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Multipotent bone marrow-derived mesenchymal stem/stromal cells (BMSCs) exhibit a finite life span after ex vivo expansion leading to cellular senescence. Many factors can contribute to this. Recently, our group has identified for the first time expression of the chemokine-like factor superfamily 8 (CMTM8) gene in cultured human BMSCs. In this study, we examine the role of CMTM8 in BMSC proliferation, migration, and differentiation. Functional studies using siRNA-mediated knockdown of CMTM8 in human BMSCs resulted in decreased capacity to undergo proliferation and migration and an increased capacity for osteogenic differentiation in vitro. Furthermore, reduced CMTM8 levels led to a decrease in the epidermal growth factor receptor (EGFR) signaling pathway during BMSC proliferation and migration, respectively. Supportive studies using retroviral mediated enforced expression of CMTM8 in BMSC resulted in an increased capacity for proliferation and migration but a decreased osteogenic differentiation potential. Collectively, these data suggest that CMTM8 promotes BMSC proliferation and BMSC migration through the EGFR/ERK1/2 pathway. This study provides insight into novel regulatory mechanisms of human BMSC growth and cell fate determination.

Multiple myeloma (MM) is a devastating cancer with a highly heterogeneous outcome. Because of the heterogeneity of myeloma cells, risk stratification is important for making therapeutic regimens. Nevertheless, no immunohistochemical predictive and prognostic marker has been constructed yet. In the present study, we explored the prognostic value of proteolipid protein 2 (PLP2) in MM patients using immunohistochemistry (IHC). We assessed PLP2 expression in bone marrow (BM) biopsy specimens obtained from 87 newly diagnosed MM (NDMM) patients. Correlations between PLP2 expression and clinicopathological features were analyzed. PLP2 expression was present in high-risk MM patients, which was increased with disease progression and poor prognosis. PLP2 was increasing in parallel with high beta-2 microglobulin ( β 2-MG) and lactate dehydrogenase (LDH). Furthermore, MM patients with low PLP2 expression could achieve a favorable treatment response. PLP2 may be a novel biomarker for prognostic prediction and a therapeutic target for anti-MM treatments., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Hua Bai et al.)

B-1a cells represent a distinct B cell population with unique phenotype, self-renewing capacity and restricted Igμ repertoire. They primarily locate in body cavity and also exist in spleen. The different subpopulations of B-1a cells are heavily affected by local environment. Our previous studies revealed that MARVEL-domain-containing membrane protein, CMTM7, was involved in B-1a cell development. Here, we focused its influence on peritoneal and splenic B-1a cells. Unlike peritoneal B-1a cells, we found that splenic Cmtm7 -/- B-1a cells expressed higher level of CD5, CD80 and CD86 compared with WT counterparts. They also exhibited an enhanced tonic BCR signals in steady state. Though the cell viability was unaffected in vitro, Cmtm7 knockout markedly promoted splenic B-1a cell apoptosis in situ, which was likely associated with down-regulation of Il-5rα. With regard to Igμ repertoire, peritoneal and splenic Cmtm7 -/- B-1a cells exhibit similar changes exemplified by the loss of V H 11 and gain of V H 12, whereas an increase in V H 1 usage and skewed J segments from J H 1 to J H 2 and J H 4 families could only be detected within splenic Cmtm7 -/- B-1a cells. Overall, these data indicate that Cmtm7 functions differently in peritoneal and splenic B-1a cells and plays a more important role in splenic cells., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: CMTM6 was identified as an important regulator of the PD-L1 protein. The role of CMTM6 in lung adenocarcinoma (LUAD) has so far remained unclear. We aimed at investigating the role of CMTM6 in LUAD at transcriptome and genomic levels and its relationship with tumor-infiltrating immune cells (TIICs)., Methods: We downloaded the data sets of LUAD from TCGA. The genomic profiles containing somatic mutations were analyzed and the transcriptome level of CMTM6 was also obtained. Gene set variation analysis (GSVA) was used to predict the pathway change. In addition, we explored the association between CMTM6 and LUAD immune infiltrates by means of CIBERSORT. The association between CMTM6 and PD-L1 mRNA was analyzed using an integrated repository portal for tumor-immune system interactions (TISIDB) and was further validated in 80 LUAD patients. Kaplan-Meier survival curve and the log-rank test was used to analyze the survival significance of CMTM6., Results: We found that CMTM6 was downregulated in LUAD. Patients with low CMTM6 expression were more likely to be frequent with somatic mutations. Moreover, GSVA analysis exhibited that CMTM6 was associated with immune responses and inflammatory activities. Specifically, a positive correlation between increased CMTM6 expression and immune infiltrating level of Dendritic cells resting, Eosinophils, Macrophages M1, Macrophages M2, Neutrophils, T cells CD4 memory activated and T cells CD4 memory resting was established. The CMTM6 expression was positively correlated with PD-L1 in both mRNA and protein level. Clinically, patients with high expression of CMTM6 tended to have a better survival., Conclusion: CMTM6 expression likely had an important effect on TIICs composition and prognosis in LUAD patients. The CMTM6 expression was positively correlated with PD-L1 in LUAD. These findings establish CMTM6 as a promising target for immunotherapeutic prospects., Competing Interests: Declaration of Competing Interest No potential conflicts of interest were disclosed., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing family (CMTM) is widely expressed in the immune system. Abnormal expression of CMTM is associated with the development of various diseases. This article summarizes the relevant research on the role of the CMTM family in immune disorders. This information will increase our understanding of pathogenesis and identify promising targets for the diagnosis and treatment of autoimmune diseases. The CMTM family is highly expressed in peripheral blood mononuclear cells. CKLF1 may be involved in the development of arthritis through its interaction with C-C chemokine receptor 4. CKLF1 is associated with the pathogenesis of lupus nephritis and psoriasis. Both CMTM4 and CMTM5 are associated with the pathogenesis of systemic lupus erythematosus. CMTM1, CMTM2, CMTM3, and CMTM6 play a role in rheumatoid arthritis, systemic sclerosis, Sjögren syndrome, and anti-phospholipid syndrome, respectively. The CMTM family has been implicated in various autoimmune diseases. Further research on the mechanism of the action of CMTM family members may lead to the development of new treatment strategies for autoimmune diseases.

This study aimed to investigate the expression and clinical significance of chemokine-like factor-like MARVEL transmembrane domain-containing family member 2 (CMTM2) in hepatocellular carcinoma (HCC) tissues. Bioinformatics analysis showed that CMTM2 was downregulated in HCC tissues and correlated with vascular invasion of HCC patients. The immunohistochemistry (IHC) method found that CMTM2 expression was negative in 29/75 (38.67%) cases of HCC tissues and 13/75 (17.33%) cases of paracancerous tissues, also showed a significantly lower expression of CMTM2 in HCC tissues than the paired paracancerous tissues ( p  < 0.05). Moreover, CMTM2 expression was significantly correlated with tumor grade of HCC patients ( p  < 0.05), but had no relationship with other clinicopathological features. In addition, multivariate logistic regression analysis indicated that tumor grade was an independent risk factor for CMTM2 expression. The survival time of HCC patients between high and low expression of CMTM2 had no difference by bioinformatics analysis, but the IHC result showed that the negative expression of CMTM2 was related to a poor prognosis of HCC patients. Further COX regression analysis showed that CMTM2 expression was an independent protective factor for the prognosis of HCC patients. We identify that CMTM2 is downregulated in HCC tissues and correlated with the prognosis of HCC patients, suggesting a potential tumor suppressor role of CMTM2 in HCC progression.

