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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., The cytokine like protein FAM3D (Fam3D in mice) is highly expressed in the digestive tract with unknown role in colon pathophysiology. Here, by using gene deficient mice, the authors show that Fam3D is critically involved in colon homeostasis, host defense against colitis-associated carcinogenesis, and the balance of microbiota.

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 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.

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. Electronic supplementary material The online version of this article (doi:10.1007/s13238-017-0421-7) contains supplementary material, which is available to authorized users.

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.

Autophagy is a multistep process that involves the degradation and digestion of intracellular components by the lysosome. It has been proved that many core autophagy-related molecules participate in this event. However, new component proteins that regulate autophagy are still being discovered. At present, we report PHF23 (PHD finger protein 23) with a PHD-like zinc finger domain that can negatively regulate autophagy. Data from experiments indicated that the overexpression of PHF23 impaired autophagy, as characterized by decreased levels of LC3B-II and weakened degradation of endogenous and exogenous autophagic substrates. Conversely, knockdown of PHF23 resulted in opposite effects. Molecular mechanism studies suggested that PHF23 interacts with LRSAM1, which is an E3 ligase key for ubiquitin-dependent autophagy against invading bacteria. PHF23 promotes the ubiquitination and proteasome degradation of LRSAM1. We also show that the PHD finger of PHF23 is a functional domain needed for the interaction with LRSAM1. Altogether, our results indicate that PHF23 is a negative regulator associated in autophagy via the LRSAM1 signaling pathway. The physical and functional connection between the PHF23 and LRSAM1 needs further investigation.

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.

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.

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.

PDCD5 is a novel apoptosis-related gene. Overexpression of PDCD5 facilitates apoptosis in various tumor cells. Here, we investigated the roles of recombinant human PDCD5 (rhPDCD5) protein in chemosensitivities of hematologic malignancies. The rhPDCD5 increased the in vitro cytotoxicity of daunorubicin (DNR), adriamycin (ADM) and As2O3 in three hematologic tumor cell lines. In vivo studies showed that DNR combined with rhPDCD5 significantly suppressed tumor growth of U937 xenograft nude mice compared with DNR alone. In conclusion, rhPDCD5 combined with the chemotherapeutic drug has greater efficacy than chemotherapy alone, and rhPDCD5 is a very promising chemosensitizer.

Cell migration plays a critical role during the development of most organisms and the process of malignant tumor metastasis. In the present study, we investigated the role of PTPIP51 (protein tyrosine phosphatase interacting protein 51) in cell motility. Overexpression of PTPIP51 induced cell elongation, increased cell migration, adhesion, and spreading, while downregulation of PTPIP51 had the opposite effects. We demonstrated here, that PTPIP51 could regulate ERK activity on Raf level, since MEK inhibitor and dominant-negative Raf-1 but not Ras could inhibit the ERK activation induced by PTPIP51. Further studies proved that PTPIP51 could interact with Raf-1 through 14–3–3, suggesting that PTPIP51 is a regulator of the Raf–MEK–ERK cascade through modulation of Raf-1 by 14–3–3. In addition, two redundant 14–3–3 binding domains in the PTPIP51 protein have been identified by deletion/mutation studies. We conclude that PTPIP51 regulates cell morphology and cell motility via interaction with Raf-1 through 14–3–3, and that PTPIP51 binds to 14–3–3 through two redundant binding domains.

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. (Cancer Sci 2008; 99: 2128–2135)

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.

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 (ΔΨm) 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. [Mol Cancer Ther 2008;7(1):222–32]

In this study, we report a novel gene, CCDC134 (coiled-coil domain containing 134), that encodes a secretory protein that can inhibit the MAPK pathway as a novel human MAPK-regulating protein. The CCDC134 mRNA contains 1280 nucleotides, encoding a protein of 229 amino acids. CCDC134 is a classical secretory protein. Expression profile analysis by Northern blot, RT-PCR, immunohistochemistry and Western blot reveals that CCDC134 is widely expressed in normal adult tissues, tumor tissues and cell lines. Functional investigation reveals that overexpression of CCDC134 and its purified protein significantly inhibit transcriptional activity of Elk1 and phosphorylation of Erk and JNK/SAPK but not p38 MAPK. Conversely, specific siRNA against CCDC134 activates Elk1 transcriptional activity and promotes Erk and JNK/SAPK phosphorylation. These results clearly indicate that CCDC134 is a novel member of the secretory family and down-regulates the Raf-1/MEK/ERK and JNK/ SAPK pathways.

