CARMA3: A potential therapeutic target in non-cancer diseases.

Inflammation is a key characteristic in the pathogenesis of cardiovascular diseases, including atherosclerosis, hypertension, and abdominal aortic aneurysms ( 65 ). In the inflammatory process, blood vessels and perivascular connective tissue are vital regulators, and congestion and increased capillary permeability are important features of inflamed tissues ( 66 ). In murine endothelial cells, CARMA3 assembles BCL10 and MALT1 to form a CBM signalosome, which triggers the activation of NF-κB induced by CXCL8/IL8, an essential chemokine involved in pro-angiogenic and pro-inflammatory processes. This process controls VEGF expression and promotes the autocrine activation of VEGF receptors ( 46 ). The CBM complex also mediates the activation of thrombin-induced NF-κB in murine endothelial cells, and CARMA3 deficiency completely blocks the thrombin-induced phosphorylation of IκB ( 41 ). Using siRNA to disrupt the CBM signalosome, the thrombin-responsive induction of adhesion molecules intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1), which are relevant to atherogenesis, are dramatically downregulated. In addition, thrombin-induced monocyte/endothelial adhesion was also reduced. With this, the CBM complex plays a key role in proximal steps of NF-κB activation as well as in downstream regulation of NF-κB target genes ( 41 ). Moreover, the CBM complex triggers the Ang II-dependent NF-κB inflammatory signaling cascade in endothelial and vascular smooth muscle cells, and then induces pro-inflammatory signals in the vessel wall, which is a vital factor of atherogenesis ( 43 ). BCL10 and MALT1 are also important in the activation of Ang II-induced NF-κB, using siRNA to knockdown BCL10 or MALT1, Ang II-induced phosphorylation of IκB is completely ablated. In BCL10-deficient mice, the extent of atherosclerosis is significantly reduced, and the incidence and severity of abdominal aortic aneurysms are significantly lower than those observed in the presence of BCL10 ( 43 ). In addition, several pro-inflammatory mediators, which are associated with atherogenesis, such as the SCF/c-Kit ligand, CD40 ligand, CXCL6, IL-1α, and Eotaxin/CCL11, have lower serum levels in BCL10-deficient mice ( 43 ). These results indicate that BCL10-deficient mice are dramatically protected against Ang II-induced atherosclerosis and abdominal aortic aneurysms. In addition, MALT1 protease activity is also critical in vascular inflammation, it mediates thrombin-induced endothelial permeability and the activation of MALT1 protease disrupts endothelial capillary tubes ( 67 ). These studies indicated the importance of CARMA3 and its binding partners in cardiovascular diseases. Asthma is broadly defined as airway inflammation implicated in hyperresponsiveness and mucus hypersecretion. NF-κB plays a key role in the pathogenesis of asthma, including airway epithelial barrier dysfunction, airway hyperreactivity and remodeling, chemokine and cytokine production ( 68 – 71 ). CARMA3 is highly expressed in human airway epithelial cells and mediates LPA-induced NF-κB activation ( 72 ). It is necessary for the production of pro-inflammatory cytokines and chemokines, such as CC-​chemokine ligand 20 (CCL20)/macrophage inflammatory protein (MIP)-3α, granulocyte-macrophage colony-stimulating factor (GM-CSF), thymic stromal lymphopoietin (TSLP), IL-8, IL-25, and IL-33, which are significantly upregulated in patients with allergic pulmonary inflammation or asthma in response to inhaled stimuli ( 72 – 74 ). In addition, in an asthma mouse model induced by ovalbumin, deletion of CARMA3 in airway epithelial cells (AECs) alleviated allergic airway inflammation ( 73 ). The CARMA3-deficient murine model not only attenuated the production of pro-inflammatory cytokines and chemokines, but also impaired dendritic cell (DC) recruitment, DC maturation, DC migration, Ag processing, and resultant T cell proliferation ( 73 , 74 ). After CARMA3-deficient mice were stimulated with Alternaria alternata , the total number and percentage of type 2 innate lymphoid cells (ILC2) in the lungs were reduced, suggesting that CARMA3 mediates the accumulation of ILC2 in the lungs after allergen exposure, which may be one mechanism by which type 2 cytokine production is attenuated in the lung and airway inflammation in CARMA3-deficient mice ( 74 ). Additionally, CARMA3 plays a vital role in pulmonary fibrosis ( 75 ). Knockdown of CARMA3 attenuates bleomycin-induced inflammation and fibrosis and protects the lung from inflammation, fibrosis, collagen fibril deposition, alveolar epithelial cell destruction, and pathological airway remodeling ( 75 ). In conclusion, CARMA3 is recognized as a pro-inflammatory molecule involved in lung inflammation, such as asthma and pulmonary fibrosis. In hepatocytes, palmitate metabolism stimulates the CBM complex to trigger NF-κB-mediated inflammation in the absence of receptor engagement. siRNA-mediated depletion of CARMA3 effectively blocked palmitate-dependent IκBα phosphorylation. Free fatty acids (FFA), which are essential for the pathogenesis of insulin resistance, are increased in the serum of obese people. Deletion of BCL10 in mice eliminates hepatic NF-κB activation induced by a high-fat diet, resulting in mitigated inflammation and protection from insulin resistance ( 76 ). Ang II is involved in tissue-repairing process by mediating inflammatory cell infiltration, myofibroblast proliferation, and collagen synthesis after liver injury ( 77 ). But in chronic liver injury, Ang II induces pathologic liver fibrosis by activating NF-κB ( 36 ). Knockout of CARMA3 effectively disrupts the activation of Ang II-induced NF-κB signaling pathway, preventing the occurrence of liver fibrosis ( 36 ). However, in an autoimmune hepatitis model induced by Con A, deletion of CARMA3 leads to aggravated liver injury and liver sinusoidal endothelial cells (LSECs) damage, indicating that CARMA3 plays a protective role in the process, which is different from previous studies. CARMA3 exerts its protective effect by preserving mitochondrial integrity, but the detailed mechanisms is still unknown ( 78 ). These results indicate that CARMA3 has diverse characters in different hepatic diseases. CARMA3 is widely expressed in various tissues such as the heart, lung, liver, intestine, and kidney ( 29 ). However, current research on CARMA3 in non-cancer diseases mainly focuses on the cardiovascular system and lung diseases, with relatively few studies on diseases of other systems. Herein, we briefly overview the role of CARMA3 in other disorders. In intestinal epithelial cells (IECs), CBM signalosomes activate the NF-κB signaling pathway triggered by platelet-activating factor (PAF) and play a pivotal role in IL-8 production ( 79 ). Subsequently, Dudeja et al. demonstrated that Lactobacillus acidophilus , a probiotic, could eliminate PAF-induced inflammation in IECs by blocking PAF-induced NF-κB activation and IL-8 production. In addition , Lactobacillus acidophilus culture supernatant (LA-CS) can attenuate the PAF-induced elevation of BCL10 mRNA and protein levels, BCL10 promoter activity, BCL10 interaction with MALT1, and PAF-induced ubiquitination of IKKγ ( 80 ). These results suggest a novel target complex for the treatment of PAF-induced inflammation in IECs. In normal human epidermal keratinocytes (NHEKs), CARMA2 is more investigated, it is expressed in keratinocytes and its mutations increase the risk of inflammatory skin disease ( 81 ). Recently, a CARMA3-dependent tonic signalosome was described, which activates MALT1 protease activity and mediates IL-17/TNF-α−induced keratinocyte inflammatory response ( 82 ). In proliferating NHEKs, the level of CARMA3 is 10-fold higher than that of CARMA2. During differentiation, CARMA2 was upregulated and CARMA3 was downregulated, indicating that CARMA3 may be involved in keratinocytes, particularly during their proliferation ( 82 ). Subsequently, knockdown of MALT1 or CARMA3 in proliferating NHEKs abrogated the cleavage of MALT1 substrates, indicating that CARMA3 is fundamental to MALT1 protease activity ( 82 ). In addition, knockout of CARMA3 led to downregulation of serpin family B member 2 (SERPINB2) at both the mRNA and protein levels, which is positively related to psoriasis severity ( 22 ). These results suggest the importance of CARMA3 in psoriasis.