Proteolipid protein 2 (PLP2) is an integral ion channel membrane protein of the endoplasmic reticulum. The protein has been shown to be highly expressed in many cancer types, but its importance in glioma progression is poorly understood. Using publicly available datasets (Rembrandt, TCGA and CGGA), we found that the expression of PLP2 was significantly higher in high-grade gliomas than in low-grade gliomas. We confirmed these results at the protein level through IHC staining of high-grade (n = 56) and low-grade glioma biopsies (n = 16). Kaplan-Meier analysis demonstrated that increased PLP2 expression was associated with poorer patient survival. In functional experiments, siRNA and shRNA PLP2 knockdown induced ER stress and increased apoptosis and autophagy in U87 and U251 glioma cell lines. Inhibition of autophagy with chloroquine augmented apoptotic cell death in U87- and U251-siPLP2 cells. Finally, intracranial xenografts derived from U87- and U251-shPLP2 cells revealed that loss of PLP2 reduced glioma growth in vivo. Our results therefore indicate that increased PLP2 expression promotes GBM growth and that PLP2 represents a potential future therapeutic target., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Lipid accumulation has been highlighted in cancer development and progression, but the exact mechanism remains unclear in renal cell carcinoma (RCC). MicroRNAs (miRNAs) have been confirmed to participate in the pathological processes of cancers, including tumour occurrence and inhibition. However, the role and mechanism of miR-765 have not been elucidated in clear cell renal cell carcinoma (ccRCC)., Methods: Using The Cancer Genome Atlas (TCGA) database and qRT-PCR, we investigated differences in miR-765 and proteolipid protein 2 (PLP2) expression, as well as their clinical relevance. To investigate the function of miR-765 and PLP2 in ccRCC, we performed in vitro and in vivo experiments to explore their biological functions in ccRCC., Findings: In this study, we showed that miR-765 was upregulated in the plasma of ccRCC patients after tumour resection. Consistently, ccRCC tissues had low expression of miR-765 when compared with corresponding non-cancerous tissues. Overexpression of miR-765 suppressed cell proliferation and metastasis in vitro and in vivo. Mechanistic studies demonstrated that PLP2 was a direct target gene of miR-765. PLP2 was highly expressed in ccRCC tissues, and high PLP2 levels were positively correlated with higher tumour stage and grade and poor prognosis. PLP2 expression was negatively correlated with the miR-765 level in patient samples. We further showed that PLP2 restrained the cell metastasis and proliferation induced by miR-765 and reduced the lipid-eliminating effects of miR-765 in renal cancer cells., Interpretation: Our findings suggest that miR-765 may function as a tumour suppressor and eliminate lipids in clear cell renal cell carcinoma by targeting PLP2., Funding: This work was funded the grants from the National Natural Scientific Foundation of China (Grant No. 81672528, 81672524, 81602218, 31741032, 81902588)., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of this manuscript., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Chemokine-like factor 1 (CKLF1) is a potential target for ischemic stroke therapy. The NOD-like receptor protein 3 (NLRP3) inflammasome has been postulated to mediate inflammatory responses during ischemic/reperfusion (I/R) injury. The compound IMM-H004 is a novel coumarin derivative that can improve cerebral I/R injury. This study aims to investigate the effects of IMM-H004 on ischemia stroke injury and further elucidate the molecular mechanisms. The standard pMCAO model of focal ischemia was used in this paper. Drugs were administered at 6 hours after ischemia, and behavioral assessment, euthanasia, and outcome measures were evaluated at 9 hours after ischemia. The effects of IMM-H004 on ischemic stroke injury were determined using 2,3,5-triphenyltetrazolium chloride (TTC) staining, behavioral tests, enzyme-linked immunosorbent assay (ELISA), and Nissl staining. Immunohistologic staining, immunofluorescence staining, quantitative RT-PCR (qPCR), western blotting, and coimmunoprecipitation (CO-IP) assays were used to elucidate the underlying mechanisms. IMM-H004 treatment provided significant protection against ischemia stroke through a CKLF1-dependent anti-inflammatory pathway in rats. IMM-H004 downregulated the amount of CKLF1 binding with C-C chemokine receptor type 4, further suppressing the activation of NLRP3 inflammasome and the following inflammatory response, ultimately protecting the ischemic brain. This preclinical study established the efficacy of IMM-H004 as a potential therapeutic medicine for permanent cerebral ischemia. These results support further efforts to develop IMM-H004 for human clinical trials in acute cerebral ischemia, particularly for patients who are not suitable for reperfusion therapy., (Copyright © 2019 Elsevier Inc. All rights reserved.)

CKLF-like MARVEL transmembrane domain-containing member (CMTM) is a new gene family first cloned and reported in 2001. The CMTM family consists of nine members including CKLF and CMTM1-CMTM8, which are located on different chromosomes. Besides exhibiting extensive chemotactic activity, the CMTM family plays an important role in the hematopoiesis system, the immune system, the cardiovascular system and the male reproductive system. Recent in-depth research has also revealed that CMTM is closely associated with the genesis, development and metastasis of tumors, displaying opposing activities in diverse human tumors. In this review, we discuss the structural and functional characteristics of the CMTM family and summarize latest research findings of the relationship between several CMTM members and non-small cell lung cancer., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Chemokine-like factor 1 (CKLF1) is a cytokine, which has a detrimental effect on the multiple disease progression. Our previous studies reported that arterial injury induced the upregulation of CKLF1 expression in artery at 7-14 days after injury. Here, using a rat carotid balloon injury model, we found that CKLF1 knockdown in the injured site abolished neointimal formation and even decreased medial area; contrarily, CKLF1 overexpression developed a thicker neointima than controls, demonstrating that CKLF1 exerted positive effects on neointimal hyperplasia and the accumulation of vascular smooth muscle cells (VSMC). The mechanism study indicated that CKLF1 reduced susceptibility to the cell cycle G2/M arrest and apoptosis, and thereby speeding up VSMC accumulation. This role of CKLF1 was tightly associated with phosphatidylinositol (PI) 3-kinase signaling pathway. CKLF1 increased the expression of four isoforms of the PI3-kinase catalytic subunits, which in turn activated its downstream targets Akt and an effector NF-κB accepted as critical transcription factors of cell survival and proliferation. Furthermore, RNA-sequencing analysis revealed that CKLF1 had wide-ranging roles in regulating the expression of genes that mainly engaged in cell apoptosis and innate immune response. Collectively, the data allow us to conclude that high level CKLF1 after artery injury switches the balance of VSMC proliferation and apoptosis through PI3K/AKT/NF-κB signaling and consequently leads to neointimal hyperplasia. The findings shed insight into new treatment strategies to limit restenosis based on CKLF1 as a future target., (Copyright © 2019. Published by Elsevier Masson SAS.)

Renal clear cell carcinoma (RCC) patients who do not achieve optimal control of progression with immune checkpoint blockade (ICB) should be further studied. Unsupervised consensus clustering was used to group 525 RCC patients based on two typical ICB pathways, CTLA-4 and pogrammed death 1 (PD-1)/programmed death-ligand 1 (PD-L1), as well as two new discovered regulators, CMTM6 and CMTM4. Three immune molecular subtypes (IMMSs) with different clinical and immunological characteristics were identified (type I, II, and III), among which there were more stage I and low-grade tumors in type I RCC than in type II and III. The proportion of males was highest in type II RCC. Overall survival of type II and III was similar (5.2 and 6 years) and statistically shorter than that of type I (7.6 years) before and after adjusting for age and gender. When conducting stratified analysis, our IMMSs were able to identify high-risk patients among middle-aged patients, males, and stage IV patients. Among the differentially expressed genes, approximately 84% were highly expressed in type II and III RCC. Genes related to ICB (CTLA-4, CD274, and PDCD1LG2) and cytotoxic lymphocytes (CD8A, GZMA, and PRF1) were all highly expressed in type II and III RCC. These results documented that patients with type II and III cancer may be more sensitive to anti-CTLA-4 therapy, anti-PD-1/PD-L1 therapy, and a combination of immunotherapies. High expression of CMTM4 in type I RCC (69%) and a statistically significant interaction of CD274 and CMTM6 indicated that CMTM4/6 might be new therapy targets for type I, who are resistant to ICB., (© 2019 Wiley Periodicals, Inc.)

Purpose: Hepatocellular carcinoma (HCC), one of the most common and deadliest malignancies worldwide, has a poor prognosis, owing to its high potential for vascular invasion and metastasis and the lack of biomarkers for early diagnosis. Thus, it must be a crucial factor for investigating therapeutic strategies for HCC to identify the functional molecular targets. Here, we reported a novel chemokine, CKLF1, that might act as a pivotal modulator in the invasion and metastasis of HCC and could serve as an attractive target for cancer therapy., Experimental Design: Bioinformatics analysis, PCR, Western blotting, and IHC were performed to detect the expression of CKLF1 in HCC. The function of CKLF1 was demonstrated by a series of in vitro and in vivo experiments. Pharmacologic treatment, flow cytometry, and Western blotting were carried out to demonstrate the potential mechanisms of CKLF1., Results: We proved that CKLF1 was overexpressed in HCC tissues and was related to tumor stage, vascular invasion, and patient survival. Then, functional assays showed that CKLF1 promoted hepatocellular carcinogenesis and metastatic potential. Finally, the IL6/STAT3 signaling pathway was involved in the mechanistic investigation. The results demonstrated that CKLF1 enhanced the progression of HCC and prevented doxorubicin-induced apoptosis through activating the IL6/STAT3 pathway., Conclusions: These data showed that CKLF1 inhibited apoptosis and promoted malignant transformation through the IL6/STAT3 pathway, and ultimately enhanced the development and metastasis of HCC. Thus, our work revealed that CKLF1 was a significant prognostic factor of HCC and might be a potential molecular therapeutic target for HCC., (©2019 American Association for Cancer Research.)

