PSMP/MSMP promotes hepatic fibrosis through CCR2 and represents a novel therapeutic target.

BACKGROUND & AIMS: C-C motif chemokine receptor 2 (CCR2) has been recognized as a promising target for the treatment of liver fibrosis. PC3-secreted microprotein (PSMP)/microseminoprotein (MSMP) is a novel chemotactic cytokine and its receptor is CCR2. In the present study we investigated the expression and role of PSMP in liver fibrosis/cirrhosis. METHODS: PSMP expression was studied in patients with fibrosis/cirrhosis and in 3 murine models of liver fibrosis, including mice treated with carbon tetrachloride (CCl4), bile-duct ligation, or a 5-diethoxycarbonyl-1,4-dihydrocollidine diet. The role of PSMP was evaluated in Psmp-/- mice and after treatment with a PSMP antibody in wild-type mice. The direct effects of PSMP on macrophages and hepatic stellate cells were studied in vitro. RESULTS: In this study, we found that PSMP was highly expressed in fibrotic/cirrhotic tissues from patients with different etiologies of liver disease and in the 3 experimental mouse models of fibrosis. Damage-associated molecular pattern molecules HMGB-1 and IL-33 induced hepatocytes to produce PSMP. PSMP deficiency resulted in a marked amelioration of hepatic injury and fibrosis. In CCl4-induced hepatic injury, the infiltration of macrophages and CCR2+ monocytes into the liver was significantly decreased in Psmp-/- mice. Consistent with the decreased levels of intrahepatic macrophages, proinflammatory cytokines were significantly reduced. Moreover, adeno-associated virus-8 vectors successfully overexpressing human PSMP in Psmp-/- mouse livers could reverse the attenuation of liver injury and fibrosis induced by CCl4 in a CCR2-dependent manner. Treatment with a specific PSMP-neutralizing antibody, 3D5, prevented liver injury and fibrosis induced by CCl4 in mice. At the cellular level, PSMP directly promoted M1 polarization of macrophages and activation of LX-2 cells. CONCLUSION: PSMP enhances liver fibrosis through its receptor, CCR2. PSMP is a potentially attractive therapeutic target for the treatment of patients with liver fibrosis. LAY SUMMARY: Our present study identifies the essential role of the protein PSMP for the development and progression of liver fibrosis in humans and mice. PSMP promotes liver fibrosis through inflammatory macrophage infiltration, polarization and production of proinflammatory cytokines, as well as direct activation of hepatic stellate cells via its receptor CCR2. A PSMP antibody can significantly reduce liver fibrosis development in vivo. These findings indicate that PSMP is a potential therapeutic target and its antibody is a potential therapeutic agent for the treatment of liver fibrosis.

FAM19A1 is a new ligand for GPR1 that modulates neural stem-cell proliferation and differentiation.

FAM19A1 is a member of the family with sequence similarity 19 with unknown function. FAM19A1 mRNA expression is restricted to the CNS. Here, we report that FAM19A1 is a classic secretory protein, and expression levels correlate with brain development, increasing from embryonic d 12.5, peaking between postnatal d (P)1 and P7 and decreasing at wk 8. The adult hippocampus is a region of FAM19A1 high expression. Recombinant FAM19A1 suppressed the proliferation and self-renewal of neural stem cells (NSCs) and altered the lineage progression of NSCs with promoted neuron differentiation and suppressed astrocyte differentiation. Although GPCR 1 (GPR1) has been reported to be expressed in the CNS, its functions in the brain remain unclear. We identified GPR1 to be a functional receptor for FAM19A1. FAM19A1 interacted with GPR1 via the N-terminal domain (GPR1-ND), and its NSC modulatory functions required the Rho-associated protein kinase (ROCK) /ERK1/2 and ROCK/signal transducer and activator of transcription 3 signaling pathways. GPR1-ND that selectively bound to FAM19A1 neutralized the effects of FAM19A1 on NSC functions. Taken together, our results show, for the first time to our knowledge, that FAM19A1 is a novel regulatory factor of the proliferation and differentiation of NSCs, and identified a novel mechanism by which GPCR mediates the effects of FAM19A1 on NSC functions that may be important for brain development and neurogenesis. Additional exploration of the functions of FAM19A1 and GPR1 in the CNS may broaden the range of therapeutic options available for major brain disorders.-Zheng, C., Chen, D., Zhang, Y., Bai, Y., Huang, S., Zheng, D., Liang, W., She, S., Peng, X., Wang, P., Mo, X., Song, Q., Lv, P., Huang, J., Ye, R. D., Wang, Y. FAM19A1 is a new ligand for GPR1 that modulates neural stem-cell proliferation and differentiation.

