Abstract 8558: Mechanical Stretch Induces the Apoptosis Regulator Puma in Vascular Smooth Muscle Cells

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Aims The expression of PUMA (p53-upregulated modulator of apoptosis), an apoptosis regulated gene, increases during endoplasmic reticulum (ER) stress. How mechanical cyclic stretch affects the regulation of PUMA in vascular smooth muscle cells (VSMCs) during apoptosis is not fully understood. We aimed to test the hypothesis that cyclical stretch enhances PUMA expression in VSMCs undergoing apoptosis. Methods and results Human VSMCs grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. An in vivo model of aorta-caval shunt in adult rats was used to investigate PUMA expression. Cyclic stretch significantly enhanced PUMA protein and mRNA expression after 18 h of stretch. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125 and interferon-γ (IFN-γ) antibody 30 min before stretch inhibited the induction of PUMA protein. Gel shift assay showed that DNA-binding activity of Interferon Regulatory Factor-1 (IRF-1) increased after stretch. SP600125, JNK siRNA and IFN-γ antibody abolished the binding activity induced by stretch. Stretch increased while PUMA-Mut plasmid, SP600125, and IRF-1 antibody abolished the promoter activity. Cyclic stretch significantly increased the interferon-γsecretion from VSMCs at 1 h and remained elevated for 6 h. Both conditioned media from stretched VSMCs and exogenous administration of IFN-γrecombinant protein to the non-stretched VSMCs increased PUMA protein expression similar to that seen after stretch. Cells in the sub-G1 fraction stained with propidium iodide and caspase 3 activity was elevated after stretch for 18 h and addition of interferon-γ. Addition of PUMA and JNK siRNA abolished the induction of sub-G1 and caspase 3 activity induced by stretch. The increases of TUNEL-positive nuclei in VSMCs induced by stretch were significantly reversed by PUMA and JNK siRNA. An in vivo model of aorta-caval shunt in adult rats also demonstrated the increased PUMA protein expression in the aorta. Conclusion Cyclic stretch enhanced PUMA expression in cultured human VSMCs. The stretch-induced PUMA is mediated by IFN-γ,JNK and IRF-1 pathway. These findings suggest that PUMA plays a role in stretch-induced VSMC apoptosis.