CARMA3 is a member of the CARMA family, which includes CARMA1, CARMA2, and CARMA3 proteins. The three CARMA proteins are encoded by different genes and expressed in different tissues. CARMA1 is encoded by CARD11 and is mainly expressed in lymphoid and hematopoietic tissues, and CARMA2 is encoded by CARD14 and expressed in mucosal and skin tissues, while CARMA3 is encoded by CARD10 and is widely expressed in tissues other than hematopoietic cells ( 1 ). Despite different expression patterns, CARMA family members appear to share functional similarity. They are regarded as molecular scaffolds that assist in recruiting and assembling signal transduction molecules. All three CARMA proteins can recruit BCL10 and MALT1 to form the CARMA-BCL10-MALT1 complex (commonly known as CBM), which regulates NF-κB activation and is involved in the occurrence of various diseases ( 1 ). CARMA1 is involved in both innate and adaptive immune systems ( 2 ) and is crucial for antigen receptor-dependent lymphocyte activation and NF-κB signaling ( 3 – 5 ). Both CARMA1 deficiency and mutations are associated with immunodeficiency ( 6 – 8 ). CARMA1 mutations can also cause atopic diseases, B cell expansion and susceptibility to B cell lymphoma ( 8 – 11 ). CARMA2 is the less-studied CARMA protein family member. CARMA2 mutations are associated with inflammatory skin disorders, especially psoriasis ( 12 ). In cancer cells, CARMA2 knockout leads to inhibition of cell proliferation and migration and decrease in cell survival ( 13 , 14 ). The function of CARMA3 is similar to that of other CARMA proteins, it mediates GPCRs- and RTKs-induced signaling pathways, which are linked to human disease pathogenesis. Despite its potential significance, a discussion on the function of CARMA3 in non-cancer diseases is lacking. Hence, this study aims to review an understanding of the molecular function, mutation, and regulation of CARMA3 and highlight recent findings in non-cancer diseases.