Functional characterization of the tumor suppressor CMTM8 and its association with prognosis in bladder cancer.

Previous research revealed that CMTM8 acts as a tumor suppressor gene in variety cancers. However, the role of CMTM8 in bladder cancer has never been reported. In this study, the expression profile of CMTM8 was examined in bladder cancer tissues and bladder cancer cell lines. The effects of CMTM8 on bladder cancer cell proliferation, apoptosis, migration, and invasion were examined. Bladder tumor tissues from 84 cases were examined for CMTM8 expression by immunohistochemistry. Disease-specific survival was investigated using a Kaplan-Meier analysis, and Cox proportional hazards analysis was assessed. Our results showed that upregulation of CMTM8 in the T24 cell line could suppress T24 cells proliferation, migration and invasion and enhance the sensitivity to Epirubicin. Kaplan-Meier analysis revealed that the expression of CMTM8 was correlated with the survival time of bladder cancer patients. Altogether, our data suggested that CMTM8 is an important tumor suppressor gene in human bladder cancer and qualified as a useful prognostic indicator for patients with bladder cancer.

PC3-secreted microprotein is a novel chemoattractant protein and functions as a high-affinity ligand for CC chemokine receptor 2.

PC3-secreted microprotein (PSMP) or microseminoprotein is a newly discovered secreted protein whose function is currently unknown. In this study, PSMP was found to possess chemotactic ability toward monocytes and lymphocytes, and its functional receptor was identified as CCR2B. PSMP was identified as a chemoattractant protein from a PBMC chemoattractant platform screen that we established. The mature secreted PSMP was able to chemoattract human peripheral blood monocytes, PBLs, and CCR2B-expressing THP-1 cells, but not peripheral blood neutrophils, even though it does not contain the classical structure of chemokines. CCR2B was identified as one receptor for PSMP-mediated chemotaxis by screening HEK293 cells that transiently expressed classical chemokine receptors; results obtained from the chemotaxis, calcium flux, receptor internalization, and radioligand-binding assays all confirmed this finding. To further identify the major function of PSMP, we analyzed its expression profile in tissues. PSMP is highly expressed in benign prostatic hyperplasia and in some prostate cancers, and can also be detected in breast tumor tissue. In response to PSMP stimulation, phosphorylated ERK levels downstream of CCR2B signaling were upregulated in the PC3 cell line. Taken together, our data collectively suggest that PSMP is a chemoattractant protein acting as a novel CCR2 ligand that may influence inflammation and cancer development.

Recombinant human PDCD5 protein enhances chemosensitivity of breast cancer in vitro and in vivo.

Resistance to paclitaxel is common for treatment of breast cancer. Programmed cell death 5 (PDCD5) accelerates apoptosis in different cell types in response to various stimuli; moreover PDCD5 has been shown to be down-regulated in many tumors. In this study, protein levels of PDCD5 were found to be up-regulated in paclitaxel-treated MDA-MB-231 breast cancer cells. MTT, CCK-8, and clonogenic assays have shown that recombinant human PDCD5 (rhPDCD5) alone could not produce an obvious growth inhibition. However, upon paclitaxel triggering apoptosis, rhPDCD5 protein potentiated chemotherapeutic drugs-induced growth arrest in MDA-MB-231, SK-BR-3, and ZR-75-1 breast cancer cells. In vivo, we use a human breast cancer xenograft model to study. We found that rhPDCD5 dramatically improves the antitumor effects of paclitaxel treatment by intraperitoneal administration. These data suggest that rhPDCD5 has the potential to use as a therapeutic agent to enhance the paclitaxel sensitivity of breast cancer cells.

Simvastatin induces estrogen receptor-alpha expression in bone, restores bone loss, and decreases ERα expression and uterine wet weight in ovariectomized rats.

