Elevated connexin-43 expression in term human myometrium correlates with elevated c-Jun expression and is independent of myometrial estrogen receptors.

Abstract

Just previous to the onset of parturition, a number of genes such as the one that codes for connexin-43 (Cx43) gap junction protein are induced in the myometrium. We have shown previously that activation of protein kinase C in human myometrial cultured cells leads to an up-regulation of cx43 transcription through an activating protein-1 element in the 5'-flanking promoter. Analyses were now performed on extracts of term myometrial tissue to test for an association between the up-regulation of cx43 expression and the expression of transcription factors and steroid hormone receptors that might regulate cx43 expression at term. Immunoblot analyses were performed on extracts of term myometrial tissue from women receiving elective or indicated cesarean sections to test for an association between the up-regulation of cx43 expression and the up-regulation of expression of the transcription factors c-Jun, c-Fos, and Sp1, which have cognate binding elements in the cx43 5'-flanking promoter. Immunoblot analysis, immunohistochemistry, and receptor binding assays were also performed to analyze the levels of progesterone receptors (PR) and estrogen receptors (ER) in the same term myometrial tissue, and these were compared to the levels in nonpregnancy myometrial tissue. The levels of PR were consistently 2- to 3-fold higher in term myometrial tissue than in nonpregnancy values and did not fluctuate during the menstrual cycle as did ER levels. Surprisingly, in term myometrium, ER was barely detectable by immunoblot and had whole cell diffuse staining by immunohistochemistry. In addition, very low levels of estrogen binding were observed in the term myometrial tissue. Treatment of primary myometrial cultures containing ER with estrogen for 3 or 48 h did not result in up-regulation of c-Jun or c-Fos proteins or in trans-activation from the proximal cx43 promoter with the activating protein-1 element. In contrast, an activated form of c-Jun protein was 10- to 18-fold higher in term myometrial tissue that also had elevated cx43 expression compared to c-Jun levels in term myometrial tissue with low cx43 expression. Likewise, c-Fos and Sp1 levels were 2-4 fold higher in term myometrial tissue with elevated cx43 expression. Although c-Fos and Sp1 proteins could be detected by immunoblot in myometrial tissue from nonpregnant women, c-Jun and Cx43 proteins could not. In summary, these results suggest that up-regulation of human myometrial cx43 gene expression at term involves induction of primarily c-jun expression through a mechanism that does not directly involve myometrial ER or the loss of PR. Peptide hormones that activate protein kinase cascades, such as the protein kinase C cascade, may be important to signal the onset of labor in humans.