Martha J. Fernstrom

Liver cell-specific transcriptional regulation of connexin32

Gap junctional intercellular communication facilitates liver homeostasis and growth control in the liver. The major gap junction protein expressed by hepatocytes is connexin32 (Cx32) and non-parenchymal hepatic cells do not express this gene. We investigated the regulation of Cx32 transcription by trans-activating factors in liver cells. Transient transfection assays using deletions of the rat Cx32 promoter (nt -753 to -33) linked to the luciferase gene were performed in MH1C1 rat hepatoma cells that express endogenous Cx32 compared with WB-F344 rat liver epithelial cells that do not. The basal promoter element was located within nt -134 to -33 and was 1.4-fold more active in MH1C1 cells than WB-F344 cells whereas the entire promoter fragment (nt -754 to -33) was four-fold more active in MH1C1 cells. Specific nuclear protein-DNA complexes that bound to Sp1 consensus sites within the basal promoter were formed using nuclear extracts from both types of cells. Additional promoter sequences increased promoter activity more strongly in MH1C1 cells than WB-F344 cells and this was correlated with the binding of hepatocyte nuclear factor-1 (HNF-1) to two HNF-1 consensus sites centered at -187 and -736. Expression of HNF-1 and binding to these elements was only observed with MH1C1 cells. Other specific protein-DNA complexes were formed, however, that included YY-1- and NF-1-containing complexes, but these were not related to promoter activity. Dexamethasone increased Cx32 promoter activity and expression in MH1C1 cells, but had little effect in WB-F344 cells and did not alter protein-DNA complex formation. These data suggest that Sp1 is responsible for Cx32 promoter basal activity, that HNF-1 determines the cell-specific expression of Cx32, and that dexamethasone increases Cx32 expression through other mechanisms.

Positive regulation of connexin32 transcription by hepatocyte nuclear factor-1α

Connexin32 (Cx32) encodes the predominant gap junction protein expressed by hepatocytes. We investigated the transcriptional control of Cx32 in expressing and nonexpressing rat liver cell lines and hypothesized that a putative hepatocyte nuclear factor-1 (HNF-1) binding site (centered at mp -187) in the liver-active, P1 promoter is essential for transcription of Cx32. HNF-1alpha was expressed by Cx32-expressing rat liver cell lines and bound the promoter at the -187 site, but was not expressed by non-Cx32-expressing hepatic lines. Stable transfection of non-Cx32-expressing WB-F344 rat liver epithelial cells with HNF-1alpha stimulated a transfected Cx32 promoter element (mp -244 to -33), binding of HNF-1alpha to the -187 site, and expression of endogenous Cx32. Site-directed mutagenesis of this HNF-1 binding site abolished HNF-1alpha binding and proximal promoter activity. Hepatic Cx32 expression was also significantly decreased in HNF-1alpha(-/-) mice. These data indicate that HNF-1alpha is a positive regulator of Cx32 expression in hepatic cells.

Lack of growth inhibition or enhancement of gap junctional intercellular communication and connexin43 expression by β-carotene in murine lung epithelial cells in vitro

The carotenoid, beta-carotene, has been examined in human trials as a possible lung cancer chemopreventive agent, but initial results indicate that the compound is ineffective. Here we have considered whether beta-carotene could enhance gap junctional intercellular communication (GJIC) and affect the growth of lung epithelial cells, since these effects may be involved in the carotenoids chemopreventive actions. In accordance with its lack of lung cancer chemopreventive activity, beta-carotene (1-10 microM; 1-5 days treatment durations) did not affect GJIC, gap junction protein (connexin43; Cx43) expression, or growth in vitro of non-transformed (C10) or neoplastic (E9 and 82-132) murine lung epithelial cells. beta-Carotene enhanced GJIC and Cx43 expression and reduced the growth of C3H10T1/2 murine fibroblasts, however. These data indicate that the effects of beta-carotene on GJIC and growth are cell-specific which may partly explain why the carotenoid is an ineffective lung cancer chemopreventive agent.