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.

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.

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.

Chemokine-like factor 1(CKLF1) is a newly cloned cytokine with three RNA splicing isoforms. It has chemotactic activities on leukocytes and plays an important role in skeletal muscle regeneration. Here we have isolated two rat homologues of human chemokine-like factors by expressed sequence tag assembly, which are designated as rat chemokine-like factor 1 and 2 (rat CKLF1, CKLF2). The full-length cDNAs of rat CKLF1 and -2 contain 523 and 682 nucleotides and the open reading frames encoding 98 and 151 amino acids, respectively. Rat CKLF1 and -2 share about 54.1 and 59.6% homologies with human CKLF1 and -2 at the amino acid level; both rat CKLF1 and -2 contain a CX3C motif at their C-terminal regions while human CKLFs have a CC motif at the same regions. Rat CKLFs are highly expressed in testis, while human CKLFs have a broad expression spectrum across multiple tissues. Recombinant rat CKLF1 can be secreted into the cell culture supernatants and has chemotactic effects on neutrophils, macrophages and lymphocytes, which is similar to human CKLF1, while recombinant rat CKLF2 has weaker chemotactic effects on these cells. These findings show that rat CKLFs have similar bioactivity with human CKLFs, although they are different in tissue distribution and contain different characteristic motifs.

Chemokine-like factor1 (CKLF1), and its three isoforms (CKLF2, 3 and 4), are recently identified human cytokines. CKLF1 is a potent chemoattractant for human leukocytes and can stimulate inflammation and the regeneration of murine skeletal muscle. CKLF2 can promote proliferation and differentiation of C2C12 muscle cells directly by inducing expression of myogenin and activating transcription factors. In the present study, we cloned CKLF murine homologues, and based on their biological and structural features, named them murine chemokine-like factor 2, 4, 5 and 6 (mCKLF2, 4, 5 and 6). mCKLF2, 4, 5 and 6 encode 152, 120, 122 and 86 amino-acid proteins, respectively. mCKLFs map to mouse chromosome 8 and have high sequence similarity to human CKLFs. Compared to human CKLFs, which have a CC motif in the C-terminal region, mCKLF2 and 4 contain a CX3C motif. Using a PCR-based approach, it appeared mCKLF2 and 5 mRNA were highly expressed in adult testis, while mCKLF4 mRNA was detected only in differentiated C2C12 cells, a pattern different from human CKLFs. Conditioned media from COS-7 cells transfected with mCKLF2 and 4 was chemotactic for mouse neutrophils, macrophages and lymphocytes. Our results show that mCKLF2, 4, 5 and 6 are four splicing variants which are homologues of human CKLFs and murine CKLFs possess distinct features compared to their human counterparts.

The effects of precipitation pH values on the microstructural characteristics of nickel hydroxide materials synthesized by a chemical precipitation method have been studied. The relationship between structural characteristics and electrochemical activity of nickel hydroxide was also examined. The structural characteristics of the synthesized β-Ni(OH) 2 , such as degree of crystallinity, crystalline lattice disorders, crystallite size and crystal growth orientation were strongly related to the pH values of the chemical precipitation reaction. The amounts of SO 4 2− , CO 3 2− and H 2 O adsorbed in crystals, and the thermal stability of the β-Ni(OH) 2 also depended on the pH. Under relatively high pH values, the synthesized nickel hydroxide materials possessed a reduced crystallite size and lower thermal stability, more crystalline defects and a higher Ni composition. All these characteristics were likely to improve the electrochemical activity of nickel hydroxide.

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.

Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing 1 (CMTM1) consists of at least 23 alternatively spliced isoforms designated CMTM1_v1-v23. In the present study, we detected CMTM1_v17 expression in multiple human normal and tumor tissues and found that CMTM1_v17 was highly expressed in testis and many tumor tissues including breast tumor. The overexpression of CMTM1_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_v17 sensitized MDA-MB-231 cells to TNF-α-induced apoptosis. We propose that CMTM1_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_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_v17 for the treatment of breast and other cancers in which CMTM1_v17 impacts cellular proliferation and survival.

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.

Using the cDNA-representative differences analysis (cDNA-RDA) approach, we identified a novel gene, TFAR19 (TF-1 cell apoptosis related gene-19), from TF-1 cells undergoing apoptosis. The human TFAR19 encodes a protein which shares significant homology to the corresponding proteins of species ranging from yeast to mice. TFAR19 exhibits a ubiquitous expression pattern and its expression is upregulated in the tumor cells undergoing apoptosis. Overexpression of TFAR19 in tumor cells enhances apoptosis triggered by growth factor or serum deprivation. We propose that TFAR19 may play a general role in the apoptotic process.

Chemokines plays a crucial role in the migration of leukocytes, which are involved in immune and inflammatory reactions with their receptors. To find novel chemotactic cytokines, we carried out genome-wide bioinformatics analysis and PBMC chemoattractant platform screen. Seven novel potential chemotactic cytokines were picked up, one of them was PC3-secreted microprotein (PSMP). The mature secreted PSMP was able to chemoattract human peripheral blood monocytes, lymphocytes and THP-1 cells. CCR2 was identified as a high-affinity receptor for PSMP by in vitro chemotaxis, calcium flux, receptor internalization, and radioligand-binding assays. Furthermore, we found that PSMP was extensively expressed at low levels in normal human tissues, but was significantly up-regulated in intestinal biopsies from patients with inflammatory bowel disease and mouse DSS-induced colitic tissue in the early stage prior to IL-6, TNF-α and CCL2 up-regulation. Moreover, the results showed that PSMP triggered inflammatory Ly6ChiCCR2+ monocyte migration from the circulation into the lamina propria in a CCR2-dependent manner and promoted the progression of inflammation. A PSMP neutralizing monoclonal antibody significantly mollified inflammation in a mouse DSS colitis model. Taken together, our study provides the first evidence that PSMP is a novel chemotactic cytokines whose receptor is CCR2 and PSMP plays a vital and irreplaceable role in triggering and promoting DSS colitis. Our finding not only reveals the function and regulating mechanism of PSMP, but also provides a promising therapeutic strategy for treating inflammatory bowel disease.

To investigate the effect of simvastatin on inducing endothelial progenitor cells (EPCs) homing and promoting bone defect repair, and to explore the mechanism of local implanting simvastatin in promoting bone formation.Simvastatin (50 mg) compounded with polylactic acid (PLA, 200 mg) or only PLA (200 mg) was dissolved in acetone (1 mL) to prepare implanted materials (Simvastatin-PLA material, PLA material). EPCs were harvested from bone marrow of 2 male rabbits and cultured with M199; after identified by immunohistochemistry, the cell suspension of EPCs at the 3rd generation (2 x 10(6) cells/mL) was prepared and transplanted into 12 female rabbits through auricular veins (2 mL). After 3 days, the models of cranial defect with 15 cm diameter were made in the 12 female rabbits. And the defects were repaired with Simvastatin-PLA materials (experimental group, n=6) and PLA materials (control group, n=6), respectively. The bone repair was observed after 8 weeks of operation by gross appearance, X-ray film, and histology; gelatin-ink perfusion and HE staining were used to show the new vessels formation in the defect. Fluorescence in situ hybridization (FISH) was performed to show the EPCs homing at the defect site.All experimental animals of 2 groups survived to the end of the experiment. After 8 weeks in experimental group, new bone formation was observed in the bone defect by gross and histology, and an irregular, hyperdense shadow by X-ray film; no similar changes were observed in control group. FISH showed that the male EPC containing Y chromosome was found in the wall of new vessels in the defect of experimental group, while no male EPC containing Y chromosome was found in control group. The percentage of new bone formation in defect area was 91.63% +/- 4.07% in experimental group and 59.45% +/- 5.43% in control group, showing significant difference (P0.05).Simvastatin can promote bone defect repair, and its mechanism is probably associated with inducing EPCs homing and enhancing vasculogenesis.

