New Genes The Gsdml The Cloning Of Gsdmdc Family, Characterization, And Functional Studies

GSDMDC (Gasdermin domain containing) protein family, which includes DFNA5, DFNA5L, MLZE and GSDM, is defined by a conserved Gasdermin domain that is approximately 400 amino acids in length. Although the precise functions of the family remain unclear, DFNA5, MLZE and GSDM have been implicated in the development and progression of cancer. The expression of the DFNA5 gene is strongly induced by both exogenous and endogenous p53. The ectopic expression of DFNA5 enhances etoposide-induced cell death in the presence of p53. Further analysis revealed that DFNA5 is expressed moderately in normal tissues but is down-regulated in gastric cancer cell lines due to methylation of the region around its transcription start site. Methylation of DFNA5 can be detected in 50% of primary gastric tumors. Silencing of DFNA5 occurs frequently in gastric cancer and may play a key role in development and progression of the cancer. MLZE is one of the genes whose expression is upregulated during the course of acquisition of metastatic potential in melanoma cells. GSDM is predominantly expressed in upper gastrointestinal tract but significantly suppressed in human gastric cancer cells. These data suggest that loss of expression of the human GSDM is required for the carcinogenesis of gastric tissue and that the gene has an activity adverse to malignant transformation of cells.In order to find new cancer-associated genes, we isolated a novel gene, termed GSDML (Gasdermin-like) gene, from a human fetal liver cDNA library. As a novel member of the GSDMDC family, the function of the GSDML gene is predicted as cancer associated. The GSDML gene is mapped to 17q21.2, which region contains the erbB2 gene and is often amplified in malignant tumors. The full-length cDNA of GSDML is 1518 bp long and contains an open reading frame (ORF) from nucleotide 72 to nucleotide 1283 capable of encoding a 403 amino acids polypeptide with a predicted molecular weight of 45 kDa. GSDML protein contains a Gasdermin domain, a potential nuclear localizing signal (amino acids 242 to 261) and two LXX(L/I)L motifs (amino acids 58 to 62, 325 to 329) wich have been found to be necessary and sufficient for interaction with the nuclear receptor .Interestingly, we also isolated 3 shorter splicing isoforms of GSDML. Dot blot analysis of multiple human tissues showed that GSDML was restrictedly expressed in salivary. Rabbit polyclonal antibody and mouse monoclonal antibody were generated against epitopical peptides from GSDML protein. Western blot analysis with the specific anti-GSDML antibody, we showed that GSDML is widely expressed in human cancer cell lines we examined, but not in NIH3T3 mouse fibroblast cell or COS7 monkey kidney cells. The shorter isofoms were also detected in HEK293, HepG2, MCF7, HNE1 and HEN2 cell lines. Interestingly, GSDML protein is expressed at a higher level in uterine cervix cancer tissue than in the adjacent cancer tissues and corresponding non-neoplastic tissues through immunohistochemistry analysis. Such difference has not been obsverved in hepatocellular carcinoma tissues, suggesting that GSDML may be involved in the initiation or progression of certain type cancers. The results also imply that GSDML may be involved in the development on progression of cancer of female reproductive system. To investigate the molecular mechanism, we hypothesized that GSDML might interact with nuclear receptors due to the fact that it contains two potential nuclear receptor binding motifs. Co-immunoprecipitation assay indicated that GSDML specifically interacts with the estrogen receptor alpha (ERα) and thyroid hormone receptor alpha (TRα) in a ligand-independent fashion, but not with the androgen receptor alpha (ARα), HNF4 alpha (HNF4α) or glucocorticoid receptor (GR). Knock-down of endogenous GSDML expression by RNA interfering (RNAi) led to the reduced activity of ERαand the sensitivity of ERαto estrogen ligand, suggesting that GSDML may be involved in the regulation of ERαactivity in vivo. This is the first linkage between GSDMDC protein family and the nuclear receptors. Furthermore, cell growth analysis through MTT measurement showed that GSDML/NIH3T3 stable transfectants are capable of growing faster than vector mock cells. However, colony formation assay revealed that GSDML cannot induce malignant transformation of NIH3T3 cells, implying a specific role of GSDML in regulation of cell growth.Taken together, we have cloned and characterized a novel member of GSDMDC family, GSDML. The structure, tissue/cell expression profiling and subcellular localization of GSDML was characterized. Our study revealed the role of GSDML in tumorigenesis and the potential mechanism, contributing to full understandings of the GSDMDC family. These results also provide the first evidence that GSDML can interact with and regulate nuclear receptor, supplying new clues for elucidating how the GSDMDC family's function.