REGULATION OF VASCULAR CONNEXIN43 GENE-EXPRESSION BY MECHANICAL LOADS

Vascular tissues respond to changes in the mechanical forces imposed o n them with changes in vasomotor tone in the short term and with struc tural remodeling in the long term. Since these responses involve inter cellular communication, we have investigated regulation of the gap jun ction proteins, connexin26 (Cx26), connexin37 (Cx37), connexin40 (Cx40 ), and connexin43 (Cx43), by mechanical loads. Results were compared w ith parallel experiments on c-fos and GAPDH. Twenty percent stretch of cultured vascular smooth muscle cells caused a 3-fold increase in Cx4 3 mRNA levels by 2 hours. Cx26 was expressed at low levels but failed to respond to stretch, and Cx37 and Cx40 were not detected. c-Sos mRNA levels increased after 30 minutes of stretch, whereas GAPDH mRNA did not change. Protein levels of Cx43 increased by 4 hours and remained e levated for 16 hours. Nuclear run-on experiments confirmed that Cx43 a nd c-fos were transcriptionally regulated by stretch, New protein synt hesis was not a requirement for the stretch-induced rise in Cx43 expre ssion, since mRNA levels were unaffected by treatment with cycloheximi de, To examine transcriptional control of Cx43, stretched and unstretc hed vascular smooth muscle cells were transfected with a variety of pr omoter-reporter gene constructs. Cx43 sequences extending from within exon 1 (+162) to -1686 in the 5'-flanking region were coupled to the c hloramphenicol acetyl transferase reporter gene. Deletions from the 5' end of these sequences differentially regulated reporter gene express ion and indicated multiple potential regulatory sites. in particular, a putative activator protein-1 site at the -42 to -48 region was requi red for basal reporter activity. None of the promoter constructs revea led stretch sensitivity, indicating that the site of transcriptional c ontrol by stretch lies outside the -1686 to +162 region, Finally, Cx43 mRNA levels were assessed in cultured endothelial cells subjected to laminar shear stress of 15 dynes/cm(2). Cx43 mRNA levels increased by approximate to 4-fold at 1 hour and remained elevated for the duration of shear force. In conclusion, both mechanical strain and fluid sheer stress caused increased expression of the gap junction protein Cx43.