To find an ideal material for repairing bone defect by local implanting simvastatin compounded with poly-lactic acid (PLA) into the radial critical size defects of rabbits, and to observe the reparative effect and type of bone formation induced by simvastatin.Twelve 4-months-old male New Zealand white rabbits (2.3-2.8 kg) with 22 mm radial critical size defects on both sides were randomized into 4 groups (all n=3). Right side and left side of every rabbit were set as controls with each other. The left defects (experimental groups) of groups A, B, and C were implanted with cylinder-like compound scaffolds containing 50, 100, and 200 mg of simvastatin (fixed with 250 mg PLA), or auto-bone graft as group D, respectively. The right defects of groups A, B, and C were implanted with scaffolds containing only 250 mg PLA. The right defects of group D were left without any treatment. Digital X-ray images of bone defects were taken 8 and 16 weeks after operation, X-ray was scored double blind and X-ray pixel value was measured. Animals were euthanized 16 weeks postoperatively. CT was applied to analyze new bone formation volume in the defects. In addition, morphological characters of new bones were observed through micro-CT and histology.X-ray films showed that the bone defect of each experimental side had much cloud-like callus, and the bone stump were not clear 8 weeks after operation; and the cortex in the defect was continuous and the medullary was recanalized 16 weeks after operation. In control sides, the cortexes were discontinuous and the ends of fractures were sclerified. At 8 and 16 weeks after operation, the X-ray scores, pixel values and the CT volume percentage of new bone in experiment sides were all significantly higher than those in control sides (P0.05). The X-ray scores of experimental sides in groups C and D were significantly higher than those in groups A and B 8 weeks after operation (P0.05), and the X-ray scores of experimental sides in groups B and D were significantly higher than those in groups A and C 16 weeks after operation (P0.05). The X-ray pixel values of experimental sides of group B were significantly higher than those of groups A, C, and D 8 weeks after operation (P0.05). The new bone formation volume of experimental side of groups B and D was higher than that of groups A and C (P0.05), and group D was significantly higher than that of group B (P0.05). Micro-CT showed bone defects of experimental sides of group B had totally healed, with connected medullary cavities and continuous bone cortex, on the contrary bone defects of control sides of group B did not healed completely. Histological observation showed better bone remodeling effects of the experimental sides than control sides, with connected medullary cavities and continuous bone cortex. And the osteogenetic type was endochondral ossification.Local implantation of simvastatin can promote repairing rabbit radial critical bone defect, 100 mg is the best dose of repairing the bone defects.

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-β-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.

Programmed cell death can be divided into apoptosis and autophagic cell death. We describe the biological activities of TMEM166 (transmembrane protein 166, also known as FLJ13391), which is a novel lysosome and endoplasmic reticulum-associated membrane protein containing a putative TM domain. Overexpression of TMEM166 markedly inhibited colony formation in HeLa cells. Simultaneously, typical morphological characteristics consistent with autophagy were observed by transmission electron microscopy, including extensive autophagic vacuolization and enclosure of cell organelles by double-membrane structures. Further experiments confirmed that the overexpression of TMEM166 increased the punctate distribution of MDC staining and GFP-LC3 in HeLa cells, as well as the LC3-II/LC3-I proportion. On the other hand, TMEM166-transfected HeLa and 293T cells succumbed to cell death with hallmarks of apoptosis including phosphatidylserine externalization, loss of mitochondrial transmembrane potential, caspase activation and chromatin condensation. Kinetic analysis revealed that the appearance of autophagy-related biochemical parameters preceded the nuclear changes typical of apoptosis in TMEM166-transfected HeLa cells. Suppression of TMEM166 expression by small interference RNA inhibited starvation-induced autophagy in HeLa cells. These findings show for the first time that TMEM166 is a novel regulator involved in both autophagy and apoptosis.

