keratinocyte proliferation in vitro, recent work has also shown that SR8278 slows tumor growth in mice in vivo and to be correlated with effects on the expression of genes involved in diverse growth factor signaling pathways [ 4 ]. Thus, SR8728 appears to be able to slow cell proliferation in a variety of different cell types. Though we found that SR8278 clearly slowed cell proliferation in several different cell lines in vitro (Fig. 2 A-D) and negatively affected the expression of proliferation genes (Fig. 1 ), we found that genetic knockout of the REV-ERBs had no impact on cell growth and proliferation, which argues that the growth inhibitory effect of SR8278 is not mediated by the REV-ERB proteins (Fig. 3 ). In contrast, using lentiviral gRNA and shRNA approaches, Yang et al recently showed that the sole knockdown of REV-ERBα expression alone slowed the growth of several (but not all) cancer cell lines in vitro [ 4 ]. Moreover, they reported that REV-ERBα knockdown led to the induction of apoptosis. However, we were able to readily generate REV-ERB single- and double-knockout cells (Fig. 3 A) and did not find that SR8278 induces apoptosis (Fig. 2 F). These results may indicate that acute knockdown of REV-ERBα or β in cancer cells does not necessarily recapitulate the effects of stable knockdown in the HaCaT keratinocytes primarily used in our work here. Thus, it may be important to compare transient knockdown versus stable knockout in other cell lines. Nonetheless, we note that double-knockout mice have been generated [ 31 ], which suggests that REV-ERB is not essential cell proliferation during mouse development. However, the situation may be different in tumor cells. As described in the introduction above, SR8278 has been reported to exert beneficial effects in a variety of experimental systems and pathological conditions and is assumed to be mediated by its effects on REV-ERBα and/or β. However, most previous studies involving SR8278 did not use genetic REV-ERB knockdown or knockout approaches to show that REV-ERB loss acts in a similar manner as the purported REV-ERB inhibitor SR8278. This is an important issue given that the REV-ERB agonist SR9009 has been shown to inhibit cell proliferation independent of REV-ERB using mice and mouse-derived cell lines [ 31 ]. Thus, our results showing that SR8278 similarly inhibits cell proliferation independent of REV-ERB suggests a common problem with the family of compounds reported to target REV-ERB. The exact target(s) of SR9009 and SR8278 remain to be determined. Though our data raise concerns on the use of SR8278, it remains possible that the effects of SR8278 on cell proliferation only occur at high concentrations and that the compound is still useful as a REV-ERB inhibitor. Nonetheless, we suggest that care should be taken when interpreting the results of experiments that lack complementary genetic knockdown or knockout approaches. Abbreviations REV-ERB reverse c-erbAa NR1D1 and NR1D2 genes (nuclear receptor subfamily 1 group D members 1 and 2) ROR retinoic acid-like orphan receptor CLOCK circadian locomotor output kaput BMAL1 brain and muscle Arnt-like protein-1 Declarations Data Availability All data generated or analyzed during this study are included in this published article. Ethics, Consent to Participate, and Consent to Publish declarations : not applicable Declaration of competing interests: We have nothing to declare Author Contribution M.G.K. wrote the main manuscript text and prepared the figures. 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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-6727727","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":463738729,"identity":"a761ef4d-63d1-44ae-a460-7987296365e2","order_by":0,"name":"Ushaswini Atluri","email":"","orcid":"","institution":"Wright State University Boonshoft School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ushaswini","middleName":"","lastName":"Atluri","suffix":""},{"id":463738730,"identity":"1d819d97-2ecf-4a1e-a693-5ed003898596","order_by":1,"name":"William Cvammen","email":"","orcid":"","institution":"Wright State University Boonshoft School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"","lastName":"Cvammen","suffix":""},{"id":463738731,"identity":"ab6b7e96-2772-4c70-aec4-ebbc14cf840c","order_by":2,"name":"Michael G. 