We previously reported that simvastatin induces estrogen receptor-alpha (ERα) in murine bone marrow stromal cells in vitro. In this study, we investigated the effect of simvastatin on ERα expression in bone and uterus in ovariectomized (OVX) rats and evaluated bone mass, bone strength, and uterine wet weight. Three-month-old Sprague-Dawley female rats received OVX or sham operation. Six weeks later, the rats were treated orally with simvastatin (5 or 10 mg/kg/day), or intraperitoneally with 17-ß-estradiol (E(2)) or a combination of simvastatin and E(2) for 6 weeks. Uterine wet weight, bone mineral density (BMD) of lumbar vertebrae, biomechanics of lumbar vertebrae, and induction of ERα expression in the bone and uterus were analyzed. The 6-week simvastatin treatment improved lumbar vertebral BMD and boosted biomechanical performance of the vertebral body compared to the OVX control, suggesting that simvastatin can treat osteoporosis caused by estrogen deficiency. More interestingly, simvastatin could increase ERα expression and synergy with estradiol in bone while antagonizing estradiol in the uterus, along with uterus atrophy and uterine wet weight decreases. In conclusion, these data suggest that simvastatin exert opposing modulatory effects on ERα expression on bone and uterus in ovariectomized rats, inducing ERα expression and synergy with estrogen to perform anabolic effects on the bones while decreasing E2 efficacy and uterine wet weight. This finding may be helpful to explain the mechanism of statin treatment in osteoporosis caused by estrogen deficiency.

NSA2, a novel nucleolus protein regulates cell proliferation and cell cycle.

NSA2 (Nop seven-associated 2) was previously identified in a high throughput screen of novel human genes associated with cell proliferation, and the NSA2 protein is evolutionarily conserved across different species. In this study, we revealed that NSA2 is broadly expressed in human tissues and cultured cell lines, and located in the nucleolus of the cell. Both of the putative nuclear localization signals (NLSs) of NSA2, also overlapped with nucleolar localization signals (NoLSs), are capable of directing nucleolar accumulation. Moreover, over-expression of the NSA2 protein promoted cell growth in different cell lines and regulated the G1/S transition in the cell cycle. SiRNA silencing of the NSA2 transcript attenuated the cell growth and dramatically blocked the cell cycle in G1/S transition. Our results demonstrated that NSA2 is a nucleolar protein involved in cell proliferation and cell cycle regulation.

Potent antitumor activities of recombinant human PDCD5 protein in combination with chemotherapy drugs in K562 cells.

Conventional chemotherapy is still frequently used. Programmed cell death 5 (PDCD5) enhances apoptosis of various tumor cells triggered by certain stimuli and is lowly expressed in leukemic cells from chronic myelogenous leukemia patients. Here, we describe for the first time that recombinant human PDCD5 protein (rhPDCD5) in combination with chemotherapy drugs has potent antitumor effects on chronic myelogenous leukemia K562 cells in vitro and in vivo. The antitumor efficacy of rhPDCD5 protein with chemotherapy drugs, idarubicin (IDR) or cytarabine (Ara-C), was examined in K562 cells in vitro and K562 xenograft tumor models in vivo. rhPDCD5 protein markedly increased the apoptosis rates and decreased the colony-forming capability of K562 cells after the combined treatment with IDR or Ara-C. rhPDCD5 protein by intraperitoneal administration dramatically improved the antitumor effects of IDR treatment in the K562 xenograft model. The tumor sizes and cell proliferation were significantly decreased; and TUNEL positive cells were significantly increased in the combined group with rhPDCD5 protein and IDR treatment compared with single IDR treatment groups. rhPDCD5 protein, in combination with IDR, has potent antitumor effects on chronic myelogenous leukemia K562 cells and may be a novel and promising agent for the treatment of chronic myelogenous leukemia.

FAM3D is essential for colon homeostasis and host defense against inflammation associated carcinogenesis.

The physiological homeostasis of gut mucosal barrier is maintained by both genetic and environmental factors and its impairment leads to pathogenesis such as inflammatory bowel disease. A cytokine like molecule, FAM3D (mouse Fam3D), is highly expressed in mouse gastrointestinal tract. Here, we demonstrate that deficiency in Fam3D is associated with impaired integrity of colonic mucosa, increased epithelial hyper-proliferation, reduced anti-microbial peptide production and increased sensitivity to chemically induced colitis associated with high incidence of cancer. Pretreatment of Fam3D-/- mice with antibiotics significantly reduces the severity of chemically induced colitis and wild type (WT) mice co-housed with Fam3D-/- mice phenocopy Fam3D-deficiency showing increased sensitivity to colitis and skewed composition of fecal microbiota. An initial equilibrium of microbiota in cohoused WT and Fam3D-/- mice is followed by an increasing divergence of the bacterial composition after separation. These results demonstrate the essential role of Fam3D in colon homeostasis, protection against inflammation associated cancer and normal microbiota composition.

