Role of Smad3 in the regulation of rat telomerase reverse transcriptase byTGFβ

Telomerase is induced in certain pathological conditions such as cancer and tissue injury and repair. This induction in fibroblasts from injured lung is repressed by trans forming growth factor beta(TGF beta) via yet unknown mechanisms. In this study, the role of Smad3 in the inhibition of telomerase reverse transcriptase ( TERT) gene transcription by TGF beta was investigated. The rat TERT (rTERT) gene promoter was cloned by PCR amplification and fused with a luciferase reporter gene. This construct was used to analyse regulation of promoter activity in. broblasts isolated from bleomycin-injured lung with induced telomerase activity. The results showed that TGFb inhibited rTERT transcription while stimulating Smad3 expression. Interestingly, TGFb also inhibited the expression of c-myc. Cotransfection with a Smad3 expressing plasmid further repressed rTERT transcription and c-myc expression, while cotransfection with the corresponding antisense Smad3 construct had the opposite effect. Mutation of an E-box in the rTERT promoter suppressed its activity, which could be further reduced by TGF beta treatment. In contrast, mutation at a Smad binding element enhanced promoter activity whose inhibition was impaired by TGF beta treatment. Thus TGFb inhibition of rTERT gene expression was directly mediated by Smad3 via the Smad binding element, while c-myc appears to primarily regulate its constitutive or induced expression.