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(A) \u003c/strong\u003eThe embedded heatmap() package in R was used to identify significantly differentially expressed genes (log2 fold change \u0026gt;1 or \u0026lt;-1, and p value \u0026lt;0.05) in HaCaT keratinocytes treated with SR8278 relative to DMSO (n=2). Genes were graphed and sorted based on the level of log2 fold-change in gene expression. The heatmap() function assigns a red or blue color to a gene based on whether the expression was higher or lower than the control, respectively. Additionally, the heatmap() function generates a neighbor joining tree which groups together like-genes based on the sort condition (log2 fold change). Gene Ontology Enrichment Analysis was used to find biological processes significantly altered because of SR8278 treatment. Two of the most significantly (p\u0026lt;0.05) changed pathways included \u003cstrong\u003e(B) \u003c/strong\u003eRegulation of cholesterol biosynthetic process and \u003cstrong\u003e(C)\u003c/strong\u003e G1/S transition of mitotic cell cycle. \u003cstrong\u003e(D-E) \u003c/strong\u003eSpecific genes of interest within the two biological processes that were the most significantly altered (p\u0026lt;0.00001) are shown. \u003cstrong\u003e(F) \u003c/strong\u003eRT-qPCR analysis of relative expression the indicated genes in cells treated with DMSO or SR8278 for 24 hr. \u003cstrong\u003e(G) \u003c/strong\u003eWestern blot analysis of the indicated proteins in cells treated for 24 or 48 hr with DMSO or SR8278.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6727727/v1/4eb5ad90ff452d8a69b916ff.jpeg"},{"id":83660025,"identity":"9c4a8d77-ecaa-48a3-a1bb-08124437d37e","added_by":"auto","created_at":"2025-05-30 09:40:01","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":192692,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSR8278 slows cell proliferation but does not induce cell death. (A) \u003c/strong\u003eHaCaT cells were treated with DMSO or 50 µM SR8278 and then cells were stained with crystal violet on the indicated days to visualize cell proliferation. \u003cstrong\u003e(B) \u003c/strong\u003eQuantitation of results from 3 independent experiments performed as in A. \u003cstrong\u003e(C) \u003c/strong\u003eMTT assays were used to monitor relative cell viability 3 days after treating HaCaT cells with the indicated concentration of SR8278. \u003cstrong\u003e(D) \u003c/strong\u003eThe indicated cell lines were treated with DMSO or SR8278 for between 3 and 9 days, and then MTT assays were performed to monitor relative cell viability. \u003cstrong\u003e(E) \u003c/strong\u003eWestern blotting was performed with cell lysates from U2OS or HeLa cells treated with DMSO or SR8278 for 2 days. \u003cstrong\u003e(F) \u003c/strong\u003eWestern blotting was used to detect PARP cleavage as a measure of apoptosis in cells treated for 24 hr with 30 µM cisplatin, 0.1% DMSO or 50 µM SR8278.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6727727/v1/adefa48b81d36b0869daf5a8.jpeg"},{"id":83660022,"identity":"b2d8b8da-69b7-43bd-886f-3a64d7ab4fe4","added_by":"auto","created_at":"2025-05-30 09:40:01","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":215759,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSR8278 slows cell proliferation independent of the REV-ERB proteins. (A) \u003c/strong\u003eThe expression of the REV-ERB proteins was examined in cell lysates from wild-type (WT) HaCaT cells and REV-ERBα, REV-ERBβ, and REV-ERBα/β double-knockout (DKO) HaCaT cell lines generated with CRISPR/Cas9 genome editing. \u003cstrong\u003e(B) \u003c/strong\u003eThe WT and REV-ERB KO cell lines were plated and then stained with crystal violet at the indicated times to quantify cell growth and proliferation. \u003cstrong\u003e(C) \u003c/strong\u003eThe six cell lines were treated with increasing concentrations of SR8278 for 3 days and then MTT assays were performed to monitor cell proliferation. \u003cstrong\u003e(D) \u003c/strong\u003eCells were treated with DMSO or SR8278 for 48 hr and then western blotting was carried out for the G1/S phase transcription factor E2F1.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6727727/v1/3ac1f911c240f9935f3118ba.jpeg"},{"id":100356414,"identity":"a6bf0625-19a5-451b-a7bb-f277f2019feb","added_by":"auto","created_at":"2026-01-16 07:08:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1280921,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6727727/v1/5c17f71d-5ca4-4ab9-9164-0e58979e2345.