[Outcome and significance of pulmonary pathological changes induced by a single intramuscular injection of chemokine-like factor 1 in mice].

OBJECTIVE: To investigate the significance of pathological changes in murine lung by a single intramuscular injection of chemokine-like factor 1 (CKLF1). METHODS: A total of 120 gender-matched BALB/c mice were randomly and evenly divided into treatment group and control group (60 in each). One hundred nanomilligram of pcDNA3.1-CKLF1-Myc-His, CKLF1-expressing plasmid, in 100 microl of pyrogen-free saline was injected into the anterior tibial muscle of mice followed by the delivery of electric pulses. Mice in the control group received 100 microg of pcDNA3.1-Myc-His in 100 microl of pyrogen-free saline. At the end of week 1, 4 and 8 respectively after injection of CKLF1, 20 mice were sacrificed in every group and the cellular profiles in bronchoalveolar lavage fluid (BALF) and the pulmonary pathological changes were observed. RESULTS: At the end of week 1 and 4 respectively after CKLF1 injection, the neutrophils [(35.0 +/- 5.2)% and (22.9 +/- 2.2)% respectively] and lymphocytes [(34.5 +/- 2.8)% and (22.0 +/- 2.0)% respectively] in BALF of the treatment group were higher than those of the control group [neutrophils: (6.7 +/- 2.2)% and (7.0 +/- 2.4)% respectively, lymphocytes: (5.9 +/- 1.6)% and (6.1 +/- 2.7)% respectively, all P < 0.01]. Pathological studies demonstrated shedding of bronchiolar epithelium, congestion and edema in interstitial tissue and inflammatory cell infiltration in mice at 1 week after CKLF1 injection. Week 4 after CKLF1 administration, the alveolar wall was shown significantly thickened with proliferation of neutrophils, macrophages and fibroblasts as well as remarked collagen deposition in the interstitium. At the end of week 8 after CKLF1 administration, the remarkable morphological changes of the lung gradually subsided and the structure of the lung returned to normal. CONCLUSIONS: CKLF1 causes injury of inflammation and remodeling in airway in mice. The pulmonary pathological changes induced by a single intramuscular injection of CKLF are reversible.

Adenovirus-expressed human hyperplasia suppressor gene induces apoptosis in cancer cells.

Hyperplasia suppressor gene (HSG), also called human mitofusin 2, is a novel gene that markedly suppresses the cell proliferation of hyperproliferative vascular smooth muscle cells from spontaneously hypertensive rat arteries. This gene encodes a mitochondrial membrane protein that participates in mitochondrial fusion and contributes to the maintenance and operation of the mitochondrial network. In this report, we showed that an adenovirus vector encoding human HSG (Ad5-hHSG) had an antitumor activity in a wide range of cancer cell lines. We further focused on the lung cancer cell line A549 and the colon cancer cell line HT-29 and then observed that Ad5-hHSG induced apoptosis both in vitro and in vivo. Confocal laser scanning microscopy and electron microscopy revealed that cells infected with Ad5-hHSG formed dose-dependent perinuclear clusters of fused mitochondria. Adenovirus-mediated hHSG overexpression induced apoptosis, cell cycle arrest, mitochondrial membrane potential (DeltaPsim) reduction and release of cytochrome c, caspase-3 activation, and cleavage of PARP in vitro. Overexpression of hHSG also significantly suppressed the growth of subcutaneous tumors in nude mice both ex vivo and in vivo. In addition, Ad5-hHSG increased the sensitivity of these cell lines to two chemotherapeutic agents, VP16 and CHX, and radiation. These results suggest that Ad5-hHSG may serve as an effective therapeutic drug against tumors.

DCUN1D3, a novel UVC-responsive gene that is involved in cell cycle progression and cell growth.

