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Oral communications
O1 CTGF EXPRESSION IS SENSITIVE TO CHANGES OF THE ACTIN CYTOSKELETON IN ENDOTHELIAL CELLS
S. Muehlich1, B. Krueger1, C. D. Garlichs2, M. Goppelt-Struebe1.1Medical Clinic IV and 2Medical Clinic II, University of Erlangen-Nuernberg, Erlangen, Germany
CTGF has been shown to be upregulated in human umbilical vein endothelial cells (HUVEC) under turbulent flow conditions. This prompted us to investigate the molecular mechanisms that might link changes in cell morphology and CTGF gene expression.
CTGF expression was studied in primary cultures of HUVEC. Manipulation of the actin cytoskeleton revealed a reverse correlation between CTGF expression and the cellular concentration of monomeric G-actin: when the actin cytoskeleton was disrupted by latrunculin, CTGF mRNA and protein synthesis decreased. When monomeric G-actin was reduced by enhanced formation of F-actin stress fibres by yasplakinolide, CTGF synthesis was increased. Similarly, disruption of microtubuli by colchicine and the subsequent increase in F-actin strongly enhanced CTGF synthesis.
To provide direct evidence for the role of G-actin in CTGF expression, endothelial cells were transfected with a construct encoding mutated G-actin, which was no longer able to polymerise into F-actin. This construct was kindly provided by G Posern and R Treisman (London, UK). Compared with mock transfected cells, actin transfected cells showed a reduced number of stress fibres. Endothelial cells are poorly transfectable cells. Therefore, the effect of the mutated G-actin on CTGF expression was assessed in individual cells by immunocytochemistry. CTGF was located to Golgi stacks. Comparison of transfected cells with neighbouring non-transfected cells showed a strong reduction of CTGF in transfected cells, which was not seen when the cells were transfected with an irrelevant vector. The molecular mechanism by which G-actin interferes with CTGF synthesis remains to be elucidated.
Similar data were obtained in a murine microvascular endothelial cell line and also in fibroblasts, indicating that independent of the cell type, CTGF expression …
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↵* These authors contributed equally to this work.