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"SR8278 inhibits cell proliferation independent of REV-ERB","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe \u003cem\u003eNR1D1\u003c/em\u003e and \u003cem\u003eNR1D2\u003c/em\u003e genes (nuclear receptor subfamily 1 group D members 1 and 2) encode the REV-ERB alpha (REV-ERBα) and REV-ERB beta (REV-ERBβ) nuclear receptor proteins, which utilize heme as a natural ligand [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] and compete with retinoic acid-related orphan receptors (RORs) for binding to ROR-response elements (RORE) in the promoters of target genes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The REV-ERBs have historically been thought to function as transcriptional repressors via the recruitment of the nuclear co-repressor (NCoR)/histone deacetylase 3 (HDAC2) complex. However, recent work indicates that in cancer cells, REV-ERBα becomes a transcriptional activator by interacting with BRD3/p300 to drive the expression of thousands of genes involved in tumorigenesis, including genes involved in MAPK and PI3K-Akt signaling [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNonetheless, the major known functions of the REV-ERBs are in circadian rhythms and metabolism [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The REV-ERBs are both transcriptionally activated by the CLOCK (circadian locomotor output kaput)-BMAL1 (brain and muscle Arnt-like protein-1) complex [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and REV-ERBα then feeds back to inhibit the transcription of both \u003cem\u003eBMAL1\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and \u003cem\u003eCLOCK\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Via interactions with UCP1 (uncoupling protein 1), REV-ERBα also regulates body temperature and enzymes involved in gluconeogenesis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, REV-ERBα interacts with apolipoproteins to regulate cholesterol metabolism [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Double-knockout of both REV-ERBs in mice leads to major disruptions in both circadian rhythms and lipid homeostasis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the physiological processes governed by REV-ERB, there has been interest in pharmacologically targeting REV-ERB [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This has led to the discovery and study of both agonists and antagonists of REV-ERB. The agonist SR9009 has been demonstrated to show beneficial effects in both healthy and diseased model systems ranging from cancer to neuroinflammation to heart failure [\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Similarly, the REV-ERB antagonist SR8278 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] has been shown in mice to promote corneal repair [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], reduce fibrosis in dystrophic muscle [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], stimulate amyloid plaque deposition [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], prevent kidney injury [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], stabilize mood disorder [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], protect against ischemia-reperfusion lung injury [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and slow tumor cell growth in mice [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Thus, these REV-ERB modulators have been reported to have favorable benefits in a variety of tissues and disease states.\u003c/p\u003e \u003cp\u003eThough skin exhibits robust circadian rhythms [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the function of REV-ERBs in skin and keratinocytes that comprise the major cell type of skin epidermis has not been extensively examined. However, prior studies have included examination of the effects of REV-ERB inhibition with SR8278 on cellular responses to UV radiation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and viral infection [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. To better understand the genes that are regulated by REV-ERB and SR8278 in keratinocytes, we performed RNA-seq analysis of SR8278-treated HaCaT keratinocytes and identified genes involved in cell proliferation and DNA synthesis as a major pathway impacted by SR8278. However, although SR8278 slowed cell proliferation in HaCaT cells and other cell lines, we show here that this effect is independent of REV-ERB.