ABSTRACT
OBJECTIVE: Transglutaminase 2 (TG2) is a multifunctional protein with an important role in vascular biology, where it is involved in cell-matrix interaction, cell attachment and cell population expansion. In efforts to elucidate the role of TG2 in endothelial cell biology, in this study, we measured several endothelial cell characteristics in cells where TG2 was specifically knocked down by RNAi. MATERIALS AND METHODS: The effect of small interfering RNA (siRNA)-TG2 on human umbilical vein endothelial cells was studied. Adhesion and cell viability were assessed by chemical reduction of MTT, and cell proliferation was analysed by flow cytometry. Apoptosis was evaluated by annexin V/PI dual staining and protein expression level was assayed by western blotting. RESULTS: We found that siRNA-TG2 reduced endothelial cell number, lead to cell adhesion deficiency, cell cycle arrest in G1 phase and induction of apoptosis. Our results show that exogenously added TG2 could reverse loss of adhesion but did not overcome the defect in cell proliferation, nor could it inhibit siRNA-TG2-induced apoptosis. CONCLUSION: We conclude that TG2 loss in endothelial cells causes reduction in cell number as a result of cell cycle arrest, flaws in adhesion and induction of apoptosis. Our results imply that reduction in cell number and increased apoptosis in response to TG2 silencing is independent of the cell adhesion process. Altogether, our findings underline the significance of TG2 in endothelial cell cycle progression and cell survival, in vitro.
Subject(s)
Apoptosis/drug effects , Cell Adhesion/drug effects , Cell Proliferation/drug effects , Endothelial Cells/drug effects , GTP-Binding Proteins/physiology , Transglutaminases/physiology , Annexin A5/metabolism , Apoptosis/genetics , Cell Adhesion/genetics , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Survival/drug effects , Cells, Cultured , Down-Regulation/drug effects , Flow Cytometry , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Dyes/metabolism , Gene Knockdown Techniques , Humans , Phalloidine/metabolism , Protein Glutamine gamma Glutamyltransferase 2 , RNA Interference/drug effects , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Umbilical Veins/cytologyABSTRACT
BACKGROUND: Coeliac disease is caused by dietary gluten, which triggers chronic inflammation of the small intestine in genetically predisposed individuals. In one quarter of the patients the disease manifests in the skin as dermatitis herpetiformis. Recently, a novel candidate gene, myosin IXB on chromosome 19p13, was shown to be associated with coeliac disease in the Dutch and Spanish populations. The same gene has previously been associated with inflammatory bowel disease, systemic lupus erythematosus and rheumatoid arthritis risk, making myosin IXB a potential shared risk factor in these inflammatory disorders. METHODS: In this study, previously reported myosin IXB variants were tested for genetic linkage and association with coeliac disease in 495 Hungarian and Finnish families and in an additional 270 patients and controls. RESULTS AND CONCLUSION: The results show significant linkage (logarithm of odds (LOD) 3.76, p = 0.00002) to 19p13 which supports the presence of a genuine risk factor for coeliac disease in this locus. Myosin IXB variants were not associated with coeliac disease in this study; however, weak evidence of association with dermatitis herpetiformis was found. The association could not explain the strong linkage seen in both phenotypes, indicating that the role of other neighbouring genes in the region cannot be excluded. Therefore, more detailed genetic and functional studies are required to characterise the role of the myosin IXB gene in both coeliac disease and dermatitis herpetiformis.
Subject(s)
Celiac Disease/genetics , Dermatitis Herpetiformis/genetics , Myosins/genetics , Alleles , Case-Control Studies , Celiac Disease/complications , Chromosomes, Human, Pair 19/genetics , Dermatitis Herpetiformis/complications , Female , Finland , Genetic Predisposition to Disease , Genetic Variation , Glutens/adverse effects , Haplotypes , Homozygote , Humans , Hungary , Inflammatory Bowel Diseases/genetics , Linkage Disequilibrium , Male , Risk FactorsABSTRACT
Histone acetylation plays a key role in the regulation of gene expression. The chromatin structure and accessibility of genes to transcription factors is regulated by enzymes that acetylate and deacetylate histones. The Sin3A corepressor complex recruits histone deacetylases and in many cases represses transcription. Here, we report that SAP30L, a close homolog of Sin3-associated protein 30 (SAP30), interacts with several components of the Sin3A corepressor complex. We show that it binds to the PAH3/HID (Paired Amphipathic Helix 3/Histone deacetylase Interacting Domain) region of mouse Sin3A with residues 120-140 in the C-terminal part of the protein. We provide evidence that SAP30L induces transcriptional repression, possibly via recruitment of Sin3A and histone deacetylases. Finally, we characterize a functional nucleolar localization signal in SAP30L and show that SAP30L and SAP30 are able to target Sin3A to the nucleolus.
