Abstract
Transforming growth factor-beta (TGF-beta) stimulates collagen synthesis and accumulation, and aberrant TGF-beta signaling is implicated in pathological organ fibrosis. Regulation of type I procollagen gene (COL1A2) transcription by TGF-beta involves the canonical Smad signaling pathway as well as additional protein and lipid kinases, coactivators, and DNA-binding transcription factors that constitute alternate non-Smad pathways. By using Affymetrix microarrays to detect cellular genes whose expression is regulated by Smad3, we identified early growth response factor-1 (EGR-1) as a novel Smad3-inducible gene. Previous studies implicated Egr-1 in cell growth, differentiation, and survival. We found that TGF-beta induced rapid and transient accumulation of Egr-1 protein and mRNA in human skin fibroblasts. In transient transfection assays, TGF-beta stimulated the activity of the Egr-1 gene promoter, as well as that of a minimal Egr-1-responsive reporter construct. Furthermore, TGF-beta enhanced endogenous Egr-1 interaction with a consensus Egr-1-binding site element and with GC-rich DNA sequences of the human COL1A2 promoter in vitro and in vivo. Forced expression of Egr-1 by itself caused dose-dependent up-regulation of COL1A2 promoter activity and further enhanced the stimulation induced by TGF-beta. In contrast, the TGF-beta response was abrogated when the Egr-1-binding sites of the COL1A2 promoter were mutated or deleted. Furthermore, Egr-1-deficient embryonic mouse fibroblasts showed attenuated TGF-beta responses despite intact Smad activation, and forced expression of ectopic EGR-1 in these cells could restore COL1A2 stimulation in a dose-dependent manner. Taken together, these findings identify Egr-1 as a novel intracellular TGF-beta target that is necessary for maximal stimulation of collagen gene expression in fibroblasts. The results therefore implicate Egr-1 in the profibrotic responses elicited by TGF-beta and suggest that Egr-1 may play a new and important role in the pathogenesis of fibrosis.
Highlights
The multifunctional cytokine transforming growth factor- (TGF-) is a key signal for regulation of connective tissue metabolism
Identification and Validation of early response gene-1 (EGR-1) as a Novel SMAD3 Target Gene in Fibroblasts—To identify cellular genes whose expression is directly induced by Smad3 in normal fibroblasts, we established a novel system for tightly regulated Smad3 expression
Increased Smad3 accumulation in isopropyl -thiogalactoside (IPTG)-induced fibroblasts was associated with its phosphorylation and nuclear accumulation, whereas no Smad3 activation could be demonstrated in human fibroblasts immortalized with telomerase reverse transcriptase (hTERT) fibroblasts transfected with empty vector (Fig. 1, A and B)
Summary
The multifunctional cytokine transforming growth factor- (TGF-) is a key signal for regulation of connective tissue metabolism. Each appears to contribute to translating TGF- signals into distinct biological responses in a cell type- and target genespecific manner These alternate signaling pathways may mediate TGF- responses completely independent of Smads, or they may modulate the amplitude and duration of ligand-dependent Smad activation or engage with the Smad pathway through intracellular cross-talk. Smads can modulate the expression and activity of other signaling pathways or induce additional transcription factors In light of these emerging observations indicating a high degree of complexity, a better understanding of the interactions of Smads with other signaling proteins, including those that may be downstream targets of Smads and contribute to TGF- signaling, is of clear significance. The in vivo expression of Egr-1 in skin fibroblasts was markedly elevated by TGF- injection in mice Together, these results identify Egr-1 as a novel TGF-/Smad target that up-regulates the expression of collagen genes and plays a critical role in mediating TGF- stimulation. Egr-1 appears to be a new and important component of the fibrotic process
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