The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

By stimulating collagen synthesis and myofibroblasts differentiation, transforming growth factor-β (TGF- β) plays a pivotal role in tissue repair and fibrosis. The early growth response-1 (Egr-1) transcription factor mediates profibrotic TGF-β responses, and its expression is elevated in biopsies from patients with scleroderma. NGF1-A-binding protein 2 (Nab2) is a conserved transcriptional cofactor that directly binds to Egr-1 and positively or negatively modulates Egr-1 target gene transcription. Despite the recognized importance of Nab2 in governing the intensity of Egr-1-dependent responses, the regulation and function of Nab2 in the context of fibrotic TGF-β signaling is unknown. Here we show that TGF-β caused a time-dependent stimulation of Nab2 protein and mRNA in normal fibroblasts. Ectopic expression of Nab2 in these cells blocked Egr-1-dependent transcriptional responses, and abrogated TGF-β-induced stimulation of collagen synthesis and myofibroblasts differentiation. These inhibitory effects of Nab2 involved recruitment of the NuRD chromatin remodeling complex to the COL1A2 promoter and were accompanied by reduced histone H4 acetylation. Mice with targeted deletion of Nab2 displayed increased collagen accumulation in the dermis, and genetic or siRNA-mediated loss of Nab2 in fibroblasts was associated with constitutively elevated collagen synthesis and accentuation of Egr-1-dependent TGF-β responses in vitro. Expression of Nab2 was markedly up-regulated in skin biopsies from patients with scleroderma, and was localized primarily to epidermal keratinocytes. In contrast, little Nab2 could be detected in dermal fibroblasts. These results identify Nab2 as a novel endogenous negative regulator of Egr-1-dependent TGF-β signaling responsible for setting the intensity of fibrotic responses. Defective Nab2 expression or function in dermal fibroblasts might play a role in persistent fibrotic responses in scleroderma

Clinical characteristics of scleroderma patients studied.

<p>M, male; F, female.</p

TGF-β stimulates Nab2 expression.

<p>Confluent cultures of human foreskin fibroblasts were incubated with TGF-β1 (10 ng/ml) for indicated periods. A, D Total RNA was isolated and analysed by real-time quantitative PCR. Results, normalized with actin, are the means±S.D. of triplicate determinations from a representative experiment. B. Whole cell lysates were subjected to Western analysis. Representative immunoblots. C. Fibroblasts were fixed and stained with anti-Nab2 antibodies, or DAPI to detect nuclei, and viewed by immunofluorescence microscopy (original magnification ×100).</p

Nab2 blocks Egr-1-dependent collagen stimulation.

<p>A. Foreskin fibroblasts were cotransfected with expression vector for wildtype or mutant Egr-1 along with Nab2 or empty vector, and 376COL1A2-luc_reporter constructs. Following incubation of cultures for 24 h, cell lysates were prepared and assayed for their luciferase activities. Results, normalized with Renilla luciferase, are expressed as means±S.D. of triplicate determinations. *p<0.005. B. Foreskin fibroblasts were infected with Ad-Egr-1m along with Ad-Nab2. Following 24 h incubation, whole cell lysates were prepared and subjected to Western analysis. Representative immunoblots.</p

Enhanced collagen stimulation in Nab2-deficient fibroblasts.

<p>Wildtype and Nab2^−/− mouse embryonic fibroblasts (MEFs) cultured in parallel were incubated with or without TGF-β1 for 24 h. A. Whole cell lysates and culture supernatants were subjected to Western analysis. Representative immunoblots. B. Total RNA was subjected to real-time qPCR analysis. Results, expressed relative to 18 s, are the means±S.D. of triplicate determinations from a representative experiment. Open boxes, untreated fibroblasts; closed boxes, TGF-β-treated fibroblasts. *p<0.005. C. MEFs were transfected with pEBS₄-luc inpresence or absence of Nab2. Following 24 h incubation, cultures were harvested and cell lysates were assayed for their luciferase activities. The results, normalized with Renilla luciferase, are the means±S.D. of triplicate determinations. D. siRNA knockdown. Normal dermal fibroblasts were transfected with Nab2 siRNA or irrelevant negative control siRNA, and incubated with TGF-β. Twenty-four h later, fibroblasts were harvested. Levels of Nab2 and collagen were determined by Western analysis of whole cell lysates.</p

Oligonucleotides used for Real-Time Quantitative PCR.

<p>Oligonucleotides used for Real-Time Quantitative PCR.</p

Increased skin collagen accumulation in Nab2 ^−/− mouse.

<p>Skin from Nab2^−/− mice and wildtype mice was harvested. A. Tissues were stained with hematoxylin and eosin (a–d) or picrosirius red (e,f). Original magnification ×100 (a, b), ×400 (c–f). B. Immunofluorescence using antibodies to α-smooth muscle actin (green). Nuclei were identified by DAPI (blue). Original magnification ×100 (a, d), ×200 (b, c, e, f). C. Total RNA was harvested and was subjected to real-time qPCR analysis. Results, expressed relative to 18S, are the means±S.D. of triplicate determinations from a representative experiment.</p

Rosiglitazone Abrogates Bleomycin-Induced Scleroderma and Blocks Profibrotic Responses Through Peroxisome Proliferator-Activated Receptor-γ

The nuclear hormone receptor, peroxisome proliferator-activated receptor (PPAR)-γ, originally identified as a key mediator of adipogenesis, is expressed widely and implicated in diverse biological responses. Both natural and synthetic agonists of PPAR-γ abrogated the stimulation of collagen synthesis and myofibroblast differentiation induced by transforming growth factor (TGF)-β in vitro. To characterize the role of PPAR-γ in the fibrotic process in vivo, the synthetic agonist rosiglitazone was used in a mouse model of scleroderma. Rosiglitazone attenuated bleomycin-induced skin inflammation and dermal fibrosis as well as subcutaneous lipoatrophy and counteracted the up-regulation of collagen gene expression and myofibroblast accumulation in the lesioned skin. Rosiglitazone treatment reduced the induction of the early-immediate transcription factor Egr-1 in situ without also blocking the activation of Smad2/3. In both explanted fibroblasts and skin organ cultures, rosiglitazone prevented the stimulation of collagen gene transcription and cell migration elicited by TGF-β. Rosiglitazone-driven adipogenic differentiation of both fibroblasts and preadipocytes was abrogated in the presence of TGF-β; this effect was accompanied by the concomitant down-regulation of cellular PPAR-γ mRNA expression. Collectively, these results indicate that rosiglitazone treatment attenuates inflammation, dermal fibrosis, and subcutaneous lipoatrophy via PPAR-γ in a mouse model of scleroderma and suggest that pharmacological PPAR-γ ligands, widely used as insulin sensitizers in the treatment of type-2 diabetes mellitus, may be potential therapies for scleroderma