Simvastatin has been shown to stimulate osteogenesis both in vitro and in vivo. However, the mechanism by which simvastatin exerts its effects is still unclear. We previously reported that simvastatin promotes bone morphogenetic protein 2 (BMP-2) expression, induces osteoblastic differentiation, and inhibits adipocytic differentiation in mouse bone marrow stromal cells (BMSCs), and that this occurs, at least in part, via a BMP-2-dependent pathway. The aim of this study was to investigate further the mechanisms by which simvastatin stimulates osteogenesis in mouse BMSCs. To determine whether simvastatin-mediated osteogenesis was dependent on BMP-2, mouse BMSCs were treated with nonimmune normal mouse IgG or BMP-2 neutralizing antibodies combined with different concentrations of simvastatin. Surprisingly, the stimulatory effect of simvastatin on alkaline phosphatase (ALP) activity was not completely blocked by neutralizing BMP-2 monoclonal antibody treatment. Interestingly, we found that estrogen receptor-alpha (ER-alpha) protein levels increased after mouse BMSCs were treated with simvastatin for 72 h in a concentration-dependent manner. Moreover, the stimulatory effect of simvastatin on ALP activity in BMSCs was blocked by the estrogen receptor agonist ICI 182,780, and cotreatment with 17-beta-estradiol and simvastatin increased ALP activities by two-to threefold in the BMSCs compared with treatment with simvastatin alone. These results suggest that simvastatin-induced in vitro osteogenesis in mouse BMSCs is mediated, at least in part, by induction of ER-alpha and not by BMP-2 alone. These results provide new insight into the mechanisms of simvastatin-induced bone formation in BMSCs.

Apoptosis is a genetically determined cell suicide program. Mitochondria play a central role in this process and various molecules have been shown to regulate apoptosis in this organelle. In the present study, we firstly identified that protein tyrosine phosphatase interacting protein 51 (PTPIP51) is a novel mitochondrial protein, which may induce apoptosis in HEK293T and HeLa cell lines. PTPIP51 transfection resulted in the externalization of phosphatidylserine (PS), activation of caspase-3, cleavage of PARP, and condensation of nuclear DNA. Further investigation revealed that PTPIP51 over-expression caused a decrease in mitochondrial membrane potential and release of cytochrome c, suggesting that it may be involved in a mitochondria/cytochrome c mediated apoptosis pathway. We also found that a putative TM domain near the N terminus of PTPIP51 is required for its targeting to mitochondria, as evidenced by the finding that deletion of the PTPIP51 TM domain prevented the protein's mitochondiral localization. Furthermore, this deletion significantly influenced the ability of PTPIP51 to induce apoptosis. Taken together, the results of the present study suggest that PTPIP51 is a mitochondrial protein with apoptosis-inducing function and that the N-terminal TM domain is required for both the correct targeting of the protein to mitochondria and its apoptotic functions.

Publisher Summary This chapter addresses the issues involved in creating databases so that family relationships is readily determined and followed. The chapter discusses the benefits and limitations of family‐based datasets and the implications of the methods in research. The principle for creating a second order dataset that makes it possible to organize information in a database based on families is also described. Such datasets provide a valuable opportunity to conduct truly unique types of research among siblings and across multiple generations. They also help to create a longitudinal dataset within a relatively short period by performing a second order data linkage. The key to create a second‐order dataset establishes identifying numbers (IDs) for all individuals in a record and establishes IDs for all records in a dataset. By doing so, one can identify various relationships within different types of families and capture the fluidity of the modern family structure. One of the benefits of a second‐order dataset is enhanced identification and tracking capacity, which is particularly important to public health officials. The enhanced tracking capacity makes it easier for health officials to ensure uninterrupted services to those who are eligible. The benefits of a second‐order data linkage imposes a high demand on computing, programming capacity, and accentuates the difficulties seen in typical data linkage projects based on individuals alone.