Chemokine-like factor (CKLF)-like, MAL and related proteins for vesicle trafficking and membrane link (MARVEL) transmembrane domain-containing family proteins (CMTMs) have significant roles in the immune system, in male reproduction, as well as in tumorigenesis. Previous studies have shown that CMTM family member 7 (CMTM7) was broadly expressed in various normal tissues, but not in lung, gastric, esophageal, pancreas, and cervix cancers. To explore its relationship with liver cancer, we examined the expression of CMTM7 in liver cancers and its correlation with clinical and pathological conditions. We found that CMTM7 expression was markedly reduced in liver cancer tissues, and negatively correlated with TNM staging and tumor metastasis. In vitro studies showed that enforced expression of CMTM7 inhibited the cell growth and migration of liver cancer cells. Further analysis revealed that CMTM7 suppressed AKT signaling and induced cell cycle arrest at the G0/G1 phase in the liver cancer cells, likely as the consequent of decreased levels of cyclin D1, cyclin-dependent kinase 4 (CDK4), and CDK6, and increased p27 expression. Thus, CMTM7 functions as a tumor suppressor in liver cancer through suppressing cell cycle progression., (© 2019 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

The chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing family (CMTM) is a gene family that has been implicated in male reproduction. CMTM4 is an evolutionarily conserved member that is highly expressed in the testis. However, its function in male fertility remains unknown. Here, we demonstrate that CMTM4 is associated with spermatogenesis and sperm quality. Using Western blotting and immunohistochemical analyses, we found CMTM4 expression to be decreased in poor-quality human spermatozoa, old human testes, and testicular biopsies with nonobstructive azoospermia. Using CRISPR-Cas9 technology, we knocked out the Cmtm4 gene in mice. These Cmtm4 knockout (KO) mice showed reduced testicular daily sperm production, lower epididymal sperm motility and increased proportion of abnormally backward-curved sperm heads and bent sperm midpieces. These mice also had an evident sub-fertile phenotype, characterized by low pregnancy rates on prolonged breeding with wild type female mice, reduced in vitro fertilization efficiency and a reduced percentage of acrosome reactions. We then performed quantitative proteomic analysis of the testes, where we identified 139 proteins to be downregulated in Cmtm4-KO mice, 100 (71.9%) of which were related to sperm motility and acrosome reaction. The same proteomic analysis was performed on sperm, where we identified 3588 proteins with 409 being differentially regulated in Cmtm4 -KO mice. Our enrichment analysis showed that upregulated proteins were enriched with nucleosomal DNA binding functions and the downregulated proteins were enriched with actin binding functions. These findings elucidate the roles of CMTM4 in male fertility and demonstrates its potential as a promising molecular candidate for sperm quality assessment and the diagnosis or treatment of male infertility., Competing Interests: The authors declare no conflict of interest., (© 2019 Liu et al.)

Background: IMM-H004 is a novel compound that has been shown to protect against cerebral ischemia/reperfusion injury in our previous works. Chemokine-like factor 1 (CKLF1) is a chemokine that exhibits increased expression in the ischemic brain. Dysregulation of microglia polarization dynamics is a mechanism of injury expansion poststroke., Purposes: The aim of present study was to investigate the effects of IMM-H004 on cell viability and microglia phenotypes in BV2 microglia suffering from oxygen-glucose deprivation/reperfusion and discussing the involvement of CKLF1 and possible mechanisms., Results: IMM-H004 protected BV2 microglia from oxygen-glucose deprivation/reperfusion-induced toxicity. We found that the expression of CKLF1 was increased in BV2 microglia with oxygen-glucose deprivation/reperfusion, and IMM-H004 decreased this specially increased expression. Moreover, oxygen-glucose deprivation/reperfusion induced the BV2 microglia to polarize toward an M1 phenotype, and IMM-H004 modulated the polarization shift from the M1 phenotype and skewed toward the M2 phenotype, followed by suppressing the excessive inflammatory response and improving recovery. CKLF1 modulated BV2 microglia toward M1 polarization and induced an inflammatory response. By using receptor inhibitors, we found that OGD/R induced microglia polarization partly through CC chemokine receptor 4. Furthermore, the Co-IP assay showed that IMM-H004 decreased the amount of CKLF1 binding to CC chemokine receptor 4 in the BV2 microglia oxygen-glucose deprivation/reperfusion model., Conclusions: IMM-H004 protects BV2 microglia against oxygen-glucose deprivation/reperfusion injury partly by modulating microglia polarization and further regulating the inflammatory response. The CKLF1/CCR4 axis may be involved in the protective effects of IMM-H004 modulating microglia polarization., (Copyright © 2019. Published by Elsevier B.V.)

Objective: To investigate whether CKLF-like MARVEL transmembrane domain-containing protein 2 (CMTM2) is involved in spermatogenesis in mice. CMTM2 is highly expressed in testis, and could possibly be a potential spermagogenesis specific gene., Methods: CMTM2-deficient mouse model was generated. Northern, RT-PCR and Western blotting analysis were performed on total RNA derived from wild-type (WT, CMTM2+/+) and CMTM2+/- (heterozygote) and CMTM2-/-(homozygote) mice to examine the CMTM2 level. The number of litters and the number of pups were counted and pregnancy rates calculated. The motility and morphology of the sperm and the histology of testes were analyzed. Serum testosterone and FSH concentrations were also measured. Standard t-tests were used and standard error of means were calculated., Results: CMTM2 was highly expressed in a finely regulated pattern in the mouse testis during spermatogenesis. The body weight of adult mice with CMTM2 deficiency was not significantly different from that of wild type mice. No obvious anatomical or behavioral abnormalities were observed. The testis of CMTM2-/- was smaller than that of CMTM2+/+ mice. The testis diameter in wild mice and CMTM2 null mice were (11.32±1.21) mm vs. (8.29±1.92) mm (P<0.05), and the weights were (101.63±2.33) mg vs. (85.22±2.84) mg (P<0.05), respectively. Female CMTM2 null mice were fertile, indicating that CMTM2 was not required for female gametogenesis. The CMTM2-/- mice produced virtually no sperm, and CMTM2+/- mice sperm count showed a significant decline. In terms of sperm morphorlogy study, more round spermatids could be observed in the heterozygote group, compared with the wild type group; while in the homozygote group, a large amount of round spermatids could be observed because of complete arrest of spermiogenesis. The hormone levels were not significantly different. The CMTM2-/- male mice were sterile due to a late, complete arrest of spermiogenesis. The organized architecture of the seminiferous epithelium of the seminiferous tubules seen in CMTM2+/+ mice was lost in CMTM2-/- mice., Conclusion: This study suggests CMTM2 is not required for embryonic development in the mouse but is essential for spermiogenesis, however, further studies are required for more detailed mechanism study.

(1) Background: Chemokine-like factor 1 (CKLF1) is a chemokine with potential to be a target for stroke therapy. Compound IMM-H004 is a novel coumarin derivative screened from a CKLF1/C-C chemokine receptor type 4 (CCR4) system and has been reported to improve cerebral ischemia/reperfusion injury. This study aims to investigate the protective effects of IMM-H004 on cerebral ischemia injury and its infectious cardiopulmonary complications in adult and aged rats from the CKLF1 perspective. (2) Methods: The effects of IMM-H004 on the protection was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining, behavior tests, magnetic resonance imaging (MRI) scans, enzyme-linked immunosorbent assay (ELISA), Nissl staining, histo-pathological examination, and cardiopulmonary function detection. Immunohistological staining, immunofluorescence staining, quantitative real-time PCR (qPCR), and western blotting were used to elucidate the underlying mechanisms. (3) Results: IMM-H004 protects against cerebral ischemia induced brain injury and its cardiopulmonary complications, inhibiting injury, and inflammation through CKLF1-dependent anti-inflammation pathway in adult and aged rats. IMM-H004 downregulates the amount of CKLF1, suppressing the followed inflammatory response, and further protects the damaged organs from ischemic injury. (4) Conclusions: The present study suggested that the protective mechanism of IMM-H004 is dependent on CKLF1, which will lead to excessive inflammatory response in cerebral ischemia. IMM-H004 could also be a therapeutic agent in therapy for ischemic stroke and cardiopulmonary complications in the aged population.