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eCell culture\u003c/b\u003e: HaCaT keratinocytes and HeLa, U2OS, and A549 cells were cultured and maintained in DMEM containing 10% FBS, an additional 2 mM L-glutamine, 10,000 U/ml penicillin, and 10,000 \u0026micro;g/ml streptomycin. Telomerase-immortalized human neonatal foreskin keratinocytes (N-TERTs) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] were grown in EpiLife medium with human keratinocyte growth supplement (HKGS) (Thermo Fisher Scientific) and penicillin/streptomycin. REV-ERBα and β-knockout (REV-ERBα/β-KO) HaCaT cells were generated by transfecting HaCaT cells with plasmids expressing Cas9 and guide RNAs targeting either REV-ERBα or REV-ERBβ and a homology template (Santa Cruz sc-401211, sc-401211-HDR, sc-402616 and sc-401616-HDR), selection with puromycin, and expansion of single cell clones. Double-KO (DKO) cells were generated by co-transfecting REV-ERBα-KO cells with the REV-ERBβ CRISPR plasmids along with pcDNA3 and then selection with geneticin. Cells were treated with DMSO vehicle (0.02\u0026ndash;0.1%) (Sigma) or with the indicated concentrations of SR8278 (Sigma) diluted from a 50 mM stock in DMSO.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAssays of cell survival\u003c/strong\u003e \u003cp\u003eMethylthiazolyldiphenyl-tetrazolium bromide (MTT) assays were used to monitor cell viability/proliferation by adding the MTT reagent to cell culture medium at a final concentration of 0.25 mg/ml, incubating for 30 min, and then solubilizing the samples in DMSO for measurement of absorbance at 570 nm on a Synergy H1 spectrophotometer (Bio-Tek). Clonogenic assays were performed by treating low numbers of cells in 6-well plates with SR8278 and then staining colonies with crystal violet 10\u0026ndash;14 days later. Increases in relative cell number were also determined by staining cells with crystal violet after various periods of time, solubilizing the dye in 1% SDS, and measuring the absorbance at 535 nm.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eRNA analyses\u003c/strong\u003e \u003cp\u003eCell pellets from treated cells were placed on ice, homogenized in TriZol, extracted with phenol, and then purified using RNeasy columns (Qiagen). RNA was reverse transcribed a QuantiTect Reverse Transcription Kit (Qiagen). Library preparation and Illumina sequencing was performed by Azenta Life Sciences. PCRs were prepared using 2X TaqMan Fast Universal PCR Master Mix and TaqMan probes targeting the indicated genes (Applied Biosystems). PCRs were run on an Azure Cielo 6 real-time PCR machine using an initial 3 min melting step at 95\u0026deg;C followed by 40 cycles of 95\u0026deg;C for 10 sec and 55\u0026deg;C for 30 sec. The ∆∆C\u003csub\u003et\u003c/sub\u003e method was used to determine fold-changes in gene expression using beta-2-microglobulin (B2M) as a housekeeping gene.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eProtein immunoblotting\u003c/strong\u003e \u003cp\u003eCells were lysed in either 1X SDS-PAGE sample buffer or ice-cold RIPA buffer, and then soluble protein lysates were separated on Tris-Glycine SDS gels. Proteins were then transferred to a nitrocellulose membrane using a semi-dry transfer apparatus. Blots were stained with 0.5% Ponceau S (Sigma) to ensure equal loading. The blots were blocked in 5% non-fat milk in TBST (Tris-buffered saline containing 0.1% Tween-20) and then probed overnight with primary antibodies from Cell Signaling Technology recognizing E2F1 (#3742), RRM2 (#65939), Cyclin E2 (#4132), PARP (#9542), or REV-ERBα (#13418) or antibodies from Santa Cruz Biotechnology recognizing PCNA (sc-56) or REV-ERBβ (sc-398252). After washing with TBST, blots were probed with HRP-coupled anti-rabbit IgG (Invitrogen) secondary antibodies for one hour at room temperature. Chemiluminescence was visualized with Clarity Western ECL substrate (Bio-Rad) using an Azure 600 western blot imager. Signals in the linear range of detection were quantified by densitometry using Image Lab (Bio-Rad) and normalized to the Ponceau S-stained membranes.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical analyses\u003c/strong\u003e \u003cp\u003eGraphPad Prism version 10 was used for all data analyses. ANOVAs and paired t-tests were used to compare treatment groups.\u003c/p\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSR8278 impacts the expression of genes involved in cell proliferation\u003c/h2\u003e \u003cp\u003eThough the small molecule SR8278 is reported to be an inhibitor of the REV-ERB transcription factors [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], the genes impacted by SR8278 treatment in human keratinocytes have not been examined. Because of our prior work with the compound in human keratinocytes in vitro [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], we treated HaCaT keratinocytes with either vehicle (0.1% DMSO) or 50 \u0026micro;M SR8278 for 24 hr and total RNA was subjected to RNA-seq analysis to identify genes differentially affected by SR8278 treatment. A total of 2,686 genes met the threshold criteria to be classified as significantly altered by SR8278 treatment, and a subset of the most differentially affected genes is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. Gene ontology enrichment analysis indicated that the genes could be classified into two major biological processes, including the regulation of cholesterol biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and of the G1/S phase transition of the mitotic cell cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The most significantly altered genes in each of these two biological pathways are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-E).