Regulation of Vascular Connexin43 Gene Expression by Mechanical Loads

From The Toronto Hospital Research Institute (B.L.L., D.B.C.), the Samuel Lunenfeld Research Institute (S.J.L.), Mount Sinai Hospital, and the Department of Laboratory Medicine and Pathobiology (D.B.C., B.L.L.), the Department of Physiology (D.B.C., S.J.L.), and the Department of Obstetrics and Gynecology (S.J.L., B.L.L.), the University of Toronto (Canada).

Correspondence to B. Lowell Langille, PhD, CCRW 1–836, The Toronto Hospital (General Division), 200 Elizabeth St, Toronto, Ontario M5G 2C4, Canada. E-mail lowell.langille{at}utoronto.ca

Abstract—Vascular tissues respond to changes in the mechanical forces imposed on them with changes in vasomotor tone in the short term and with structural remodeling in the long term. Since these responses involve intercellular communication, we have investigated regulation of the gap junction proteins, connexin26 (Cx26), connexin37 (Cx37), connexin40 (Cx40), and connexin43 (Cx43), by mechanical loads. Results were compared with parallel experiments on c-fos and GAPDH. Twenty percent stretch of cultured vascular smooth muscle cells caused a 3-fold increase in Cx43 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-fos mRNA levels increased after 30 minutes of stretch, whereas GAPDH mRNA did not change. Protein levels of Cx43 increased by 4 hours and remained elevated for 16 hours. Nuclear run-on experiments confirmed that Cx43 and c-fos were transcriptionally regulated by stretch. New protein synthesis was not a requirement for the stretch-induced rise in Cx43 expression, since mRNA levels were unaffected by treatment with cycloheximide. To examine transcriptional control of Cx43, stretched and unstretched vascular smooth muscle cells were transfected with a variety of promoter-reporter gene constructs. Cx43 sequences extending from within exon 1 (+162) to -1686 in the 5'-flanking region were coupled to the chloramphenicol acetyl transferase reporter gene. Deletions from the 5' end of these sequences differentially regulated reporter gene expression and indicated multiple potential regulatory sites. In particular, a putative activator protein-1 site at the -42 to -48 region was required for basal reporter activity. None of the promoter constructs revealed stretch sensitivity, indicating that the site of transcriptional control 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/cm². Cx43 mRNA levels increased by 4-fold at 1 hour and remained elevated for the duration of shear force. In conclusion, both mechanical strain and fluid shear stress caused increased expression of the gap junction protein Cx43.

Key Words: vascular remodeling • gap junction • cell stretch • shear stress

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