Fli-1 Inhibits Collagen Type I Production in Dermal Fibroblasts via an Sp1-dependent Pathway

Abstract

Fibrosis is characterized by the excessive deposition of extracellular matrix (ECM), especially collagen. Because Ets factors are implicated in physiological and pathological ECM remodeling, the aim of this study was to investigate the role of Ets factors in collagen production. We demonstrate that the expression of collagenous proteins and collagen ␣2(I) (COL1A2) mRNA was inhibited following stable transfection of Fli-1 in dermal fibroblasts. Subsequent analysis of the COL1A2 promoter identified a critical Ets binding site that mediates Fli-1 inhibition. In contrast, Ets-1 stimulates COL1A2 promoter activity. In vitro binding assays demonstrate that both Fli-1 and Ets-1 form DNA-protein complexes with sequences present in COL1A2 promoter. Furthermore, Fli-1 binding to the COL1A2 is enhanced via Sp1-dependent interaction. Studies using Fli-1 dominant interference and DNA binding mutants indicate that Fli-1 inhibition is mediated by both direct (DNA binding) and indirect (via protein-protein interaction) mechanisms and that Sp1 is an important mediator of the Fli-1 function. Furthermore, experiments using the Gal4 system in the context of different cell types as well as experiments with the COL1A2 promoter in different cell lines demonstrate that the direction and magnitude of the effect of Fli-1 is promoter-and cell context-specific. We propose that Fli-1 inhibits COL1A2 promoter activity by competition with Ets-1. In addition, we postulate that another factor (co-repressor) may be required for maximal inhibition after recruitment to the Fli-1-Sp1 complex. We conclude that the ratio of Fli-1 to Ets-1 and the presence of co-regulatory proteins ultimately control ECM production in fibroblasts.

Ets proteins in biological control and cancer

“…1). Furthermore, different Ets factors may have reciprocal functions: Ets1 and Fli1 differentially regulate the collagen α2(I) promoter [Czuwara-Ladykowska et al, 2001;Kubo et al, 2003]. Thus, the precise balance or "regulated imbalance" between cancer/metastasispromoting and-inhibiting Ets factor activities, which differentially regulate specific target genes, contributes to tissue homeostasis or tumor progression, respectively.…”

“…Significantly, our recent studies have identified two novel Ets targets that implicate Ets functions in stroma remodeling: tenascin-C and collagen type I [Shirasaki et al, 1999;Czuwara-Ladykowska et al, 2001, 2002Jinnin et al, in press]. Interestingly, the activities of Fli1 and Ets1 toward the expression of these target genes are modulated by acetylation in a TGFβ-dependent manner.…”

Persistent Down-Regulation of Fli1, a Suppressor of Collagen Transcription, in Fibrotic Scleroderma Skin

“…Our previous studies, 20 as well as experiments using adenoviral expression of Fli1, strongly suggest that Fli1 is a potent inhibitor of collagen biosynthesis. We therefore asked whether the signaling pathways known to modulate collagen gene expression control expression levels of Fli1.…”

“…20 To gain further insights into the Fli1 function, an adenoviral vector expressing Fli1 was generated to ensure efficient gene delivery into dermal fibroblasts. To assess the effect of Fli1 on newly synthesized collagenous proteins at the basal level and in response to TGF-␤, conditioned media from cells metabolically labeled with 3 H-proline were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.…”

“…20 Fli1, a member of the Ets family of transcription factors, has been shown to play roles in hematopoiesis, embryonic development, and vasculogenesis. [21][22][23][24] Collective evidence indicates that Ets transcription factors are the important mediators of cellular programs involved in extracellular matrix degradation, 25 and are frequently dysregulated in diseases characterized by abnormal matrix turnover, including invasive tumors and arthritis.…”