The Effects Of Arg133/Leu Mutation In LMNA On Adipocytedifferentiation And Transcriptome And Lamin A/C Binding Proteins

Background and ObjectiveLaminopathies is a kind of rare human heredopathia caused by mutations in the LMNA gene and defect in the lamin A/C protein.We report a new condition in a 26-yearold female,characterized by generalized lipoatrophy commencing at the onset of puberty,severe insulin resistance and diabetes,hypertriglyceridemia,a fatty liver,focal segmental glomerulosclerosis(FSGS)and hypertension.A heterozygous CGG to CTG transversion was found at LMNA gene codon 133.This mutation lead to an arginine to leucine sudstitution.Her 6-yr-old son carried the same mutation but has not(yet)developed a lipodystrophic phenotype.This study aims to investigate the effects of LMNA R133 L on the nuclear structures of adipocytes(3T3-L1 cells)or glomerular mesangial cells(SV40 MES 13 cells),especially in terms of adipocyte differentiation,global genomic expression,and the insulin-signalling pathway,and look for the downstream potential target protein.MethodsLMNA WT and LMNA R133 L mutant cell models were obtained by infecting 3T3-L1 cells or SV40 MES 13 cells with LMNA WT and LMNA R133 L mutant lentiviruses.The morphology of cell nuclei along with the expression of lamin protein were observed by immunofluorescence.The negative control,LMNA WT and LMNA R133 L mutant preadipocytes were screened by puromycin,and then the stable screening cells were induced to differentiation and the genes expression related to differentation were detected by quantitive real-time PCR.Western Blot was used to confirm the successful transfection and detect the activity of insulin signaling pathways.Gene Chip Mouse One Array Plus 2.0 microarray was used for studying global gene expression.The LMNA WT and LMNA R133 L mutant nucleoproteins were extracted and the differential lamin A/C binding nucleoproteins between the two groups were searched by protein pull-down experiment and analysed by mass spectrometry.Results 1.In pre-adipocytes or glomerular mesangial cells overexpressing LMNA R133 L,thenuclei showed disturbed shapes,varied in size,and exhibited nuclear membraneinvagination or blebbing.Immunofluorescence staining revealed that the A-typelamin meshwork was irregularly distributed around some nuclear envelopes.LaminB staining was also be in-homogeneous in LMNA R133 L cells compared to control orLMNA WT cells.2.The differentiation from 3T3-L1 pre-adipocytes stably expressing LMNA R133 L intoadipocytes was impaired;the expression of genes implicated in adipocytedifferentiation(including PPARγ,SREBP-1c,and Nipbl)were decreased.Theexpression of genes encoding extracellular matrix components was inhibited,including Col1α1,Col4α1,Col6α1,MMP2,and Adamts-7.And the angiogenesisgenes encoding Angpt1 and Rora,were significantly downregulated in LMNA R133Lcells compared to control and LMNA WT cells.3.Insulin-mediated activation of both the PI3K/Akt pathway and(downstream)associated Glut-4 membrane translocation were significantly suppressed inadipocytes stably expressing LMNA R133 L.The expression of PPARγ was alsosuppressed.4.The 3T3-L1 preadipocytes were transiently infected by the LMNA WT and LMNAR133L lentivirus.Total RNA was extracted after 4 days differentiation and thenanalyzed by One-array.The differential gene expression pathway between LMNA WTand LMNA R133 L cells were mainly related to metabolic pathways analyzed byKEGG pathway.The most prominent differences in terms of the gene ontologies ofmolecular function and biological processes were in genes implicated in RNA binding,the cellular response to DNA damage,and DNA repair.5.The 3T3-L1 preadipocytes were transiently infected by the LMNA WT and LMNAR133L lentivirus and then the nucleoproteins were extracted.Pull-down test wascarried out with lamin A/C antibody and Flag-tagged antibody to screen thedifferenent lamin A/C binding nucleoproteins between LMNA WT and LMNA R133Lcells.It was found that R133 L mutation reduced the binding of lamin A/C andAHNAK,Western Blot and immunofluorescence showed the expression of AHNAKin LMNA R133 L cells was significantly reduced.ConclusionsLMNA R133 L mutation associated with lipodystrophic features is likely due to the impaired adipocyte renewal,which was related to DNA damage and DNA repair disorders,probably secondary to the reduction of lamin A/C-AHNAK binding.