Vascular endothelial (VE) cadherin is a key component of endothelial adherens junctions (AJs) and plays an important role in maintaining vascular integrity. Endocytosis of VE-cadherin regulates junctional strength and a decrease of surface VE-cadherin reduces vascular stability. However, disruption of AJs is also a requirement for vascular sprouting. Identifying novel regulators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we evaluated the angiogenic potential of (CKLF-like MARVEL transmembrane domain 4) CMTM4 and assessed in which molecular pathway CMTM4 is involved during angiogenesis. Using a 3D vascular assay composed of GFP-labeled HUVECs and dsRED-labeled pericytes, we demonstrated in vitro that siRNA-mediated CMTM4 silencing impairs vascular sprouting. In vivo, CMTM4 silencing by morpholino injection in zebrafish larvae inhibits intersomitic vessel growth. Intracellular staining revealed that CMTM4 colocalizes with Rab4 + and Rab7 + vesicles, both markers of the endocytic trafficking pathway. CMTM4 colocalizes with both membrane-bound and internalized VE-cadherin. Adenovirus-mediated CMTM4 overexpression enhances the endothelial endocytic pathway, in particular the rapid recycling pathway, shown by an increase in early endosomal antigen-1 positive (EEA1 + ), Rab4 + , Rab11 +  , and Rab7 +  vesicles. CMTM4 overexpression enhances membrane-bound VE-cadherin internalization, whereas CMTM4 knockdown decreases internalization of VE-cadherin. CMTM4 overexpression promotes endothelial barrier function, shown by an increase in recovery of transendothelial electrical resistance (TEER) after thrombin stimulation. We have identified in this study a novel regulatory function for CMTM4 in angiogenesis. CMTM4 plays an important role in the turnover of membrane-bound VE-cadherin at AJs, mediating endothelial barrier function and controlling vascular sprouting.

Numerous studies have shown that CMTM family members have a variety of important roles in the occurrence and progression of cancer. CMTM7 has also been reported to be down-regulated in some digestive system tumors, but the expression patterns and pathological role of CMTM7 in gastric cancer remains unclear. In this study, we found that both CMTM7 and SOX10 were significantly down-regulated in gastric cancer tissues compared with paracancerous tissues, and the expression pattern of CMTM7 and SOX10 were strongly correlated (r = 0.6455, p < 0.001). Further, through bioinformatics technology and luciferase assay, we identify that SOX10 can be a transcriptional regulator of CMTM7 to mediate the expression of CMTM7 in gastric cancer. In addition, we found silencing the expression of CMTM7 can increase the proliferation and tumorigenesis of gastric cancer cells in vivo and in vitro. More interestingly, overexpression of SOX10 in cell lines stably silencing CMTM7 expression significantly inhibited the proliferation and tumor growth of gastric cancer. Therefore, our results demonstrate that CMTM7 as a tumor suppressor is down-regulated in gastric cancer, and SOX10 can regulate the proliferation and tumor formation of gastric cancer by regulating the expression of CMTM7., (Copyright © 2018. Published by Elsevier Inc.)

Chemokines are signaling proteins that are secreted to induce chemotaxis during an immunological response. However, the functions of transmembrane-type chemokine-like factor (CKLF) and the CMTM (CKLF-like MARVEL transmembrane domain containing) protein family remain to be determined. In this study, we focused on the testis-specific mouse CMTM gene cluster ( Cmtm1 , Cmtm2a and Cmtm2b ) and generated CRISPR/Cas9-mediated mutant mice to examine their physiological functions. Although Cmtm1 mutant mice were fertile, Cmtm2a and Cmtm2b double mutant mice had defects in male fertility due to impaired sperm function. We found that co-expression of sperm membrane proteins CMTM2A and CMTM2B is required for male fertility and affects the localization of the sperm membrane protein ADAM3 in regulating sperm fertilizing ability., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

In this study, we investigated how miR-10b-3p regulated the proliferation, migration, invasion in hepatocellular carcinoma (HCC) at both in vitro and in vivo levels. CMTM5 was among the differentially expressed genes (data from TCGA). The expression of miR-10b-3p and CMTM5 was detected by qRT-PCR and Western blot (WB). TargetScan was used to acquire the binding sites. Dual-luciferase reporter gene assay was used to verify the direct target relationship between miR-10b-3p and CMTM5. WB analysis proved that miR-10b-3p suppressed CMTM5 expression. Furthermore, proliferation, invasion and migration of HCC cells were measured by MTT assay, colony formation assay, transwell assay and wound-healing assay, respectively. Kaplan-Meier plotter valued the overall survival of CMTM5. Finally, xenograft assay was also conducted to verify the effects of miR-10b-3p/CMTM5 axis in vivo. Up-regulation of miR-10b-3p and down-regulation of CMTM5 were detected in HCC tissues and cell lines. CMTM5 was verified as a target gene of miR-10b-3p. The overexpression of CMTM5 contributed to the suppression of the proliferative, migratory and invasive abilities of HCC cells. Moreover, the up-regulation of miR-10b-3p and down-regulation of CMTM5 were observed to be associated with worse overall survival. Lastly, we have confirmed the carcinogenesis-related roles of miR-10b-3p and CMTM5 in vivo. We concluded that the up-regulation of miR-10b-3p promoted the progression of HCC cells via targeting CMTM5., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Objective: This study investigated miR-422a and PLP2 expressions in breast cancer cells and breast cancer stem cells (BCSCs). Besides, their influences on polymorphism changes were observed., Methods: Flow cytometry and fluorescence-activated cell sorting was performed and CD24 - /CD44 + cells were sorted from breast cancer cells and recognized as BCSCs. Microarray was applied to search for the differentially expressed miRNAs and mRNAs between MCF7 and BCSCs. The aberrant expression of miR-422a and PLP2 was further confirmed by RT-qPCR and the direct targeted relationship was verified by dual-luciferase reporter assay. After in vitro transfection, the expression of miR-422a and PLP2 were manipulated and biological functions of BMSCs were compared with CCK-8, colony formation and sphere formation assay. The tumorigenesis ability of transfected BMSCs was also investigated in NOD/SCID tumor mice models., Results: BMSCs were successfully established from MCF7 cells and miR-422a expression was downregulated while PLP2 level decreased in BMSCs. MiR-422a directly targets the 3'UTR of PLP2 and suppressed its expression. Besides, the up-regulation of miR-422a contributed to weakened ability of proliferation and microsphere formation of BMSCs, while PLP2 overexpression facilitated those biological abilities. Tumorigenesis of BMSCs in mice models was impaired by either overexpression of miR-442a or silencing of PLP2., Conclusion: Up-regulation of miR-422a attenuated microsphere formation, proliferation and tumor formation of breast cancer stem cells via suppressing the PLP2 expression.

CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) is considered to be a tumor suppressor gene in multiple types of malignancies. Previous studies have indicated that CMTM3 suppresses metastasis and epithelial-mesenchymal transition (EMT) in gastric cancer. However, its role in gastric cancer cell proliferation has rarely been discussed. Moreover, the regulatory mechanisms of CMTM3 in gastric cancer remain unclear. In this study, RT‑qPCR and IHC were used to assess the expression of CMTM3 and miR‑135b‑5p in gastric cancer tissues and cell lines. We found that the expression of miR‑135b‑5p was negatively associated with CMTM3 in gastric cancer tissues, and we verified that miR‑135b‑5p directly targeted CMTM3 in gastric cancer cells by dual-luciferase reporter assay. CCK8 assay, Transwell assay and flow cytometric analysis were conducted to examine the functions of CMTM3 and miR‑135b‑5p in vitro. Our results demonstrated that the overexpression of CMTM3 or the suppression of miR‑135b‑5p using an inhibitor suppressed SGC‑7901 gastric cancer cell proliferation, invasion and cell cycle progression, and promoted SGC‑7901 cell apoptosis. Furthermore, a BALB/c nude mouse subcutaneous xenograft model was used to verify the function of miR‑135b‑5p and CMTM3. Our results revealed that miR‑135b‑5p inhibitor significantly suppressed SGC‑7901 cell tumorigenesis in vivo. In addition, IHC revealed that CMTM3 expression was markedly increased in tumors infected with miR‑135b‑5p inhibitor lentivirus. On the whole, the findings of the present study suggest that the overexpression of miR‑135b‑5p inhibits CMTM3 expression, and promotes gastric cancer progression and metastasis. Our findings provide a novel therapeutic target for gastric cancer.