DCUN1D3 (DCN1, defective in cullin neddylation 1, domain containing 3) was found during the process of high throughput screening of novel human genes associated with serum response element (SRE) pathway activation. The DCUN1D3 gene is highly conserved among vertebrates. Human DCUN1D3 complementary DNA (cDNA) encodes 304 amino acids with an apparent molecular mass of 34 kDa. However, there has been no report about the function of DCUN1D3. This study detected that DCUN1D3 was broadly expressed in several tumor tissues and cultured cell lines; however, UVC irradiation of different doses significantly increased DCUN1D3 expression level in these cancer cell lines. Over-expression of the DCUN1D3 inhibits cell growth in HeLa. When the DCUN1D3 gene was silenced by siRNA in UVC-treated HeLa, the cell cycle in S phase was remarkably blocked; furthermore, the UVC-induced cell death was inhibited. In addition, DCUN1D3 localized mainly in the cytoplasm and perinuclear, but after UVC treatment, the DCUN1D3 gradually entered the nucleus. All the results above indicate that DCUN1D3 is a novel UVC-response gene involved in cell cycle regulation and cell survival.

CMTM3 can affect the transcription activity of androgen receptor and inhibit the expression level of PSA in LNCaP cells.

CMTM is a novel family of proteins linking chemokines and TM4SF. Several members of this family are highly expressed in testes and regulate androgen receptor (AR) transcription activity. One member of this family, CMTM3, has the highest expression level in testes and contains one leucine zipper and two LXXLL motifs. As assessed with the dual-luciferase reporter assay, overexpression of CMTM3 represses AR transactivation, while knocking down it can increase AR transactivation. Moreover, CMTM3 inhibits prostate-specific antigen (PSA) expression in LNCaP cells at both mRNA and protein levels with no obvious influence on AR expression. Taken together, CMTM3 may play some roles in the maturation and maintenance of the male reproduction.

LRRC25 plays a key role in all-trans retinoic acid-induced granulocytic differentiation as a novel potential leukocyte differentiation antigen.

Leukocyte differentiation antigens (LDAs) play important roles in the immune system, by serving as surface markers and participating in multiple biological activities, such as recognizing pathogens, mediating membrane signals, interacting with other cells or systems, and regulating cell differentiation and activation. Data mining is a powerful tool used to identify novel LDAs from whole genome. LRRC25 (leucine rich repeat-containing 25) was predicted to have a role in the function of myeloid cells by a large-scale "omics" data analysis. Further experimental validation showed that LRRC25 is highly expressed in primary myeloid cells, such as granulocytes and monocytes, and lowly/intermediately expressed in B cells, but not in T cells and almost all NK cells. It was down-regulated in multiple acute myeloid leukemia (AML) cell lines and bone marrow cells of AML patients and up-regulated after all-trans retinoic acid (ATRA)-mediated granulocytic differentiation in AML cell lines and acute promyelocytic leukemia (APL; AML-M3, FAB classification) cells. Localization analysis showed that LRRC25 is a type I transmembrane molecule. Although ectopic LRRC25 did not promote spontaneous differentiation of NB4 cells, knockdown of LRRC25 by siRNA or shRNA and knockout of LRRC25 by the CRISPR-Cas9 system attenuated ATRA-induced terminal granulocytic differentiation, and restoration of LRRC25 in knockout cells could rescue ATRA-induced granulocytic differentiation. Therefore, LRRC25, a potential leukocyte differentiation antigen, is a key regulator of ATRA-induced granulocytic differentiation.

Knockout of MARCH2 inhibits the growth of HCT116 colon cancer cells by inducing endoplasmic reticulum stress.

Membrane-associated RING-CH protein 2 (MARCH2), a member of the MARCH family, functions in vesicle trafficking and autophagy regulation. In this study, we established MARCH2 knockout HCT116 cell lines using CRISPR/Cas9-mediated genome editing to evaluate the role of MARCH2 in colon cancer in vitro and in vivo. Knockout of MARCH2 suppressed cell proliferation, and promoted autophagy, apoptosis and G2/M phase cell cycle arrest. These effects were associated with activation of endoplasmic reticulum (ER) stress. In addition, loss of MARCH2 sensitized HCT116 cells to the chemotherapy drugs etoposide and cisplatin. Moreover, we analyzed the clinical significance of MARCH2 in human colon carcinoma (n=100). High MARCH2 expression was significantly associated with advanced clinicopathological features and poorer overall survival in colon carcinoma. MARCH2 expression correlated negatively with expression of the unfolded protein response molecule p-PERK in colon cancer. Collectively, these data reveal a relationship between MARCH2, ER stress and colon cancer, and indicates MARCH2 may have an important role in the development and progression of colon cancer.

