Chemokine-like Factor Superfamily Research Articles

Current Opinions on the Relationship Between CMTM Family and Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a typically malignant tumor in the digestive system. The mortality of HCC ranks third place in the world, second only to lung cancer and colorectal cancer. For the characteristics of high invasiveness, high metastasis, high recurrence rate as well as short survival time, HCC treatment has always been difficult in clinical practice. Many causes have contributed to the appearance of these features, including insidious onset, high degree of malignancy, lack of effective early molecular diagnostic markers, and disease prediction models. The human chemokine-like factor superfamily (CMTMs) is a new gene family consisting of CKLF and CMTM1-CMTM8. CMTMs have a marvel domain which can activate and chemotaxis immune cells. Many studies have reported that CMTMs are involved in the regulation of cell growth and development, and play an important role in the malignant progression of the immune system and reproductive system, especially in the development of tumors. In this review, we summarized the structure and function of the human CMTMs, the relationship between its family members and HCC, the prognostic value, potential functions, and mechanisms in HCC. CMTMs could provide a new diagnostic and therapeutic target in clinical practice for patients with HCC.

CMTM6 and PD-1/PD-L1 overexpression is associated with the clinical characteristics of malignancy in oral squamous cell carcinoma

This study aimed to evaluate the expression of chemokine-like factor superfamily 6 (CMTM6) and programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) in oral squamous cell carcinoma (OSCC) and to further explore its clinical significance in OSCC. Samples of 44 OSCC and paracancerous tissues were investigated. We estimated the expression of the 3 proteins by immunohistochemistry and further detected mRNA expression by quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemistry results demonstrated that the positive expression of CMTM6 and PD-1/PD-L1 in OSCC tissues was significantly higher than that in paracancerous tissues. Statistical significance was found between the 2 groups (all P < .05). Moreover, PD-L1 expression was related to OSCC clinical stage and lymph node metastasis (P < .05). The qRT-PCR results confirmed that the relative expression of CMTM6 and PD-1/PD-L1 mRNA in OSCC tissues was significantly higher than that in paracancerous tissues (all P < .05), and Spearman rank correlation showed that there was a significant relationship between mRNA and protein expression (all P < .05). CMTM6 and PD-1/PD-L1 were upregulated in OSCC, and CMTM6 may play a synergistic role with PD-1/PD-L1 in the immune pathway. Therefore, we believe that CMTM6 and PD-1/PD-L1 will become checkpoints for immunotherapy of OSCC.

CMTM8 Is a Suppressor of Human Mesenchymal Stem Cell Osteogenic Differentiation and Promoter of Proliferation Via EGFR Signaling.

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.

CMTM family proteins 1-8: roles in cancer biological processes and potential clinical value.

The CKLF-like MARVEL transmembrane domain containing (CMTM) family of genes comprises CKLF and CMTM1–8 (previously called chemokine-like factor superfamily 1–8, CKLFSF1–8). The CMTM family proteins contain a structurally conserved MAL and related proteins for vesicle trafficking and membrane linking (MARVEL) domain. Dysregulated expression of multiple CMTM family members is a common feature in many human cancer types. CMTM proteins control critical biological processes in cancer development, including growth factor receptor activation and recycling, cell proliferation, apoptosis, metastasis, and immune evasion. Emerging in vivo and in vitro evidence indicates that the mechanisms of action of most CMTM proteins are complex and multifactorial. This review highlights new findings regarding the roles of CMTM1–8 in cancer, particularly in tumor growth, metastasis, and immune evasion. Additionally, the potential clinical value of CMTMs as novel drug targets or biomarkers is discussed.