Nanocrystalline nickel hydroxide powder was modified by the planetary ball milling (PBM), and the physical properties of both the ball-milled and un-milled nickel hydroxides were characterized by scanning electron microscopy, specific surface area (BET), particle size distribution and X-ray diffraction. It was found that the ball milling processing could obviously increase the surface area, break up the agglomeration, decrease the particle and crystallite size, and reduce the crystallinity of p-Ni(OH)2, which were advantageous to the improvement of the electrochemical activity of Ni(OH)2. The ball-milled nanocrystalline Ni(OH)2 was then used to alter the microstructure of pasted nickel electrodes and improve the distribution of the active material in the porous electrode substrate. Electrochemical performances of pasted nickel electrodes with an addition of ball-milled Ni(OH)2 to spherical Ni(OH)2 as the active material were investigated, and were compared with those of the pure spherical Ni(OH)2 electrodes. Charge/discharge tests showed that the addition of ball-milled Ni(OH)2 could enhance the charging efficiency, specific discharge capacity, discharge voltage and high-rate capability of pasted nickel electrodes. This performance improvement could be attributed to a more compact electrode microstructure and lower electrochemical impedance, as indicated by scanning electron microscopy and electrochemical impedance spectroscopy. Thus, it was an effective method to modify the microstructure and improve the electrochemical properties of pasted nickel electrodes by adding an appropriate amount of ball-milled nanocrystalline Ni(OH)2 to spherical Ni(OH)2 as the active material.

Chemokine-like factor1 (CKLF1), and its three isoforms (CKLF2, 3 and 4), are recently identified human cytokines. CKLF1 is a potent chemoattractant for human leukocytes and can stimulate inflammation and the regeneration of murine skeletal muscle. CKLF2 can promote proliferation and differentiation of C2C12 muscle cells directly by inducing expression of myogenin and activating transcription factors. In the present study, we cloned CKLF murine homologues, and based on their biological and structural features, named them murine chemokine-like factor 2, 4, 5 and 6 (mCKLF2, 4, 5 and 6). mCKLF2, 4, 5 and 6 encode 152, 120, 122 and 86 amino-acid proteins, respectively. mCKLFs map to mouse chromosome 8 and have high sequence similarity to human CKLFs. Compared to human CKLFs, which have a CC motif in the C-terminal region, mCKLF2 and 4 contain a CX3C motif. Using a PCR-based approach, it appeared mCKLF2 and 5 mRNA were highly expressed in adult testis, while mCKLF4 mRNA was detected only in differentiated C2C12 cells, a pattern different from human CKLFs. Conditioned media from COS-7 cells transfected with mCKLF2 and 4 was chemotactic for mouse neutrophils, macrophages and lymphocytes. Our results show that mCKLF2, 4, 5 and 6 are four splicing variants which are homologues of human CKLFs and murine CKLFs possess distinct features compared to their human counterparts.

To investigate the effect of interleukin-1 receptor antagonist gene therapy on type II collagen induced arthritis in DBA/1 mice.Plasmid pcDI-IL-1ra that expresses IL-1ra in eukaryotes was constructed by inserting human IL-1ra cDNA into the eukaryotic expression vector pcDI. Eukaryotes were transfected with the plasmid pcDI-IL-1ra in vivo and in vitro. The expression of 8 IL-1ra was examined by ELISA and immunohistochemistry. Type II collagen was used to induced arthritis in 32 DBA/1 mice. This plasmid was injected into the muscles of DBA/1 mice with arthritis induced by type II collagen by gene gun (20 micro g/mouse) and into the muscle of 8 mice by intramuscular injection (200 micro g/mouse). After the administration, the condition of arthritis was observed. The serum IL-ira was examined 6 and 12 days after administration. The expression of IL-ira in muscles was tested by computerized imaging analysis.PCR and DNA sequencing proved the accuracy of the inserted fragment. ELISA and immunohistochemistry detected high expression of IL-ira in vivo and in vitro. The absorbance ( A ) 490 value of IL-ira in the mouse muscle was 0.52 +/- 0.03 in gene gun group, and 0.48 +/- 0.02 in intramuscular injection group, all higher than that in the control group (0.41 +/- 0.02,P0.01 and P0.05). The serum IL-ira values in the gene gun group and in tramuscular injection group 6 days and 12 days after therapy were all significantly higher than that in the control group (all P0.01; and P0.01 and P0.05). Since the 6 th day after therapy, the redness and swelling of joints in both therapies groups were alleviated. Pathological examination made 12 days after therapy showed relief at different degrees of the infiltration of inflammatory cells, hyperplasia of synovia, bone infiltration, and cartilage destruction, especially in the gene gun group.Gene therapy of IL-ira via non-virus eukaryotic expression vactor, especially by gene gun, is effective in treating arthritis induced by type II collagen.