The PSMP-CCR2 interactions trigger monocyte/macrophage-dependent colitis.

Monocytes/macrophages have been found to be an important component of colitis. However, the key chemokine that initiates the CCR2+ monocytes migration from circulation to colitis tissue remains to be undiscovered. PC3-secreted microprotein (PSMP) is a novel chemokine whose receptor is CCR2. The physiological and pathological functions of PSMP have not yet been reported. In this study, PSMP was found to be expressed in colitis and colonic tumor tissues from patients and significantly up-regulated in mouse DSS-induced colitis tissues. PSMP overexpression in the colon aggravated the DSS-induced colitis and the anti-PSMP neutralizing antibody mollified the colitis by reducing macrophage infiltration and inhibiting the expression of IL-6, TNF-α and CCL2. Furthermore, we demonstrated that lipopolysaccharide and muramyl dipeptide induced PSMP expression in the colonic epithelial cells. PSMP was up-regulated in the initial stage prior to IL-6, TNF-α and CCL2 up-regulated expression in DSS colitis and promoted the M1 macrophages to produce CCL2. PSMP chemo-attracted Ly6Chi monocytes in a CCR2 dependent manner via in situ chemotaxis and adoptive transfer assays. Our data identify PSMP as a key molecule in ulcerative colitis, which provides a novel mechanism of monocyte/macrophage migration that affects gut innate immunity and makes PSMP a potential target for controlling colitis.

CMTM8 is Frequently Downregulated in Multiple Solid Tumors.

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.

Overexpression of the novel human gene, nuclear apoptosis-inducing factor 1, induces apoptosis.

Apoptosis is a genetically determined cell suicidal program that plays critical roles in many physiological and pathological processes. In this study, we report the cloning and characterization of a novel human gene, nuclear apoptosis-inducing factor 1 (NAIF1), overexpression of which induces apoptosis in cells. Human NAIF1 is located on chromosome 9q34.11 and encodes 327 amino acids with a homeodomain-like region and two nuclear localization signals at its N-terminal region. NAIF1 is conserved across diverse species, including human, mouse, crab-eating macaque, dog, chicken and frog, and shares no obvious homology to any known genes or proteins. Northern blot analysis revealed wide expression of NAIF1 mRNA throughout human tissues. NAIF1 was predominantly localized in the nucleus. Overexpression of NAIF1 inhibited cell growth and induced apoptosis. Furthermore, NAIF1 transfection caused both decreases in mitochondrial membrane potential and caspase-3 activation. In summary, NAIF1 is a nuclear protein that induces apoptosis when overexpressed.

Chemokine-like factor 1 is a functional ligand for CC chemokine receptor 4 (CCR4).

Chemokine-like factor 1 (CKLF1) exhibits chemotactic effects on leukocytes. Its amino acid sequence shares similarity with those of TARC/CCL17 and MDC/CCL22, the cognate ligands for CCR4. The chemotactic effects of CKLF1 for CCR4-transfected cells could be desensitized by TARC/CCL17 and markedly inhibited by PTX. CKLF1 induced a calcium flux in CCR4-transfected cells and fully desensitized a subsequent response to TARC/CCL17, and TARC/CCL17 could partly desensitize the response to CKLF1. CKLF1 caused significant receptor internalization in pCCR4-EGFP transfected cells. Taken together, CKLF1 is a novel functional ligand for CCR4.

Characterization and expression profile of CMTM3/CKLFSF3.

CMTM/CKLFSF is a novel family of proteins linking chemokines and TM4SF. In humans, these proteins are encoded by nine genes, CKLF and CMTM1-8/CKLFSF1-8. Here we report the characteristics and expression profile of CMTM3/CKLFSF3. Human CMTM3/CKLFSF3 has a high sequence identity among various species and similar characteristics as its mouse and rat homologues. Established by results both of RT-PCR and Quantitative Real-time PCR, the gene is highly transcribed in testis, leukocytes and spleen. For further verification, we generated a polyclonal antibody against human CMTM3/CKLFSF3 and found that the protein is highly expressed in the testis and some cells of PBMCs. Therefore, CMTM3/CKLFSF3 is an evolutionarily conserved gene that may have important roles in the male reproductive system and immune system. Further studies are necessary to validate its functions in the two systems.