Regulation of the connexin 43 promoter in the brook trout testis: Role of the thyroid hormones and cAMP

Gap junctions are critical for spermatogenesis. They are composed of integral proteins, the connexins. In mammals, a loss of Cx43 expression results in the inhibition of spermatogenesis. We have shown that Cx43 is expressed in the Sertoli cells of rainbow trout and that cAMP and triiodothyronine (T3) regulate testicular Cx43 expression in brook trout testis. The objective of this study was to determine if cAMP and T3 act at the level of the cx43 promoter to regulate its expression. A 607 bp 5′ flanking sequence of the cx43 promoter was obtained by Genome Walking. A TATA box was predicted to be located between positions −36 and −30 relative to the transcriptional initiation site. 5′-Rapid amplification of cDNA ends indicated a single transcriptional start site. Single C/EBP (−164 to −156) and tr-beta (−112 to −107) response elements were identified and electrophoretic mobility shift assays indicated the presence of competitive protein binding sites at each region. Immortalized rainbow trout gonadal cell line (RTG-2) which express cx43 and tr-beta transcripts were transfected with a vector containing the Cx43 promoter inserted into a luciferase expression vector. Transactivation of the reporter genes was stimulated by either cAMP or T3. Sequential deletion and point mutations in either the C/EBP or tr-beta response element indicated that T3 but not cAMP directly induced luciferase transactivation of the luciferase gene by acting on different sites of the Cx43 promoter. Together, these data indicate that T3 stimulates cx43 expression via direct regulation of gene transcription.

Research highlights
► Cloning of the brook trout Cx43 promoter by Genome Walking.
► Identification of a single transcriptional start site by rapid amplification of cDNA ends (RACE).
► Electrophoretic mobility shift assays showing thyroid hormone and cAMP-induced protein binding to the T3-receptor-beta and C/EBP response elements, respectively.
► Luciferase constructs transfected in RGT2 cells show transactivation of the Cx43 promoter by both cAMP and T3.
► Point mutations show that T3 but not cAMP directly regulates the transactivation of the Cx43 gene.