Role of Smad3 in the regulation of rat telomerase reverse transcriptase by TGFβ

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Abstract
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 transforming growth factor β (TGFβ) via yet unknown mechanisms. In this study, the role of Smad3 in the inhibition of telomerase reverse transcriptase (TERT) gene transcription by TGFβ 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 fibroblasts isolated from bleomycin-injured lung with induced telomerase activity. The results showed that TGFβ inhibited rTERT transcription while stimulating Smad3 expression. Interestingly, TGFβ 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 TGFβ treatment. In contrast, mutation at a Smad binding element enhanced promoter activity whose inhibition was impaired by TGFβ treatment. Thus TGFβ 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.