Over the past few years, mutations in CARD11 and CARD14 have been studied extensively. CARD11 mutations are linked to lymphoproliferative disorders and immunodeficiencies, whereas CARD14 variants are associated with several inflammatory skin disorders ( 83 , 84 ). However, the consequences of CARD10 mutations in humans remain unclear. De Boer et al. previously conducted a genome-wide survey and first identified the CARD10 polymorphism rs6000782 as a likely risk factor for type1 autoimmune hepatitis (AIH) in Dutch and German populations ( 85 ), which Motawi et al. also concluded with a cohort of Egyptian children ( 86 ). However, these results could not be replicated in a Japanese population ( 87 ). This discrepancy may result from several factors: (1) ethnic variations, (2) different sample sizes, and (3) different experimental conditions and methods. In addition, CARD10 rs9607469 and CARD10 rs9610775 are associated with primary open-angle glaucoma and amnestic mild cognitive impairment, respectively ( 88 – 90 ). Apart from CARD10 polymorphism, Hang et al. were the first to identify two in-frame deletions in CARD10 among 101 cases of ovarian endometriosis, which may cause the disease ( 91 ). Luo et al. used exome sequencing to identify the causative genes of two siblings, who are affected by autoimmune deficiency from a consanguineous family. They identified a new homozygous missense mutation (c.1258C>T; p. R420C) of CARD10 , which is implicated in rare diseases, repeated infections, Crohn’s disease, allergic diseases, and other disorders ( 92 ). Among the above mentioned CARD10 mutations, rs9610775 and R420C are missense variants and are located in exons 4 and 7 of CARD10 respectively. Both mutations alter the CARMA3 CC domain, which is associated with BCL10 aggregation and activation, suggesting that the rs9610775 and R420C mutations affect CBM assembly and the subsequent NF-κB activation ( 90 – 93 ). To date, no study has reported the direct effect of CARD10 mutations on NF-κB signaling. However, Said et al. found that the NF-κB p65 level is increase in the AIH patients with CARD10 polymorphism rs6000782, suggesting that the CARD10 polymorphism rs6000782 may cause disease by affecting NF-κB signaling and the expression levels of downstream genes ( 86 ). Further studies should be conducted to elucidate the exact effect of CARD10 mutations on the NF-κB signaling pathway. All the functions of CARMA3 in non-cancer diseases were summarized in Figure 3 . Overview of CARMA3 in non-cancer diseases. CBM complex are implicated in the pathogenesis of cardiovascular diseases, pulmonary diseases, liver-related diseases, intestinal disease and psoriasis; carma3 mutations are related to type1 autoimmune hepatitis, primary open-angle glaucoma, mild cognitive impairment, ovarian endometriosis and immunodeficiency and autoimmunity.