Pathophysiologic mechanism of CMTM5 low expression in multiple myeloma progression

目的探索趋化素样因子超家族成员CMTM5表达对多发性骨髓瘤（MM）细胞增殖活性的影响及其机制。方法去甲基化药物地西他滨处理MM细胞系U266细胞，采用实时荧光定量PCR法检测处理前后U266细胞CMTM5、caspase3、caspase9的表达水平并分析其相关性；pcDNA3.1质粒转染U266细胞使CMTM5过表达后，采用CCK-8法检测细胞增殖活性变化。结果①与对照组相比，U266细胞中CMTM5、caspase3和caspase9的mRNA表达水平与地西他滨呈浓度和时间依赖性，随浓度加大和时间延长表达水平增高更加显著（P值均<0.01）；U266细胞中CMTM5表达水平与caspase3（r=0.937）、caspase9（r=0.945）呈正相关（P值均<0.001）。②过表达质粒转染的U266细胞，其CMTM5表达水平为133.10±10.35，较空白对照组（0.63±0.14）和质粒对照组（0.64±0.11）显著增高，差异有统计学意义（P值均<0.001）；CMTM5过表达U266细胞组72 h细胞增殖活性为0.89±0.08，较对照组（1.32±0.02，t=5.005，P=0.008）和空载质粒组（1.30±0.03，t=4.700，P=0.009）显著降低，差异有统计学意义。结论MM细胞中CMTM5的缺失能够被去甲基化药物地西他滨逆转，其变化水平与caspase3、caspase9呈正相关。过表达CMTM5能够抑制U266细胞增殖。

Research Advances in Chemokine-like Factor Super Family Member 8.

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.

Human CMTM2/CKLFSF2 enhances the ligand-induced transactivation of the androgen receptor

CKLF (chemokine-like factor)-like MARVEL (MAL and related proteins for vesicle trafficking and membrane link domain) transmembrane domain containing (CMTM) is a novel gene family. One member of this family, CMTM2, also named chemokine-like factor superfamily 2 (CKLFSF2), is expressed highly in the testis and moderately in the prostate, marrow and peripheral blood cells. However, the function of human CMTM2 remains unknown. Here, we found that CMTM2 was upregulated in 5α-dihydrotestosterone (DHT)-treated LNCaP cells. We investigated the relationship between CMTM2 and the androgen receptor. Our results showed that CMTM2 enhanced DHT-mediated androgen receptor (AR) transactivation and the expression of prostate specific antigen (PSA). We also observed that CMTM2 enhanced the AR protein level, which was reversed by silencing endogenous CMTM2 expression, which suggested that CMTM2 might play an important role in maintaining the AR protein level. We also found that CMTM2 suppressed Akt activation. A previous study showed that Akt could phosphorylate AR at Ser210 and Ser790 and lead to AR ubiquitylation and degradation as well as suppression of AR activity. Taken together, suppressing Akt activation and increasing the AR protein level might be one of the mechanisms for the CMTM2-mediated enhancement of AR transactivation.

Expression and localization of CKLFSF2 in human spermatogenesis

To investigate the expression and subcellular localization of chemokine-like factor superfamily 2 (CKLFSF2) in human testis and its potential role in spermatogenesis. A specific polyclonal antibody against CKLFSF2 was raised. The expression and cellular localization of CKLFSF2 in the seminiferous tubules was checked by immunohistochemistry method. Also, in situ hybridization was applied to localize the mRNA distribution. The EGFP-CKLFSF2 fusion protein was expressed in COS-7 cells to localize its subcellular location in vitro. In addition, the abnormal expression of CKLFSF2 in testes of patients with male infertility was assayed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry methods. Having a close correlation with spermatogenesis defects, CKLFSF2 was specifically expressed in meiotic and post-meiotic germ cells, which were localized to the endoplasmic reticulum (ER) near the Golgi apparatus. CKLFSF2 could play important roles in the process of meiosis and spermiogenesis, and might be involved in the vesicular transport or membrane apposition events in the endoplasmic reticulum.

Molecular cloning and identification of mouse Cklfsf2a and Cklfsf2b, two homologues of human CKLFSF2