The programmed cell death 5 (PDCD5) protein is a novel protein related to regulation of cell apoptosis. In this report, we demonstrate that the level of PDCD5 protein expressed in cells undergoing apoptosis is significantly increased compared with normal cells, then the protein translocates rapidly from the cytoplasm to the nucleus of cells. The appearance of PDCD5 in the nuclei of apoptotic cells precedes the externalization of phosphatidylserine and fragmentation of chromosome DNA. This phenomenon is parallel to the loss of mitochondrial membrane potential, independent of the feature of apoptosis-inducing stimuli and also independent of the cell types and the apoptosis modality. In conclusion, the nuclear translocation of PDCD5 is a universal earlier event of the apoptotic process, and may be a novel early marker for apoptosis.

Cytokines are small proteins that have an essential role in the immune and inflammatory responses. The repertoire of cytokines is becoming diverse and expanding. Here we report the identification and characterization of a novel cytokine designated as chemokine-like factor 1 (CKLF1). The full-length cDNA of CKLF1 is 530bp long and a single open reading frame encoding 99 amino acid residues. CKLF1 bears no significant similarity to any other known cytokine in its amino acid sequence. Expression of CKLF1 can be partly inhibited by interleukin 10 in PHA-stimulated U937 cells. Recombinant CKLF1 is a potent chemoattractant for neutrophils, monocytes and lymphocytes; moreover, it can stimulate the proliferation of murine skeletal muscle cells. These results suggest that CKLF1 might have important roles in inflammation and in the regeneration of skeletal muscle.

Tip60 is a histone acetyltransferase (HAT) involved in the acetyltransferase activity and the cellular response to DNA damage. Here, we show that programmed cell death 5 (PDCD5), a human apoptosis-related protein, binds to Tip60 and enhances the stability of Tip60 protein in unstressed conditions. The binding amount of PDCD5 and Tip60 is significantly increased after UV irradiation. Further, PDCD5 enhances HAT activity of Tip60 and Tip60-dependent histone acetylation in both basal and UV-induced levels. We also find that PDCD5 increases Tip60-dependent K120 acetylation of p53 and participates in the p53-dependent expression of apoptosis-related genes, such as Bax. Moreover, we demonstrate the biological significance of the PDCD5-Tip60 interaction; that is, they function in cooperation to accelerate DNA damage-induced apoptosis and knockdown of PDCD5 or Tip60 impairs their apoptosis-accelerating activity, mutually. Consistent with this, PDCD5 levels increase significantly on DNA damage in U2OS cells, as does Tip60. Together, our findings indicate that PDCD5 may play a dual role in the Tip60 pathway. Specifically, under normal growth conditions, PDCD5 contributes to maintaining a basal pool of Tip60 and its HAT activity. After DNA damage, PDCD5 functions as a Tip60 coactivator to promote apoptosis.

Chemokine-like factor 1 (CKLF1) is a novel cytokine first cloned from U937 cells. It contains different splicing forms and has chemotactic effects on a wide spectrum of cells both in vitro and in vivo; it can also stimulate the regeneration of skeletal muscle cells in vivo, but the mechanism remains unclear. To probe the myogenesis function of CKLF2, which is the largest isoform of CKLFs, C2C12 murine myoblasts were stably transfected with human CKLF2 eukaryotic expression vector. Compared with control vector transfected C2C12 cells, CKLF2 overexpression causes accelerated myoblast proliferation as determined by cell counting and [3H]TdR incorporation assays. In addition, CKLF2 overexpression also promotes cell differentiation, which was determined by higher expression levels of myogenin, creatine kinase, myosin and the accelerated myoblast fusion. Further analysis also indicates that CKLF2 could activate the transcription activity of the bHLH/MyoD and MEF2 families. Finally, DNA synthesis and myotube formation could also be promoted by growing C2C12 cells in conditioned media from CKLF2-transfected cells. These findings strongly suggest a role for human CKLF2 in regulation of skeletal muscle myogenesis.