Tight junctions are required for the formation of tissue barriers and function as suppressors of signalling mechanisms that control gene expression and cell behaviour; however, little is known about the physiological and developmental importance of such signalling functions. Here, we demonstrate that depletion of MarvelD3, a transmembrane protein of tight junctions, disrupts neural crest formation and, consequently, development of neural crest-derived tissues during Xenopus embryogenesis. Using embryos and explant cultures combined with a small molecule inhibitor or mutant mRNAs, we show that MarvelD3 is required to attenuate JNK signalling during neural crest induction and that inhibition of JNK pathway activation is sufficient to rescue the phenotype induced by MarvelD3 depletion. Direct JNK stimulation disrupts neural crest development, supporting the importance of negative regulation of JNK. Our data identify the junctional protein MarvelD3 as an essential regulator of early vertebrate development and neural crest induction and, thereby, link tight junctions to the control and timing of JNK signalling during early development.

The clinical benefit for patients with diverse types of metastatic cancers that has been observed upon blockade of the interaction between PD-1 and PD-L1 has highlighted the importance of this inhibitory axis in the suppression of tumour-specific T-cell responses. Notwithstanding the key role of PD-L1 expression by cells within the tumour micro-environment, our understanding of the regulation of the PD-L1 protein is limited. Here we identify, using a haploid genetic screen, CMTM6, a type-3 transmembrane protein of previously unknown function, as a regulator of the PD-L1 protein. Interference with CMTM6 expression results in impaired PD-L1 protein expression in all human tumour cell types tested and in primary human dendritic cells. Furthermore, through both a haploid genetic modifier screen in CMTM6-deficient cells and genetic complementation experiments, we demonstrate that this function is shared by its closest family member, CMTM4, but not by any of the other CMTM members tested. Notably, CMTM6 increases the PD-L1 protein pool without affecting PD-L1 (also known as CD274) transcription levels. Rather, we demonstrate that CMTM6 is present at the cell surface, associates with the PD-L1 protein, reduces its ubiquitination and increases PD-L1 protein half-life. Consistent with its role in PD-L1 protein regulation, CMTM6 enhances the ability of PD-L1-expressing tumour cells to inhibit T cells. Collectively, our data reveal that PD-L1 relies on CMTM6/4 to efficiently carry out its inhibitory function, and suggest potential new avenues to block this pathway.

Objective: To study the expression of transmembrane protein CMTM2 in the testis and sperm of adult males and to approach the potential function of the protein in the male reproductive system., Methods: The expression of CMTM2 in human testis and sperm was confirmed by Western blot. Immunohistochemical staining was used for detecting CMTM2 localization in the testis tissue, TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction, that is, immunofluorescent staining was used for detecting CMTM2 localization in both the testis and sperm before and after the acrosome reaction., Results: CMTM2 was presented in both human testis and sperm. In the testis, CMTM2 immunoreactive particles were observed mainly in the membrane of the different stages of spermatogenic cells. In the human sperm, its immunoreactivity was restrictively localized to the posterior head where sperm-egg fusion occurred, and the CMTM2 localization was not affected by sperm acrosome reaction. CMTM2 was widely expressed in seminiferous tubules of the human testis, mainly in the cell membranes of spermatogenic cells, which was consistent with the previous reports. The immunofluorescence performed on frozen human testis slides showed similar findings with immunohistochemistry, which gave weight to the localization of CMTM2 in the cell membranes of spermatogenic cells at different stages. TRITC-CMTM2 and FITC-Hoechst double immunofluorescence staining was performed to examine the subcellular localization of CMTM2 in the human sperm before and after acrosome reaction. CMTM2 was localized at the posterior head of sperm before and after acrosome reaction. The localization and expression of CMTM2 were not affected by sperm acrosome reaction., Conclusion: Expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. The expression of CMTM2 in the male reproductive system of the adult human exhibits cell- and region-specific patterns, which suggests that they may play an important role in spermatogenesis and sperm-egg fusion. However, it still remains to be further elucidated about the definite role of CMTM2 in male reproductive system and the process of spermatogenesis. And in vitro fertilization experiments are needed to confirm the role of CMTM2 in fertilization in future.

Aspirin is widely used in the prevention of cardiovascular diseases, but the antiplatelet responses vary from one patient to another. To validate aspirin response related transcripts and illustrate their roles in predicting cardiovascular events, we have quantified the relative expression of 14 transcripts previously identified as related to high on-aspirin platelet reactivity (HAPR) in 223 patients with coronary artery disease (CAD) on regular aspirin treatment. All patients were followed up regularly for cardiovascular events (CVE). The mean age of our enrolled population was 75.80±8.57years. HAPR patients showed no significant differences in terms of co-morbidities and combined drugs. Besides, the relative expression of HLA-DQA1 was significantly lower in low on-aspirin platelet reactivity (LAPR) patients, when compared with HAPR and high normal (HN) group (p=0.028). What's more, the number of arteries involved, HAPR status and the relative expression of CLU, CMTM5 and SPARC were independent risk factors for CVE during follow up (p<0.05). In addition, overexpression of CMTM5 attenuated endothelial cells (ECs) migration and proliferation, with significantly decreased phosphorylated-Akt levels, while its inhibition promoted these processes in vitro (p<0.05).Our study provides evidence that circulating transcripts might be potential biomarkers in predicting cardiovascular events. CMTM5 might exert anti-atherosclerotic effects via suppressing migration and proliferation in the vessel wall. Nevertheless, larger-scale and long-term studies are still needed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: Decrease in VE-cadherin adherens junctions reduces vascular stability, whereas disruption of adherens junctions is a requirement for neovessel sprouting during angiogenesis. Endocytosis plays a key role in regulating junctional strength by altering bioavailability of cell surface proteins, including VE-cadherin. Identification of new mediators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we assessed the function of CMTM3 (CKLF-like MARVEL transmembrane domain 3), which we have previously identified as highly expressed in Flk1 + endothelial progenitor cells during embryonic development., Approach and Results: Using a 3-dimensional coculture of human umbilical vein endothelial cells-GFP (green fluorescent protein) and pericytes-RFP (red fluorescent protein), we demonstrated that siRNA-mediated CMTM3 silencing in human umbilical vein endothelial cells impairs angiogenesis. In vivo CMTM3 inhibition by morpholino injection in developing zebrafish larvae confirmed that CMTM3 expression is required for vascular sprouting. CMTM3 knockdown in human umbilical vein endothelial cells does not affect proliferation or migration. Intracellular staining demonstrated that CMTM3 colocalizes with early endosome markers EEA1 (early endosome marker 1) and Clathrin + vesicles and with cytosolic VE-cadherin in human umbilical vein endothelial cells. Adenovirus-mediated CMTM3 overexpression enhances endothelial endocytosis, shown by an increase in Clathrin + , EEA1 + , Rab11 + , Rab5 + , and Rab7 + vesicles. CMTM3 overexpression enhances, whereas CMTM3 knockdown decreases internalization of cell surface VE-cadherin in vitro. CMTM3 promotes loss of endothelial barrier function in thrombin-induced responses, shown by transendothelial electric resistance measurements in vitro., Conclusions: In this study, we have identified a new regulatory function for CMTM3 in angiogenesis. CMTM3 is involved in VE-cadherin turnover and is a regulator of the cell surface pool of VE-cadherin. Therefore, CMTM3 mediates cell-cell adhesion at adherens junctions and contributes to the control of vascular sprouting., (© 2017 American Heart Association, Inc.)