\u003c/p\u003e \u003cp\u003eBecause of our interest in DNA metabolism, we decided to use Taqman-based RT-qPCR to confirm a subset of the genes identified with RNA-seq.\u0026nbsp;As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, SR8278 induced a significant decrease in the expression of several genes involved in the G1/S phase transition and cell proliferation, including the pro-S phase transcription factor E2F1, the dNTP synthesizing gene RRM2, the cyclin-dependent kinase regulator Cyclin E2 (CCNE2), and the DNA synthesis factor PCNA. Western blot analysis further confirmed that SR8278 treatment for 24 or 48 hr led to reduced expression of these genes at the protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSR8278 slows cell proliferation in multiple cell lines\u003c/h3\u003e\n\u003cp\u003eTo determine whether the reduced expression of these gene products by SR8278 treatment is correlated with slower cell growth, we treated HaCaT keratinocytes with SR8278 and then visualized cell growth by staining cells with crystal violet. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and quantified in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, SR8278 significantly inhibited cell growth and proliferation. Though somewhat dose-dependent, only a high concentration of SR8278 (50 \u0026micro;M) caused a statistically significant decrease in cell proliferation after 3 days of treatment as measured by MTT assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTo confirm these results with other cell lines, we treated telomerase-immortalized N-TERT keratinocytes, A549 lung carcinoma, U2OS osteosarcoma, and HeLa cervical cancer cells with either vehicle or DMSO and performed MTT assays to assess cell proliferation. SR8278 treatment resulted in lower cell proliferation in all tested cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Similarly, western blot analysis showed that SR8278 induced significant reductions in the expression of both RRM2 and Cyclin E in both USOS and HeLa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Finally, to investigate whether the reduced cell proliferation is associated with increased apoptosis, we treated HaCaT cells with either DMSO, SR8278, or the anti-cancer drug cisplatin. Only cisplatin induced a significant cleavage of the caspase substrate protein PARP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Thus, the anti-proliferative effect of SR8278 does not appear to be due to the induction of apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSR8278 slows cell proliferation independent of REV-ERB\u003c/h3\u003e\n\u003cp\u003eTo provide genetic evidence that SR8278 acts via either REV-ERBα or REV-ERBβ, we used CRISPR/Cas9 genome editing to create HaCaT cell lines lacking expression of one or both REV-ERB proteins. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, western blot analysis showed that single knockout was associated with a corresponding increase in the expression of the other REV-ERB protein. Furthermore, we were also able to generate REV-ERBα/β double-knockout (DKO) cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Though the purported REV-ERB antagonist SR8278 slowed cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), we noted no difference in cell growth rate between single- or double-knockout cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicating that genetic loss of REV-ERB does not impact growth rate of HaCaT cells. Moreover, when we treated the single- and double-knockout cells with different concentrations of SR8278, we observed similar inhibition of cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Consistent with these results, SR8278 caused a similar decrease in E2F1 protein expression in all the cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). We conclude that the effect of SR8278 on cell proliferation is independent of the REV-ERB proteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRelated to our work here on the effects of SR8278 on slowing keratinocyte proliferation in vitro, recent work has also shown that SR8278 slows tumor growth in mice in vivo and to be correlated with effects on the expression of genes involved in diverse growth factor signaling pathways [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Thus, SR8728 appears to be able to slow cell proliferation in a variety of different cell types. Though we found that SR8278 clearly slowed cell proliferation in several different cell lines in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D) and negatively affected the expression of proliferation genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), we found that genetic knockout of the REV-ERBs had no impact on cell growth and proliferation, which argues that the growth inhibitory effect of SR8278 is not mediated by the REV-ERB proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, using lentiviral gRNA and shRNA approaches, Yang et al recently showed that the sole knockdown of REV-ERBα expression alone slowed the growth of several (but not all) cancer cell lines in vitro [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Moreover, they reported that REV-ERBα knockdown led to the induction of apoptosis. However, we were able to readily generate REV-ERB single- and double-knockout cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and did not find that SR8278 induces apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results may indicate that acute knockdown of REV-ERBα or β in cancer cells does not necessarily recapitulate the effects of stable knockdown in the HaCaT keratinocytes primarily used in our work here. Thus, it may be important to compare transient knockdown versus stable knockout in other cell lines. Nonetheless, we note that double-knockout mice have been generated [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which suggests that REV-ERB is not essential cell proliferation during mouse development. However, the situation may be different in tumor cells.\u003c/p\u003e \u003cp\u003eAs described in the introduction above, SR8278 has been reported to exert beneficial effects in a variety of experimental systems and pathological conditions and is assumed to be mediated by its effects on REV-ERBα and/or β. However, most previous studies involving SR8278 did not use genetic REV-ERB knockdown or knockout approaches to show that REV-ERB loss acts in a similar manner as the purported REV-ERB inhibitor SR8278. This is an important issue given that the REV-ERB agonist SR9009 has been shown to inhibit cell proliferation independent of REV-ERB using mice and mouse-derived cell lines [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Thus, our results showing that SR8278 similarly inhibits cell proliferation independent of REV-ERB suggests a common problem with the family of compounds reported to target REV-ERB. The exact target(s) of SR9009 and SR8278 remain to be determined. Though our data raise concerns on the use of SR8278, it remains possible that the effects of SR8278 on cell proliferation only occur at high concentrations and that the compound is still useful as a REV-ERB inhibitor. Nonetheless, we suggest that care should be taken when interpreting the results of experiments that lack complementary genetic knockdown or knockout approaches.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eREV-ERB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereverse c-erbAa\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNR1D1 and NR1D2 genes (nuclear receptor subfamily 1 group D members 1 and 2)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eretinoic acid-like orphan receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLOCK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecircadian locomotor output kaput\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBMAL1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebrain and muscle Arnt-like protein-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics, Consent to Participate, and Consent to Publish declarations\u003c/strong\u003e: not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests:\u0026nbsp;\u003c/strong\u003eWe have nothing to declare\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.G.K. wrote the main manuscript text and prepared the figures. All authors generated data and reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors wish to thank the WSU Proteome Analysis Laboratory for use of equipment.