Human chemokine-like factor superfamily ( CKLFSF) is a novel gene family comprising CKLF and CKLFSF1–8. Among them, CKLFSF2 is highly expressed in testis and may play important roles in male reproduction. Besides, it is very active during evolution and has two counterparts in mouse. For further study, we cloned the two mouse genes by EST assembly and RT-PCR methods and designated them as mouse Cklfsf2a and Cklfsf2b. Their predicted open-reading frames (ORFs) that encode 169 and 210 amino acids, respectively, were obtained; and their predicted full-length molecular sizes that are approximately 1.2 kb for mCklfsf2a and 0.9 kb for mCklfsf2b were confirmed by Northern blot analysis. Mouse Cklfsf2a and Cklfsf2b show similarities with human CKLFSF2 in the expression patterns that are abundant in testis, hematopoietic and immune tissues; as well as in the chromosome localizations that neighbor CKLFSF1 and 3. Their putative protein products have 47.6 and 45.5% identities with hCKLFSF2, respectively; both of them contain four potential transmembrane regions and MARVEL domains, which are also similar with hCKLFSF2. Functionally, they all can affect the transcriptional activity of androgen receptor in PC-3 and HeLa cells, but mCklfsf2a is a repressor while mCklfsf2b and hCKLFSF2 are enhancers. Taken together, we conclude that mouse Cklfsf2a and Cklfsf2b are two homologues of human CKLFSF2. Studies on them would provide much help in further investigation of the latter.

Regulation of EGF receptor signaling by the MARVEL domain-containing protein CKLFSF8

It is known that chemokine-like factor superfamily 8 (CKLFSF8), a member of the CKLF superfamily, has four putative transmembrane regions and a MARVEL domain. Its structure is similar to TM4SF11 (plasmolipin) and widely distributed in normal tissue. However, its function is not yet known. We show here that CKLFSF8 is associated with the epidermal growth factor receptor (EGFR) and that ectopic expression of CKLFSF8 in several cell lines suppresses EGF-induced cell proliferation, whereas knockdown of CKLFSF8 by siRNA promotes cell proliferation. In cells overexpressing CKLFSF8, the initial activation of EGFR was not affected, but subsequent desensitization of EGF-induced signaling occurred rapidly. This attenuation was correlated with an increased rate of receptor endocytosis. In contrast, knockdown of CKLFSF8 by siCKLFSF8 delayed EGFR endocytosis. These results identify CKLFSF8 as a novel regulator of EGF-induced signaling and indicate that the association of EGFR with four transmembrane proteins is critical for EGFR desensitization.

CKLFSF2 is highly expressed in testis and can be secreted into the seminiferous tubules

CKLFSF2 is a member of the chemokine-like factor superfamily (CKLFSF), a novel gene family containing CKLF and CKLFSF1– 8. Using a combination of data mining and polymerase chain reactions, we determined the full cDNA sequence and genomic structure of human CKLFSF2, a 4-exon gene encoding 248 amino acids and spanning approximately 8.8 kb on chromosome 16q22.1. Expression profile analyses indicated that CKLFSF2 is expressed in a limited number of tissues. Specifically, immunohistochemistry indicated that CKLFSF2 is highly expressed in testis, mainly in spermatogonia and the seminiferous tubular fluid. Subcellular localization experiments suggested that CKLFSF2 is equally distributed in the cytoplasm, and Western blot analysis revealed that overexpressed CKLFSF2 is secreted into the supernatant of cultured cells. The data therefore strongly suggest that CKLFSF2 is a secreted protein that may be functionally relevant during spermatogenesis.

A functional promoter region of the CKLFSF2 gene is located in the last intron/exon region of the upstream CKLFSF1 gene

The genes for CKLFSF1 (chemokine-like factor super family member 1) and CKLFSF2 (chemokine-like factor super family member 2) are very closely linked, within 312 bp of each other. Here, we present evidence that the last intron/exon region of the CKLFSF1 gene contains a novel eukaryotic promoter capable of directing the expression of the downstream gene, CKLFSF2. We identified two segments of the upstream region of the CKLFSF2 gene, 2146 bp (−2134/+12, relative to ATG +1) and 1483 bp (−2134/−652), that were capable of efficiently driving expression of a linked reporter gene upon transient transfection into several kinds of cell lines. The 1483 bp segment exhibited more than a two-fold increase in luciferase activity relative to the 2146 bp segment. By analyzing 5′-deletion mutants of the 1483 bp segment, we identified a 195 bp segment (−846/−625) located in the last intron/exon region of the CKLFSF1 gene that was critical for promoter activity. DNA decoy experiments revealed that a 122 bp (−846/−725) fragment markedly inhibited CKLFSF2 mRNA transcription. Furthermore, we found that the putative promoter region of the CKLFSF2 gene is separated from the transcription start site by about 500 bp. Accumulating reports suggest that introns have many functions, including the modulation of regulation and structure. This work provides evidence that a eukaryotic gene promoter sequence from one gene located in an intron/exon of another.