Previous studies have demonstrated that overexpression of CMTM8 inhibits cell growth and induces apoptosis in multiple types of cancer cells, whereas the downregulation of CMTM8 induces the epithelial-to-mesenchymal (EMT)-like phenotype in hepatocyte carcinoma cells, implying its important roles in tumorigenesis and tumor metastasis. No extensive studies on the expression of CMTM8 in either normal or tumorous human tissues have been reported to date. Here, using real-time quantitative polymerase chain reaction, we analyzed CMTM8 expression in multiple normal human tissue samples. Moreover, by applying high-throughput immunohistochemical staining of tissue microarrays with homemade anti-CMTM8 antibodies, we studied CMTM8 expression in carcinoma samples and adjacent normal samples of 6 types of human tissues. CMTM8 is widely expressed in many normal human tissues and is frequently downregulated or absent in multiple solid tumors (liver, lung, colon, rectum, esophagus, stomach). χ tests revealed a significant negative correlation between CMTM8 expression and tumorigenesis: liver, lung (squamous carcinoma), colon, rectum, P<0.0001; esophagus, P<0.001; stomach, P<0.01. Real-time quantitative polymerase chain reaction analysis of samples from esophageal carcinomas and the adjacent normal tissues revealed that CMTM8 mRNA levels are reduced in carcinomas compared with normal tissues, indicating that CMTM8 is potentially downregulated at the mRNA level (P<0.01). This is the first extensive study of CMTM8 expression in both normal and tumorous human tissues. Our findings strongly supported the potential role of CMTM8 as a novel tumor suppressor and may shape further functional studies on this gene.

CMTM3 (CKLF-like MARVEL transmembrane domain containing 3), a tumor suppressor gene, is involved in multiple types of malignancies. CMTM3 knockdown promotes metastasis of gastric cancer via the STAT3/Twist1/EMT signaling pathway. Strong epidermal growth factor receptor1 (EGFR) expression is significantly associated with tumor metastasis and poor outcomes of gastric cancer patients. In this paper, we show that CMTM3 suppresses epidermal growth factor (EGF)-mediated migration and STAT3 signaling, downregulates EGFR expression via accelerating EGFR degradation in gastric cancer cells. CMTM3 colocalizes with early endosome markers Rab5 and EEA1. Co-immunoprecipitation (Co-IP) assay further confirms that CMTM3 interacts with Rab5. More importantly, CMTM3 markedly increases Rab5 activity. The suppressive effects of CMTM3 on EGFR expression and EGF-mediated migration can be abrogated by the siRNA against Rab5. Finally, we found that the C-terminal region of CMTM3 plays more important roles in the tumor suppressive effects of CMTM3. Overall, this study demonstrates that CMTM3 decreases EGFR expression, facilitates EGFR degradation, and inhibits the EGF-mediated tumorigenicity of gastric cancer cells via enhancing Rab5 activity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Considering neoadjuvant chemotherapy (NAC) prior to surgery could shrink and reduce the primary tumor and distant micro-metastases to reduce the high relapses rates, NAC has been an accepted therapeutic management for patients with non-small cell lung cancer (NSCLC). CMTM1&#95;v17 is highly expressed in human testis tissues and solid tumor tissues but relatively low expression was obtained in the corresponding normal tissues. This study aims to investigate the significance of CMTM1&#95;v17 in NSCLC and its association with platinum-based NAC efficacy., Methods: 31 pairs of tumor tissues before and after NAC and 78 resected tumor tissues after NAC were utilized for immunohistochemistry (IHC) staining of CMTM1&#95;v17 protein. The correlation between CMTM1&#95;v17 expression and chemotherapy efficacy was analyzed. The prognostic value of CMTM1&#95;v17 index for disease-free survival (DFS) and overall survival (OS) was analyzed using Kaplan-Meier survival and multivariable Cox regression., Results: CMTM1&#95;v17 expression was related to treatment effect and outcome in tumor tissues after NAC not before NAC from 31 cases of NSCLC. We identified that high CMTM1&#95;v17 expression was associated with low objective remission rate (ORR) (P = 0.008) and poor prognosis (the median OS: 35.1 months vs 65.6 months, P = 0.0045; the median DFS: 17.27 months vs 35.54 months, P = 0.0207) in the 31 patients. Next, we detected CMTM1&#95;v17 expression to confirm correlation between this protein status and clinical characteristics in 78 NSCLC patients with NAC treatment. The upregulation of CMTM1&#95;v17 had a higher SD rate (P = 0.007) and worse outcome (the median OS: 41.0 months vs 80.6 months, P = 0.0028; the median DFS: 33.4 vs 64.8 months, P = 0.0032). COX multivariate analysis indicated that CMTM1&#95;v17 is an independent prognostic risk factor on patients who have received NAC (OS: HR = 3.642, P = 0.002; DFS:HR = 3.094, P = 0.002)., Conclusions: CMTM1&#95;v17 expression is significantly associated with chemoresistance and poor prognosis of the early stage NSCLC patients who have received NAC.

Microcystin-LR (MC-LR) is one of the most toxic members of microcystins released by freshwater cyanobacterial. The major mechanism of MC-LR toxicity has been attributed to its inhibition of protein phosphatases 1 (PP1) and 2A (PP2A). In our prior research, α4 protein, a regulator of PP2A, was found not only crucial for PP2A regulation but also for the overall response of HEK 293 cells encountering MC-LR. To explore the role of α4 in MC-LR toxicity via PP2A regulation, here, HEK 293 cells overexpressing α4 protein were exposed to MC-LR and PP2A, cytoskeletal organization, and cytoskeleton-related proteins were investigated. The results showed that PP2A activity decreased and PP2A/C subunit expression and phosphorylation at Tyr307 increased significantly in the group exposed to high MC-LR. Vimentin IF became concentrated and formed perinuclear bundles. However, the assembly of actin filament and microtubules remained unchanged and the expression and phosphorylation of the cytoskeleton-related proteins HSP27 and VASP did not increase significantly. Some of these results differ from those of our previous study in which normal HEK293 cells were exposed to MC-LR. Our results indicate that elevated α4 expression confers some resistance to MC-LR-induced cytoskeletal change These new findings provide helpful insights into the mechanism of MC-LR toxicity and the role of α4 in regulating PP2A function. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 255-264, 2017., (© 2016 Wiley Periodicals, Inc.)

Chemokine-like factor super family member (CMTM) is a novel generic family firstly reported by Peking University Center for Human Disease Genomics. CMTM8 is one member of this family and has exhibited tumor-inhibiting activities. It can encode proteins approaching to the transmembrane 4 superfamily. CMTM8 is down-regulated in most carcinoma cell lines and tissues. Over-expression of CMTM8 may inhibit the proliferation,migration,and invasion of carcinoma cells. However,the exact mechanism of its anti-tumor activity remains unclear. CMTM8 may be involved in various signaling pathways governing the occurrence and development of tumors. CMTM8 may be a new target in the gene therapies for tumors,while further studies on CMTM8 and its anti-tumor mechanisms are warranted.

Objective: To investigate whether chemokine like factor (CKLF)-like myelin and lymphocyte and related proteins for vesicle trafficking and membrane link (MARVEL) transmembrane domain-containing protein 2 (CMTM2) is involved in spermatogenesis in varicocele induced subfertility rats and to discuss the possible mechanisms., Methods: Forty male SD rats (body weight: 220-330 g, age: 6-7 weeks) were randomly divided into 4 groups: varicocele for 4 weeks, varicocele for 12 weeks, sham operation for 4 weeks and sham operation for 12 weeks, with 10 rats in each group. These rats were introduced by partially ligating left kidney veins for the experimental groups, and the sham surgery groups as controls were executed with exactly the same surgery as in the experimental groups except for the ligation. The rats in control and experimental groups for 4 and 12 weeks were killed after laparotomy at the end of 4 and 12 weeks, respectively, the left testes and epididymis were taken out for counting the sperm, observing the seminiferous tubule change and immunochemistry for CMTM2. The changes included sperm density and motility, the outer diameter and inner diameter change and the changes of epithelium and the CMTM2 expression in immunochemistry., Results: Compared with the control groups, the sperm density [(63.9±7.1)×10 6 /mL vs.(74.3±5.0)×10 6 /mL] and motility [(58.7%±7.9%) vs.(66.1%±4.3%)] were reduced slightly in group of varicoele for 4 weeks, respectively (t=1.432, 1.563; P=0.076, 0.059, respectively). Varicocele significantly caused a decrease in sperm concentration [(40.5±7.2) ×10 6 /mL vs.(71.1±4.5)×10 6 /mL] and motility [(35.2%±8.5%)vs. (63.4%±4.1%)] at 12 weeks, compared with the related sham groups (t=3.754, 3.933; P=0.004, 0.002, respectively). Additionally, testis CMTM2 exhibited the same disparity, that is, the CMTM2 protein expression in varicocele group was significantly reduced, with the ratio of sham group to varicocele group at the end of 12 weeks 2.3±0.4 (t=1.978; P=0.039). In the evaluation of seminiferous tubules diameter, the external [(198.2±10.2) μm vs. (255.8±12.7) μm, t=2.125, P=0.003] and epithelium diameter [(54.1±1.5) μm vs. (75.5±4.1) μm, t=2.246, P=0.021] were decreased compared with the sham-related groups and previous varicocele groups. In all the varicocele groups, all types of sperm motility decreased compared with the related sham-operated group (P<0.05)., Conclusion: This study suggests varicocele has a detrimental effect on CMTM2 levels and decreases spermatogonia cell number, seminiferous tubules diameter, and sperm indices. CMTM2 is associated with sperm changes in rats with varicocele, and further studies are needed to study the mechanism.