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYin L, Wu N, Curtin JC, Qatanani M, Szwergold NR, Reid RA, et al. Rev-erbalpha, a heme sensor that coordinates metabolic and circadian pathways. Science 2007;318:1786\u0026ndash;9. https://doi.org/10.1126/science.1150179.\u003c/li\u003e\n\u003cli\u003eRaghuram S, Stayrook KR, Huang P, Rogers PM, Nosie AK, McClure DB, et al. Identification of heme as the ligand for the orphan nuclear receptors REV-ERBalpha and REV-ERBbeta. Nat Struct Mol Biol 2007;14:1207\u0026ndash;13. https://doi.org/10.1038/nsmb1344.\u003c/li\u003e\n\u003cli\u003eGomatou G, Karachaliou A, Veloudiou OZ, Karvela A, Syrigos N, Kotteas E. The Role of REV-ERB Receptors in Cancer Pathogenesis. Int J Mol Sci 2023;24. https://doi.org/10.3390/ijms24108980.\u003c/li\u003e\n\u003cli\u003eYang Y, Zhang X, Cai D, Zheng X, Zhao X, Zou JX, et al. Functional inversion of circadian regulator REV-ERB\u0026alpha; leads to tumorigenic gene reprogramming. Proc Natl Acad Sci U S A 2024;121:e2411321121. https://doi.org/10.1073/pnas.2411321121.\u003c/li\u003e\n\u003cli\u003eCho H, Zhao X, Hatori M, Yu RT, Barish GD, Lam MT, et al. Regulation of circadian behaviour and metabolism by REV-ERB-\u0026alpha; and REV-ERB-\u0026beta;. Nature 2012;485:123\u0026ndash;7. https://doi.org/10.1038/nature11048.\u003c/li\u003e\n\u003cli\u003eTakahashi JS. Transcriptional architecture of the mammalian circadian clock. Nature Reviews Genetics 2017;18:164\u0026ndash;79. https://doi.org/10.1038/nrg.2016.150 [doi].\u003c/li\u003e\n\u003cli\u003eGuillaumond F, Dardente H, Gigu\u0026egrave;re V, Cermakian N. Differential control of Bmal1 circadian transcription by REV-ERB and ROR nuclear receptors. J Biol Rhythms 2005;20:391\u0026ndash;403. https://doi.org/10.1177/0748730405277232.\u003c/li\u003e\n\u003cli\u003eCrumbley C, Burris TP. Direct regulation of CLOCK expression by REV-ERB. PLoS One 2011;6:e17290. https://doi.org/10.1371/journal.pone.0017290.\u003c/li\u003e\n\u003cli\u003eGerhart-Hines Z, Feng D, Emmett MJ, Everett LJ, Loro E, Briggs ER, et al. The nuclear receptor Rev-erb\u0026alpha; controls circadian thermogenic plasticity. Nature 2013;503:410\u0026ndash;3. https://doi.org/10.1038/nature12642.\u003c/li\u003e\n\u003cli\u003eCoste H, Rodr\u0026iacute;guez JC. Orphan nuclear hormone receptor Rev-erbalpha regulates the human apolipoprotein CIII promoter. J Biol Chem 2002;277:27120\u0026ndash;9. https://doi.org/10.1074/jbc.M203421200.\u003c/li\u003e\n\u003cli\u003eWang S, Li F, Lin Y, Wu B. Targeting REV-ERB\u0026alpha; for therapeutic purposes: promises and challenges. 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Rev-erb regulation of cholesterologenesis. Biochem Pharmacol 2017;131:68\u0026ndash;77. https://doi.org/10.1016/j.bcp.2017.02.006.\u003c/li\u003e\n\u003cli\u003eSolt LA, Wang Y, Banerjee S, Hughes T, Kojetin DJ, Lundasen T, et al. Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists. Nature 2012;485:62\u0026ndash;8. https://doi.org/10.1038/nature11030.\u003c/li\u003e\n\u003cli\u003eSitaula S, Zhang J, Ruiz F, Burris TP. Rev-erb regulation of cholesterologenesis. Biochem Pharmacol 2017;131:68\u0026ndash;77. https://doi.org/10.1016/j.bcp.2017.02.006.\u003c/li\u003e\n\u003cli\u003eXue Y, Liu P, Wang H, Xiao C, Lin C, Liu J, et al. Modulation of Circadian Rhythms Affects Corneal Epithelium Renewal and Repair in Mice. Invest Ophthalmol Vis Sci 2017;58:1865\u0026ndash;74. https://doi.org/10.1167/iovs.16-21154.\u003c/li\u003e\n\u003cli\u003eWelch RD, Billon C, Valfort A-C, Burris TP, Flaveny CA. Pharmacological inhibition of REV-ERB stimulates differentiation, inhibits turnover and reduces fibrosis in dystrophic muscle. Sci Rep 2017; 7:17142. https://doi.org/10.1038/s41598-017-17496-7.\u003c/li\u003e\n\u003cli\u003eLee J, Kim DE, Griffin P, Sheehan PW, Kim D-H, Musiek ES, et al. Inhibition of REV-ERBs stimulates microglial amyloid-beta clearance and reduces amyloid plaque deposition in the 5XFAD mouse model of Alzheimer\u0026rsquo;s disease. Aging Cell 2020;19:e13078. https://doi.org/10.1111/acel.13078.\u003c/li\u003e\n\u003cli\u003eGuo L, Zhang T, Wang F, Chen X, Xu H, Zhou C, et al. Targeted inhibition of Rev-erb-\u0026alpha;/\u0026beta; limits ferroptosis to ameliorate folic acid-induced acute kidney injury. Br J Pharmacol 2021;178. https://doi.org/10.1111/bph.15283.\u003c/li\u003e\n\u003cli\u003eWang Y, Wang Z, Wu Z, Chen M, Dong D, Yu P, et al. Involvement of REV-ERB\u0026alpha; dysregulation and ferroptosis in aristolochic acid I-induced renal injury. Biochem Pharmacol 2021;193. https://doi.org/10.1016/j.bcp.2021.114807.