Promoter activity of the rat connexin 43 gene in NRK cells.

Cellular communication mediated by gap junctions plays a major role in organ function. Gap junction channels are formed by the organization of polypeptide subunits, termed connexins (Cx), on the cell surface of adjacent cells. One mechanism to regulate gap-junctional communication is by change in Cx expression. In the present study, the promoter region of the rat Cx43 gene was characterized. Nested deletions of the 5' flanking region of the first Cx43 exon were coupled to the human growth hormone gene and transfected into normal rat kidney (NRK) cells, that express this gene constitutively. The minimal region of the Cx43 gene that showed maximal promoter activity was localized within 110 bp upstream of the transcriptional initiation site. One particular subregion that contains a Sp-1 binding site (located within 98--93 bp from the transcriptional initiation site) was found to sustain Cx43 promoter activity to the same extent as that of the 110 bp promoter region. Mutations of this Sp-1 binding site abolished transcriptional activity and DNA-protein interactions. These observations suggest that the Sp-1 binding site plays a major role in the basal transcriptional activity of Cx43 gene in NRK cells.

Expression of HSP70 is impaired at the transcriptional level in stressed murine neuroblastoma cells.

Although the expression of heat shock or stress proteins (hsps) is a well conserved response to stress, the accumulation of these proteins is different between various cell-types. Particularly, cells of neuronal origin show a reduced expression of Hsp70 after stress. The possible mechanism of this reduced Hsp70 expression was studied in thermally stressed murine neuroblastoma cells (N18). These cells showed no detectable levels of Hsp70 or Hsp70 mRNA after heat shock. Hsp70 transcription was not detectable after stress. However, heat shock transcription factor 1 (HSF1) is active in these cells under stress conditions. Cells transiently transfected with the chloramphenicol acetyltransferase (CAT) gene under control of the human heat shock promoter showed a stress-dependent expression of CAT, suggesting that the cells contain the factors necessary for the expression of Hsp70. Integration of the foreign human heat shock promoter into genomic DNA did not affect its transcriptional inducibility. These results suggest that the impairment of Hsp70 expression in N18 cells is due to the environment (chromatin structure, methylation pattern) of the Hsp70 locus.

Thermotolerant cells show an attenuated expression of Hsp70 after heat shock.

Expression of heat shock proteins (hsps) results in the protection of cells from subsequent stresses. However, hsps are also toxic when present within cells for a prolonged time period. Thus, the expression of hsps should be tightly regulated. In the present study, the expression of Hsp70 after heat shock was compared between thermotolerant cells, which contain a large concentration of Hsp70, and nonthermotolerant cells (naive). Accumulation of Hsp70, assessed by Western blotting, was negligible when thermotolerant cells were heat-shocked a second time. Hsp70 transcription was similar between thermotolerant and naive cells during heat shock. However, Hsp70 transcription was attenuated more rapidly in thermotolerant than naive cells immediately upon return to non-heat shock conditions. In addition, Hsp70 mRNA stability was reduced in thermotolerant cells as compared with naive cells following the stress. New synthesis of Hsp70 and the efficiency of Hsp70 mRNA translation were similar between thermotolerant and naive cells during the post-stress period. These results suggest that thermotolerant cells limit Hsp70 expression by transcriptional and pretranslational mechanisms, perhaps to avoid the potential cytotoxic effect of these proteins.

Downregulation of connexin 43 gene expression in rat heart during inflammation. The role of tumour necrosis factor.

Gap junctions form channels that mediate the communication between adjacent cells. Alterations in gap junction function and/or expression are believed to contribute to cardiac dysfunction such as those observed in septic patients. The expression of connexin 43 (Cx43), the subunit component of the most abundant cardiac gap junction, was analysed in rat heart during inflammation induced by the administration of bacterial lipopolysaccharide (LPS). Cx43 mRNA levels were found to be dramatically (>50%) and rapidly (2 h) reduced in the heart after injection of LPS (1 mg/kg). To investigate the possible mechanism of the decrease in Cx43 expression during inflammation, the promoter region of this gene was cloned. The basal Cx43 promoter activity was observed within 224-134 bp of the transcriptional initiation site after transfection into a rat myoblast cell-line (H9c2). The Cx43 promoter activity was found to be reduced by incubation of the transfected cells with serum obtained from LPS-treated rats. Moreover, Cx43 promoter activity was also decreased upon incubation with tumour necrosis factor alpha. These results suggest that Cx43 expression in the heart can be modulated by circulating cytokines. These observations may have important implications in the depression of heart function observed in septic patients.