Disruption of blood-brain barrier (BBB) and subsequent edema are major contributors to the pathogenesis of ischemic stroke, and the current clinical therapy remains unsatisfied. Chemokine-like factor 1 (CKLF1), as a novel C-C chemokine, plays important roles in immune response. The expression of CKLF1 increased after focal cerebral ischemia and inhibition of CKLF1 activity showed neuroprotective effect by alleviating infiltration of neutrophil and neuron apoptosis in cerebral ischemia. However, few studies have focused on the role of CKLF1 on BBB integrity. The objective of present study was to investigate the role of CKLF1 on BBB integrity by applying anti-CKLF1 antibodies in rat focal cerebral ischemia and reperfusion model. Brain water content, Evans blue leakage and the expression of aquaporin-4 (AQP-4), matrix metalloproteinase-9 (MMP-9), Zonula Occludens-1 (ZO-1) and Occludin were measured. After treatment with anti-CKLF1 antibody, brain water content and Evans blue leakage in ipsilateral hemisphere were decreased in a dose-dependent manner at 24h after reperfusion, but not changed in contralateral hemisphere. Anti-CKLF1 antibody reduced the expression of AQP-4 and MMP-9, and upregulated the expression of ZO-1 and Occludin. These results suggest that CKLF1 is involved in BBB disruption after reperfusion. Inhibition of CKLF1 protects against cerebral ischemia by maintaining BBB integrity, possibly via inhibiting the expression of AQP-4 and MMP-9, and increasing the expression of tight junction protein., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

CMTM3 (CKLF-like MARVEL transmembrane domain containing 3) possesses tumor suppressor properties in multiple types of malignancies. Restoration of CMTM3 significantly inhibits the metastasis of gastric cancer, and its expression level is correlated with prognosis. However, the physiological effects and the mechanism of CMTM3 remain unknown. Here, we suppress CMTM3 expression by shRNA to explore its endogenous effects and its mechanism of action in gastric cancer. Stable knockdown of CMTM3 promotes cell migration, invasion and tumor metastasis, increases MMP2 expression and enhances MMP2 activity. CMTM3 inhibits EMT along with the upregulation of E-cadherin and the downregulation of N-cadherin, Vimentin and Twist1. It has no obvious effects on Zeb1 and Snail. CMTM3 suppresses the phosphorylation of STAT3 but not Akt. More importantly, the EMT phenotype and cell migration induced by CMTM3 knockdown can be reversed by the Jak2/STAT3 inhibitor JSI-124 or by siRNA against STAT3 or Twist1. Overall, this study demonstrates that knockdown of CMTM3 promotes the metastasis of gastric cancer through the STAT3/Twist1/EMT pathway., Competing Interests: The authors declare that there are no conflicts of interest.

Uroplakins (UPs) are major differentiation products of urothelial umbrella cells and play important roles in forming the permeability barrier and in the expansion/stabilization of the apical membrane. Further, UPIa serves as a uropathogenic Escherichia coli receptor. Although it is understood that UPs are delivered to the apical membrane via fusiform vesicles (FVs), the mechanisms that regulate this exocytic pathway remain poorly understood. Immunomicroscopy of normal and mutant mouse urothelia show that the UP-delivering FVs contained Rab8/11 and Rab27b/Slac2-a, which mediate apical transport along actin filaments. Subsequently a Rab27b/Slp2-a complex mediated FV-membrane anchorage before SNARE-mediated and MAL-facilitated apical fusion. We also show that keratin 20 (K20), which forms a chicken-wire network ∼200 nm below the apical membrane and has hole sizes allowing FV passage, defines a subapical compartment containing FVs primed and strategically located for fusion. Finally, we show that Rab8/11 and Rab27b function in the same pathway, Rab27b knockout leads to uroplakin and Slp2-a destabilization, and Rab27b works upstream from MAL. These data support a unifying model in which UP cargoes are targeted for apical insertion via sequential interactions with Rabs and their effectors, SNAREs and MAL, and in which K20 plays a key role in regulating vesicular trafficking., (© 2016 Wankel et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Chemokine-like factor 1 (CKLF-1) is a novel cytokine which have a crucial role in immune and inflammatory responses. In this study, the expression level of CKLF-1 was measured to assess the difference between keloid patients and people without keloid. Fifty samples were taken from 30 patients: 10 keloid patients; 10 scar patients; and 10 patients without obvious scarring. Patients were randomly selected from the hospitalized patients of Peking Union Medical College Hospital from September 2013 to July 2015. Five groups of samples were established: keloid samples from keloid patients (K); normal skin samples from keloid patients (KS); scar samples from scar patients (C); normal skin samples from scar patients (CS); and normal skin samples from patients without obvious scarring (S). Hematoxylin and eosin (H&E) staining was used to observe morphological changes. CKLF-1, IL-6, IL-8, IL-18, and TGF-β were detected by immunohistochemical and western blot technology. The expression of CKLF-1's mRNA was also measured by the real-time quantitative polymerase chain reaction (RT-qPCR). Compared to the K group, the other 4 groups presented significantly less inflammatory infiltration and lower expression levels of CKLF-1, IL-6, IL-8, IL-18, and TGF-β. Among the 3 normal skin groups, the expression level of CKLF-1 was significantly higher in the KS group than in the CS or S group. The mRNA expression was also obvious in the K and KS groups. CKLF-1 and other inflammatory factors were overexpressed in the samples from keloid patients, indicating that the formation of keloid may be related to inflammation and that CKLF-1 may play an important role in this process.

Chemokine-like factor 1 (CKLF1) is a newly cloned chemotactic cytokine with CCR4 being its functional receptor. Recent evidence demonstrates a role of CKLF1 in arthritis. The aim of this study was to quantify the expression of CKLF1 as well as assess the correlation between CKLF1 and plasma acute-phase markers. Synovium was obtained from 16 osteoarthritis (OA), 15 rheumatoid arthritis (RA) and 10 ankylosing spondylitis (AS) patients undergoing total joint arthroplasty, with other 11 patients treated for meniscal tears during sport accidents serving as normal controls. Levels of CKLF1 and CCR4 mRNA were detected by qRT-PCR, and the expression of CKLF1 was investigated by immunohistochemistry staining, subsequently analyzed with semiquantitative scores. Plasma acute-phase markers of inflammation were determined by ELISA. CKLF1 was found with a particularly up-regulated expression in synovim from AS and RA patients, and CCR4 mRNA levels increased in RA patients, not in OA or AS patients. Elevated levels of plasma markers of inflammation including CRP, ESR and D-dimer were observed in RA. Further, significantly positive correlations between relative expression levels of CKLF1 and CRP/ESR in RA patients and a positive correlation between CKLF1 and ESR in AS patients were found. There was no detectable correlation between CKLF1 and plasma D-dimer. This study confirms an increased but different level of CKLF1 in RA, OA and AS patients, all significantly higher than that in controls. Additionally, the significant positive correlations between CKLF1 levels and CRP/ESR in RA and between CKLF1 and ESR suggest that CKLF1 might contribute to the inflammation state and clinical symptoms in these rheumatic diseases. Further studies are required to investigate the utility of targeting specific CKLF1 for symptom control or disease modification in RA and AS.

CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of genes first reported at international level by Peking University Human Disease Gene Research Center. The gene products are between chemokines and the transmembrane-4 superfamily. Loaceted in several human chromosomes, CMTMs, which are unregulated in kinds of tumors, are potential tumor suppressor genes consisting of CKLF and CMTM1 to CMTM8. CMTMs play important roles in immune, male reproductive and hematopoietic systems. Also, it has been approved that CMTM family has strong connection with diseases of autoimmunity, haematopoietic system and haematopoietic system. The in-depth study in recent years found the close relation between CMTMs and umorigenesis, tumor development and metastasis. CMTM family has a significant clinical value in diagnosis and treatment to the diseases linking to tumor and immune system.

The dysregulation of epidermal growth factor receptor (EGFR) signaling has been well documented to contribute to the progression of non-small cell lung cancer (NSCLC), the leading cause of cancer death in the world. EGF-stimulated EGFR activation induces receptor internalization and degradation, which plays an important role in EGFR signaling. This process is frequently deregulated in cancer cells, leading to enhanced EGFR levels and signaling. Our previous study on CMTM7 is only limited to a brief description of the relationship of overexpressed CMTM7 with EGFR-AKT signaling. The biological functions of endogenous CMTM7 and its molecular mechanism remained unclear. In this study, we show that the stable knockdown of CMTM7 augments the malignant potential of NSCLC cells and enhances EGFR-AKT signaling by decreasing EGFR internalization and degradation. Mechanistically, CMTM7 knockdown reduces the activation of Rab5, a protein known to be required for early endosome fusion. In NSCLC, the loss of CMTM7 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, our findings highlight the role of CMTM7 in the regulation of EGFR signaling in tumor cells, revealing CMTM7 as a novel molecule related to Rab5 activation.

Downregulation of CKLF-like MARVEL transmembrane domain-containing member 3 (CMTM3) has been reported in a number of human tumors. However, the role of CMTM3 in oral squamous cell carcinoma (OSCC) remains largely unknown. In this study, we showed that the expression of CMTM3 was significantly reduced in OSCC cell lines and primary tumor specimens (P < 0.001). Methylation-specific PCR showed hypermethylation in CMTM3 promoter in a significant proportion of tumor tissues (61 %). The expression of CMTM3 was associated with T stage, lymph node metastasis, tumor node metastasis (TNM) stage, and recurrence of OSCC patients (P < 0.05, n = 201). More importantly, CMTM3 expression was associated with the prognosis of OSCC patients (P < 0.001) and was an independent prognostic factor (hazard ratio = 0.593, 95 % confidence interval, 0.272-1.292; P = 0.039). Overexpression of CMTM3 inhibited the growth and migration of OSCC cells. In vivo experiments also showed that the growth of OSCC xenografts in nude mice was significantly inhibited by CMTM3 overexpression. These findings indicate that downregulation of CMTM3 due to promoter hypermethylation contributed to the proliferation and migration of OSCC cells and suggest that CMTM3 is an independent prognostic factor for the evaluation of the survival of OSCC patients.

Objective: To investigate the expression patterns of CKLF-like MARVEL transmembrane domain containing 5 (CMTM5), a novel tumor suppressor, and epidermal growth factor receptor (EGFR) in prostate cancer (PCa) tissues and cells and to analyze the relationship between CMTM5 and EGFR in PCa., Methods: The expression patterns of CMTM5 and EGFR in PCa tissues and cells were detected by immunohistochemistry and Western blot, respectively., Results: CMTM5 was highly expressed in 75% (27/36) of benigh prostatic hyperplasia (BPH) tissues but 35.9% (23/64) of PCa tissues (P<0.001). There was a significant difference of CMTM5 expression between the two groups of PCa tissues with different Gleason scores (P=0.003), though its expression was not related to the age, clinical stage, and metastatic situation (P>0.05). EGFR was highly expressed in 57.8% (37/64) of PCa tissues, it had statistical significance between EGFR and CMTM5 expressions in PCa tissues. Furthermore, 23 cases (35.9%) had low CMTM5 expression and high EGFR expression. Western blot showed that CMTM5 was undetectable in PCa cells, in which the EGFR expression was upregulated., Conclusion: The loss of CMTM5 may participate in the progression of PCa resulting from deregulated EGFR.

Purpose: A novel tumor suppressor gene CKLF-like MARVEL transmembrane domain-containing member 3 (CMTM3) is reduced or undetectable in many kinds of cancers and relates tumor malignant features. We detected its role in prostate cancer for possibility of target therapy as accumulating evidence has shown that CMTM3 is a promising tumor suppressor gene (TSG) for gene therapy., Methods: The expression of CMTM3 detected in prostate tissue microarray, specimens and cell lines were evaluated by immunohistochemistry and semi-quantitative PCR and Western blot, respectively. After being transfected with CMTM3 adenovirus or vector (mock), the proliferation and migration and invasion of LNCaP cells were detected by transwell assay and matrigel assay, respectively. Furthermore, the effects of CMTM3 on tumor growth were performed in nude mice xenograft in vivo., Results: We found CMTM3 was reduced in PCa tissues and cells compared with BPH tissues, and its expression in PCa tissues was related to the Gleason score. Moreover, after being transfected with adenovirus, ectopic expression of CMTM3 in LNCaP cells led to significant inhibition of cell proliferation and migration and invasion compared with the control (P < 0.05), which may be attributed to decreased Erk1/2 activity as p-Erk1/2 was remarkably reduced when CMTM3 was overexpressed. Finally, restoration of CMTM3 significantly suppressed xenograft tumor growth in vivo (P < 0.01).

The novel CKLF-like Marvel Transmembrane Domain-containing gene family (CMTM) consists of 8 members (CMTM1-8). As little is known about the oncogenic impact of these genes, we aimed to systematically investigate the relevance of CMTMs to glioblastoma pathogenesis. We performed mRNA expression analyses and survival correlations in glioblastoma patients. Moreover, we analyzed the impact of RNAi-based silencing and overexpression of CMTM family genes on tumor cell proliferation and invasion in vitro. CMTMs appeared to be widely regulated in the group of glioblastomas relative to non-neoplastic brain (NB) tissue (significant upregulation for CMTM2, 3, and 6 and significant downregulation for CMTM 4 and 8). For CMTM1, 5 and 7, we found aberrant expression levels in individual tumors. Functionally, CMTM1, 3, and 7 promoted tumor cell invasion, while CMTM1 additionally enhanced cell proliferation. In a large clinically annotated dataset, higher CMTM1 and 3 expression was significantly correlated with shorter overall survival. Our data thus suggest CMTM1 and 3 as priority targets in glioblastomas. Using a human phosphokinase protein expression profiling assay, we can provide first insights into signalling of these two genes that might be conveyed by growth factor receptor, Src family kinase and WNT activation., (© 2015 Wiley Periodicals, Inc.)

Tight junctions (TJ) regulate the paracellular passage of ions and molecules through the paracellular pathway and maintain plasma membrane polarity in epithelial and endothelial cells. Apart from these canonical functions, several proteins of the TJ have been found in recent years to regulate gene expression. This function is found in proteins that shuttle between the nucleus and TJs, and in integral TJ proteins. In this review, we will describe these proteins and their known mechanisms of gene regulation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing 1 (CMTM1) consists of at least 23 alternatively spliced isoforms designated CMTM1&#95;v1-v23. In the present study, we detected CMTM1&#95;v17 expression in multiple human normal and tumor tissues and found that CMTM1&#95;v17 was highly expressed in testis and many tumor tissues including breast tumor. The overexpression of CMTM1&#95;v17 in the breast cancer cell line MDA-MB-231 promoted cell proliferation and resistance to tumor necrosis factor-α (TNF-α)-induced apoptosis. Moreover, siRNA-mediated silencing of CMTM1&#95;v17 sensitized MDA-MB-231 cells to TNF-α-induced apoptosis. We propose that CMTM1&#95;v17 may be a novel potential target for therapy in breast cancer patients. The present study provides insight into a novel mechanism by which CMTM1&#95;v17 enhances cellular proliferation and abrogates TNF-α-induced apoptosis. These findings also have implications for clinical practice as they highlight the potential for therapeutic targeting of CMTM1&#95;v17 for the treatment of breast and other cancers in which CMTM1&#95;v17 impacts cellular proliferation and survival.