Regulation of Vascular Connexin43 Gene Expression by Mechanical Loads

Abstract

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 (ϩ162) to Ϫ1686 in the 5Ј-flanking region were coupled to the chloramphenicol acetyl transferase reporter gene. Deletions from the 5Ј 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 Ϫ42 to Ϫ48 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 Ϫ1686 to ϩ162 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 Ϸ4-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. (Circ Res. 1998;82:786-793.)

Control and Coordination of Vasomotor Tone in the Microcirculation

“…It is important to note that Gap27 and 18α-glycyrrhetinic acid are two well-known gap junction blockers, but they also block connexin-formed hemichannels, which indicates that hemichannels may also be involved in the development of the myogenic response. In any case, the involvement of Cx43-based channels in the control of vasomotor tone is consistent with the finding that tensile stretch increased the expression of this connexin as well as gap junction intercellular communication in vascular smooth muscle cells (Cowan et al, 1998). Interestingly, this response was mediated by the formation of reactive oxygen species (Cowan et al, 1998;Cowan et al, 2003), which has been reported to contribute to the initiation of the myogenic constriction in mouse-tail arterioles (Nowicki et al, 2001).…”

Multiple Connexin Expression in Regenerating Arterial Endothelial Gap Junctions

“…Apart from mechanical injury, other factors are reported to affect endothelial connexins. For example, increased mechanical load enhances Cx43 expression, 23 and treatment with tumor necrosis factor-␣, a cytokine promoting endothelial cell migration, leads to upregulation of Cx43 and downregulation of Cx37 and Cx40 in human umbilical vein endothelial cells. 25 In addition, Cx43 and Cx37 in cultured bovine aortic endothelial cells are reported to be differentially regulated by cell density and growth status 21 ; although levels of Cx43 mRNA and protein were high in subconfluent cells and low in confluent cells, the opposite applied to Cx37 mRNA, which was present only in low amounts until the cultures became confluent.…”

“…6,18 -21 The specific pattern of connexin expression may also be differentially affected by physical and chemical factors, such as mechanical load, blood sugar level, growth factors, and cytokines. [21][22][23][24][25] However, current knowledge about the role of each connexin in the endothelial cell is limited, and most studies have been limited to examination of 1 or 2 connexins. Previous studies of regenerating vascular endothelium in animal models have been confined to morphometric measurement of the size and number of gap junctions, and corresponding in vitro studies have been confined to Cx43.…”

Mechanotransduction in endothelial cell migration

“…In gap junctions, laminar shear stress transiently increases the expression of connexin 43, while disturbed flow sustains the induction of connexin 43 but disorganizes the cell-cell communication [Cowan et al, 1998;Gabriels and Paul, 1998;DePaola et al, 1999]. In tight junctions, shear stress increases occludin phosphorylation and decreases occludin expression, which may be responsible for the increase of hydraulic conductivity of an EC monolayer [DeMaio et al, 2001].…”