Smad-Independent Transforming Growth Factor-β Regulation of Early Growth Response-1 and Sustained Expression in Fibrosis

Abstract

Transforming growth factor-␤ (TGF-␤) plays a key role in scleroderma pathogenesis. The transcription factor early growth response-1 (Egr-1) mediates the stimulation of collagen transcription elicited by TGF-␤ and is necessary for the development of pulmonary fibrosis in mice. Here, we report that TGF-␤ causes a time-and dose-dependent increase in Egr-1 protein and mRNA levels and enhanced transcription of the Egr-1 gene via serum response elements in normal fibroblasts. The ability of TGF-␤ to stimulate Egr-1 was preserved in Smad3-null mice and in explanted Smad3-null fibroblasts. The response was blocked by a specific mitogen-activated protein kinase kinase 1 (MEK1) inhibitor but not by an ALK5 kinase inhibitor. Furthermore, MEK1 was phosphorylated by TGF-␤, which was sufficient to drive Egr-1 transactivation. Stimulation by TGF-␤ enhanced the transcriptional activity of Elk-1 via the MEK-extracellular signal-regulated kinase 1/2 pathway. Bleomycininduced scleroderma in the mouse was accompanied by increased Egr-1 accumulation in lesional fibroblasts. Furthermore, biopsies of lesional skin and lung from patients with scleroderma showed increased Egr-1 levels, which were highest in early diffuse disease. Moreover, both Egr-1 mRNA and protein were elevated in explanted scleroderma skin fibroblasts in vitro. Together, these findings define a Smadindependent TGF-␤ signal transduction mechanism that underlies the stimulation of Egr-1, demonstrate for the first time sustained Egr-1 up-regulation in fibrotic lesions and suggests that Egr-1 has a role in the induction and progression of fibrosis.

The Early Growth Response Gene Egr2 (Alias Krox20) Is a Novel Transcriptional Target of Transforming Growth Factor-β that Is Up-Regulated in Systemic Sclerosis and Mediates Profibrotic Responses

“…Each of these molecules is known to be abnormally expressed, and play a role, in the pathogenesis of fibrosis and SSc. 49 In previous studies, 14 Egr-1 similarly induced the expression of fibrotic genes.…”

“…14,18 Most primer pairs span two exons, except the intronless 18S ribosomal RNA. Primers were tested by dissociation curve analysis to ensure only a single amplicon.…”

“…14 In the present studies, we focused our attention on the regulation of Egr-2. The results of qPCR showed that TGF-␤2 stimulated Egr-2 mRNA expression in foreskin fibroblasts in a time-and dose-dependent manner ( Figure 1, A and B).…”

“…In previous studies 14 investigating the expression and function of Egr-1 in the context of fibrogenesis, we showed that Egr-1 was required for TGF-␤-induced fibroblast activation and played an important role in both physiological and pathological fibrogenesis. The expression of Egr-1 was rapidly induced by TGF-␤ in skin and lung fibroblasts, and Egr-1 was sufficient to stimulate the transcription of COL1A2 and other TGF-␤-inducible genes.…”

“…The expression of Egr-1 was rapidly induced by TGF-␤ in skin and lung fibroblasts, and Egr-1 was sufficient to stimulate the transcription of COL1A2 and other TGF-␤-inducible genes. 14,15 Moreover, mice with targeted deletion of Egr-1 were protected from bleomycin-induced fibrosis. 16 In contrast to Egr-1, the expression, regulation, and physiological roles of Egr-2 in connective tissue metabolism remain incompletely understood.…”