Role Of Nuclear Receptor TLX In Islet ? Cells Survival

Objective To investigate the expression of nuclear receptor TLX in murine pancreatic islets and pancreatic ? cell line (MIN6). And to determine whether TLX affects basal ? cells functions such as proliferation, apoptosis, and insulin production. Also to screening the direct target gene of TLX, and to investigate the regulation mechanism of TLX in ? cells biological behavior.Methods Immunofluorescence was performed in murine pancreas and pancreatic ? cell line (MIN6) to detecte TLX expression. MIN6 cells were infected with lentiviral vectors carrying mouse TLX sequence to create TLX overexpression cells and also infected with lentiviral vectors carrying shRNA sequence to create TLX knockdown cells. The effects of TLX on cell viability, proliferation, cell cycle, apoptosis, insulin secretion, and its regulated genes were analyzed by MTT assay, EdU assay, flow cytometric, Quantitative real time transcriptase QRT-PCR, and ELISA. Then gene expression profiling analysis were performed to identify the differentially expressed genes between TLX overexpression cells and control cells and bioinformatical analysis were used to screening the candidate target gene of TLX. The interaction of TLX and target gene was analyzed by chromatin immunoprecipitation (ChIP). The expression level of target gene was analyzed by QRT-PCR and Western-blot in TLX overexpression cells and TLX knockdown cells. Finally, target gene expression was restored in TLX overexpression MIN6 cells and cell viability, proliferation, cell cycle, apoptosis were analyzed by MTT assay, EdU assay, and flow cytometric.Results TLX protein is expressed in murine pancreatic islets and pancreatic endocrine cell line (MIN6), and there were more TLX positive cells in the MIN6 cell line than in mouse islet cells. Overexpression of TLX in MIN6 cells lead to decreased sensitivity to lipotoxicity, increased proliferation, higher percentage of cells exiting G1 into S-phase, and lower rates of apoptosis. In contrast, knockdown of TLX in MIN6 cells resulted in increased sensitivity to lipotoxicity, decreased proliferation, a partial G0/G1 cell-cycle arrest and higher rates of apoptosis. However, TLX did not improve basal and glucose-stimulated insulin secretion in MIN6 cells but, also did not cause any functional impairment. Further gene expression profiling analysis revealed that overexpression of TLX in MIN6 cells causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. Bioinformatical analysis show that Gadd45a was a candidate target gene of TLX and TLX-Gadd45a interaction was validated by chromatin immunoprecipitation. Moreover, The expression level of Gadd45a in downregulated TLX overexpression cells and upregulated TLX knockdown cells. Finally, compared to TLX overexpression MIN6 cells, restoring Gadd45a expression in TLX overexpression MIN6 cells resulted in increased sensitivity to lipotoxicity, decreased proliferation and higher rates of apoptosis.Conclusion TLX was expressed in the pancreatic ? cells. TLX may control pancreatic ? cells proliferation and survival by directly suppress Gadd45a transcription. Overexpression of TLX may protect ? cells from lipotoxicity, which may serve as a target for the development of novel therapies for diabetes.