Molecular cloning and characterization of chemokine-like factor super family member 1 (CKLFSF1), a novel human gene with at least 23 alternative splicing isoforms in testis tissue*1

Molecular cloning and characterization of chemokine-like factor super family member 1 (CKLFSF1), a novel human gene with at least 23 alternative splicing isoforms in testis tissue*1

Molecular cloning and characterization of chemokine-like factor super family member 1 ( CKLFSF1), a novel human gene with at least 23 alternative splicing isoforms in testis tissue

Chemokine-like factor (CKLF) was isolated from PHA-stimulated U937 cells. It is composed of 152 amino acids and located on chromosome 16q22. Utilizing bioinformatics, based on CKLF cDNA and protein sequences, in combination with experimental validation, we identified a novel gene designated chemokine-like factor super family member 1 ( CKLFSF1). CKLFSF1 maps on chromosome 16q22, and the full-length gene comprises of seven exons and six introns. Using RACE-PCR, we identified two potential alternative transcription start sites, 1A and 1B. Northern blot and RT-PCR analysis demonstrated that CKLFSF1 is predominantly expressed in human testis tissue, with only lower levels of expression in many other human tissues. RT-PCR and cDNA sequencing identified 23 alternatively spliced isoforms of CKLFSF1 in the testis tissue, which encode protein variants ranging from 36 to 169 amino acids in length. Immunohistochemistry analysis demonstrated that CKLFSF1 proteins are highly expressed in spermatocyte and in tissue fluid of human testes tissue. In light of these findings, we propose that CKLFSF1 may play an important role in spermatogenesis or testicular development.

Last intron of the chemokine-like factor gene contains a putative promoter for the downstream CKLF super family member 1 gene

The genes for chemokine-like factor ( CKLF) and four chemokine-like factor super family members ( CKLFSF1–4) are tightly linked on chromosome 16, with only 325 bp separating CKLF and CKLFSF1. We used Northern blotting and RT-PCR to show that these two genes are expressed independently of one another. We then used a novel computational promoter prediction method based on the interaction among transcription factor binding sites (TFBSs) to identify a putative promoter region for the CKLFSF1 gene. Our method predicted a promoter region in the last intron of the upstream gene, CKLF. We PCR amplified the predicted promoter region and used a luciferase assay to show that the region was able to drive the luciferase gene. DNA decoy experiments indicated that 214 bp fragment neighboring the TATA box markedly inhibited CKLFSF1 gene expression. Sequence analysis of the region revealed a typical TATA box (TATATAA) and multiple potential transcription factor binding sites, providing further evidence for this being a functional promoter for CKLFSF1. This work provides the first evidence of a promoter from one gene located in an intron of another.

Identification of eight genes encoding chemokine-like factor superfamily members 1–8 ( CKLFSF1– 8) by in silico cloning and experimental validation

TM4SF11 is only 102 kb from the chemokine gene cluster composed of SCYA22, SCYD1, and SCYA17 on chromosome 16q13. CKLF maps on chromosome 16q22. CKLFs have some characteristics associated with the CCL22/MDC, CX3CL1/fractalkine, CCL17/TARC, and TM4SF proteins. Bioinformatics based on CKLF2 cDNA and protein sequences in combination with experimental validation identified eight novel genes designated chemokine-like factor superfamily members 1– 8 ( CKLFSF1– 8). CKLFSF1– 8 form gene clusters; the sequence identities between CKLF2 and CKLFSF1–8 are from 12.5 to 39.7%. Most of the CKLFSFs have alternative RNA splicing forms. CKLFSF1 has a CC motif and higher sequence similarity with chemokines than with any of the other CKLFSFs. CKLFSF8 shares 39.3% amino acid identity with TM4SF11. CKLFSF1 links the CKLFSF family with chemokines, and CKLFSF8 links it with TM4SF. The characteristics of CKLFSF2–7 are intermediate between CKLFSF1 and CKLFSF8. This indicates that CKLFSF represents a novel gene family between the SCY and the TM4SF gene families.