Although the structure of CARMA3 has been elucidated, the regulation of CARMA3 remains largely unknown. Several microRNAs (miRNAs) reportedly play important roles in regulating CARMA3 levels ( Figure 1A ). miRNA146a is upregulated in the serum and tissues of abdominal aortic aneurysm (AAA) patients and inhibits CARMA3 expression in human umbilical vein endothelial cells ( 19 ). It inhibits NF-κB activation by targeting CARD10 through negative feedback ( 20 ), and miR-146a knockdown significantly suppressed lipopolysaccharide-induced cell migration and tube formation ( 21 ). Silencing of miR-146a/b that directly targets CARD10 , resulted in inflammation in psoriatic skin ( 22 ). miR-181b has also been identified as an important regulator of arterial thrombosis that directly targets CARD10 in endothelial cells (ECs). Knockdown of CARMA3 reduces thrombin-induced gene expression and NF-κB signaling, while overexpression can effectively rescue miR-181b-mediated inhibition of NF-κB target genes, such as VCAM-1 , ICAM-1 , and E-SELECTIN ( 23 ). Additionally, CEBPE, a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, regulates CARD10 transcription ( 24 ), as shown in Figure 1A . Chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR), luciferase reporter assay and electrophoretic mobility shift assay confirmed that CEBPE binds regulatory elements upstream of CARD10 (-1.5 kb and -7kb). In immature granulocytes from CEBPE-knockout mice, the expression of CARMA3 was significantly lower than that at the same stage of differentiation in wild-type mice ( 24 ). Some studies reported that A20 regulated the NF-κB signaling pathway and downregulated proinflammatory cytokines expression ( 25 – 27 ). A20 negatively regulates CARMA3- and BCL10-mediated NF-κB activation via its deubiquitylation activity ( 28 ). It directly deubiquitylates IKKγ/NEMO and/or TRAF6, thereby suppressing NF-κB activation by perturbing assembly of the complex comprising CARMA3, BCL10 and IKKγ/NEMO ( 28 ). These results indicate that CARMA3 is regulated at the transcriptional and CBM complex formation level by different regulators. All the CARMA3 regulators reported have been listed in Table 1 . List of CARMA3 regulators.

CARMA3, also known as CARD10 or BCL10-interacting membrane protein 1 (Bimp1), was discovered in 2001 ( 15 ). The CARD10 gene is located on human chromosome 22q13.1 and consists of 20 exons. Its mRNA and coding sequence (CDS) lengths are 4111nt and 3099nt respectively. It encodes a 1032-amino acid protein, which comprises five functional domains: a caspase recruitment domain (CARD) domain, a coiled-coil (CC) domain, a PDZ domain, an SH3 domain, and a guanylate kinase-like (GUK) domain ( 15 ), as shown in Figure 1 . The CARD domain is a protein module composed of six or seven antiparallel alpha helices. It is necessary for BCL10 recruitment, which is essential for NF-κB signaling ( 15 ). Close to CARD domain is a CC domain, it is composed of one or more alpha-helical peptides that are intertwined in a super-helical fashion ( 16 ). CC domain can mediate self-association, leading to the aggregation and activation of BCL10 in response to upstream signals ( 15 , 17 ). The Membrane-associated GUK (MAGUK) domain comprises a PDZ domain, SH3 domain, and GUK domain ( 18 ). The PDZ and SH3 domains of CARMA3 are essential for NF-κB activation, which is not affected by deletion of GUK domain ( 15 ). Gene structure of CARD10 and protein structures of CARMA1, CARMA2, and CARMA3. (A) The CARD10 gene consists of 20 exons. Its mRNA and CDS lengths are 4111 and 3099nt respectively. CEBPE is a transcription factor, that targets the promoter region of CARD10 . microRNA -146a/b and microRNA-181b regulate CARD10 by acting on the 3-’UTR of CARD10 mRNA. (B) All the three CARMA proteins are comprised of five functional domains: a CARD domain, a CC domain, a PDZ domain, an SH3 domain, and a GUK domain.

CARMA3 is widely expressed in a variety of tissues ( 29 ) and plays key roles in the pathological process of human diseases ( 37 ). To date, most of the existing literature on the role of CARMA3 in diseases has focused on a variety of cancers. Relatively fewer studies investigated the role of CARMA3 in non-cancer diseases. In addition, most studies focused on airway epithelia and the cardiovascular system while searching in the literature for association between non-cancer disease and CARMA3. Considering that CARMA3 affects cell inflammation, proliferation, survival, migration, and other cell processes ( 55 , 58 , 64 , 94 ), it should be taken into account that CARMA3 could be involved in other non-cancer diseases, such as urinary system diseases, nervous system diseases, and bone and joint disorders. Further studies exploring these areas are needed. Current studies on the regulation of CARMA3 mainly focus on the transcriptional level, with no investigations on phosphorylation, ubiquitination, and acetylation of CARMA3 proteins. To further understand the contribution of CARMA3 in diseases, its regulation at the protein level is worthy of investigation. Furthermore, CARMA3-dependent signal transmission plays a fundamental role in pathophysiology of various diseases, indicating that generating molecular tools, such as novel inhibitors specific to CARMA3 and small peptides target to CBM signalosome, would be immeasurable in treating CARMA3-related disorders.

WX and ZJ conducted the review, and ZG, WX, and YZ drafted the manuscript. All the authors have written, reviewed and approved the manuscript.