\u003c/li\u003e\n\u003cli\u003eKim J, Park I, Jang S, Choi M, Kim D, Sun W, et al. Pharmacological Rescue with SR8278, a Circadian Nuclear Receptor REV-ERB\u0026alpha; Antagonist as a Therapy for Mood Disorders in Parkinson\u0026rsquo;s Disease. Neurotherapeutics 2022; 19:592\u0026ndash;607. https://doi.org/10.1007/s13311-022-01215-w.\u003c/li\u003e\n\u003cli\u003eChu S-J, Liao W-I, Pao H-P, Wu S-Y, Tang S-E. Targeting Rev-Erb\u0026alpha; to protect against ischemia-reperfusion-induced acute lung injury in rats. Respir Res 2023; 24:247. https://doi.org/10.1186/s12931-023-02547-7.\u003c/li\u003e\n\u003cli\u003eDuan J, Greenberg EN, Karri SS, Andersen B. The circadian clock and diseases of the skin. FEBS Lett 2021; 595:2413\u0026ndash;36. https://doi.org/10.1002/1873-3468.14192.\u003c/li\u003e\n\u003cli\u003eLubov JE, Cvammen W, Kemp MG. The Impact of the Circadian Clock on Skin Physiology and Cancer Development. Int J Mol Sci 2021;22. https://doi.org/10.3390/ijms22116112.\u003c/li\u003e\n\u003cli\u003eCvammen W, Kemp MG. The REV-ERB antagonist SR8278 modulates keratinocyte viability in response to UVA and UVB radiation. Photochem Photobiol 2024;100:1864\u0026ndash;73. https://doi.org/10.1111/php.13930.\u003c/li\u003e\n\u003cli\u003eKirchner SJ, Lei V, Kim PT, Patel M, Shannon JL, Corcoran D, et al. An aging-susceptible circadian rhythm controls cutaneous antiviral immunity. JCI Insight 2023;8. https://doi.org/10.1172/jci.insight.171548.\u003c/li\u003e\n\u003cli\u003eDickson MA, Hahn WC, Ino Y, Ronfard V, Wu JY, Weinberg RA, et al. Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 2000; 20:1436\u0026ndash;47.\u003c/li\u003e\n\u003cli\u003eKojetin D, Wang Y, Kamenecka TM, Burris TP. Identification of SR8278, a synthetic antagonist of the nuclear heme receptor REV-ERB. ACS Chem Biol 2011; 6:131\u0026ndash;4. https://doi.org/10.1021/cb1002575 [doi].\u003c/li\u003e\n\u003cli\u003eDierickx P, Emmett MJ, Jiang C, Uehara K, Liu M, Adlanmerini M, et al. SR9009 has REV-ERB\u0026ndash;independent effects on cell proliferation and metabolism. Proc Natl Acad Sci U S A 2019;116. https://doi.org/10.1073/pnas.1904226116.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cell proliferation, small molecule, circadian, REV-ERB, DNA synthesis, nuclear receptor","lastPublishedDoi":"10.21203/rs.3.rs-6727727/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6727727/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe small molecule SR8278 was initially identified as an antagonist of the REV-ERB (reverse c-ERBAa) nuclear receptor proteins, which play an important role in metabolism and circadian rhythms. Though SR8278 has been shown to have beneficial physiological effects in a variety of preclinical disease contexts, its impact on gene expression and cell proliferation in keratinocytes has not previously been examined.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAn RNA-seq analysis was used to identify genes differentially impacted by SR8278 treatment in human HaCaT keratinocytes, which was confirmed by RT-qPCR and western blotting. Cell growth and viability assays were further used to examine cell proliferation in HaCaT and other cell lines. CRISPR/Cas9 genome editing was used to generate cells lacking REV-ERBα and β.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eRNA-seq analysis indicated genes involved in the G1/S transition of the cell cycle were significantly impacted by SR8278 treatment, which was confirmed via RT-qPCR and western blotting. Cell proliferation assays showed that SR8278 slowed cell growth but did not induce apoptosis. Finally, the knockout of the REV-ERBs did not impact the effect of SR8278 on gene expression and cell proliferation.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWe conclude that the anti-proliferative effects of SR8278 are not mediated by the REV-ERB proteins, and thus care should be taken when interpreting studies involving this compound unless complementary genetic approaches are also shown.\u003c/p\u003e","manuscriptTitle":"SR8278 inhibits cell proliferation independent of REV-ERB","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-30 09:39:57","doi":"10.21203/rs.3.rs-6727727/v1","editorialEvents":[{"type":"communityComments","content":3}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c4a0c643-7cb1-47b2-8ce1-782f62d518a4","owner":[],"postedDate":"May 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-08T18:24:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-30 09:39:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6727727","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6727727","identity":"rs-6727727","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}