An essential role for CCAAT/enhancer binding protein beta in bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is characterized by inflammation, genesis of myofibroblasts, and abnormal tissue repair. Despite extensive research, its pathogenesis remains incompletely understood. Previously, the transcription factor CCAAT/enhancer binding protein beta (C/EBPbeta) was found to be a key regulator of myofibroblast differentiation in vitro, and to be involved in the acute phase and inflammatory responses. In an attempt to test the role of C/EBPbeta in the development of pulmonary fibrosis, experiments using C/EBPbeta null mice and their wild-type littermates were conducted. Our findings indicated that, compared to wild-type mice, animals deficient in C/EBPbeta showed significantly reduced fibrotic lesions and collagen deposition in the lung upon endotracheal injection of bleomycin. Further studies on the mechanisms by which C/EBPbeta regulates fibrosis indicated that knockout of C/EBPbeta attenuates inflammatory cytokine expression in bleomycin-treated mice. The reduced alpha-smooth muscle actin gene expression in either isolated lung fibroblasts or lung tissue from bleomycin or saline-treated C/EBPbeta deficient mice suggests that C/EBPbeta regulates myofibroblast differentiation during fibrosis. Consistent with this finding, cells from C/EBPbeta deficient mice exhibited higher proliferative rates than those from wild-type mice. These data suggest that C/EBPbeta plays an essential role in pulmonary fibrosis and that this role appears to be multifactorial with respect to cytokine expression, cell differentiation, and proliferation.

Role of Smad3 in the regulation of rat telomerase reverse transcriptase by TGFbeta.

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 beta (TGFbeta) via yet unknown mechanisms. In this study, the role of Smad3 in the inhibition of telomerase reverse transcriptase (TERT) gene transcription by TGFbeta 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 TGFbeta inhibited rTERT transcription while stimulating Smad3 expression. Interestingly, TGFbeta 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 TGFbeta treatment. In contrast, mutation at a Smad binding element enhanced promoter activity whose inhibition was impaired by TGFbeta treatment. Thus TGFbeta 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.