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

Abstract

Although the early growth response-2 (Egr-2, alias Krox20) protein shows structural and functional similarities to Egr-1, these two related early-immediate transcription factors are nonredundant. Egr-2 plays essential roles in peripheral nerve myelination, adipogenesis, and immune tolerance; however, its regulation and role in tissue repair and fibrosis remain poorly understood. We show herein that transforming growth factor (TGF)-β induced a Smad3-dependent sustained stimulation of Egr2 gene expression in normal fibroblasts. Overexpression of Egr-2 was sufficient to stimulate collagen gene expression and myofibroblast differentiation, whereas these profibrotic TGF-β responses were attenuated in Egr-2-depleted fibroblasts. Genomewide transcriptional profiling revealed that multiple genes associated with tissue remodeling and wound healing were up-regulated by Egr-2, but the Egr-2-regulated gene expression profile overlapped only partially with the Egr-1-regulated gene profile. Levels of Egr-2 were elevated in lesional tissue from mice with bleomycin-induced scleroderma. Moreover, elevated Egr-2 was noted in biopsy specimens of skin and lung from patients with systemic sclerosis. These results provide the first evidence that Egr-2 is a functionally distinct transcription factor that is both necessary and sufficient for TGF-β-induced profibrotic responses and is aberrantly expressed in lesional tissue in systemic sclerosis and in a murine model of scleroderma. Together, these findings suggest that Egr-2 plays an important nonredundant role in the pathogenesis of fibrosis. Targeting Egr-2 might represent a novel therapeutic strategy to control fibrosis.

Figure 1

TGF-β stimulates Egr-2 expression. Confluent foreskin fibroblasts (A, B, and D) or healthy adult skin and lesional SSc skin fibroblasts (C) were incubated in serum-free media with TGF-β2 (10 ng/mL, unless otherwise indicated) for the indicated periods (A and D) or for 120 minutes (B and C). A through C: Total RNA was subjected to qPCR. The results represent the mean ± SEM of triplicate determinations from three independent experiments. *P < 0.05; ***P < 0.001. D: Whole cell lysates were subjected to Western analysis. Representative autoradiograms are shown. E: NIH-3T3 fibroblasts were transiently transfected with Egr-2–luc and incubated with TGF-β2 for 24 hours. Cell lysates were assayed for their luc activities.

Figure 2

Sustained Egr-2 stimulation by TGF-β. Foreskin fibroblasts at confluence were incubated in serum-free media with TGF-β (10 ng/mL or as indicated) for the indicated periods (A and B) or for 24 hours (C). A: qPCR. The results represent the mean ± SEM of triplicate determinations from three independent experiments. B: Western analysis. C: Confocal microscopy. Cells were fixed and immunostained with antibodies to Egr-2 (green), type I collagen as a positive control (red), or DAPI (blue). Representative immunofluorescence photomicrographs are shown. Original magnification, ×100.

Figure 3

Stimulation of Egr-2 expression via Smad3 and extracellular signal–regulated kinase 1/2. A: Confluent fibroblasts were preincubated for 30 minutes with SB431542 (SB), U0126, or LY294002 (LY), followed by TGF-β (10 ng/mL) for a further 120 minutes before RNA isolation. qPCR was performed. The results represent the mean ± SEM of triplicate determinations from three independent experiments. B: Fibroblasts were transiently transfected with Smad3-specific siRNA or scrambled control siRNA in parallel and incubated with TGF-β (10 ng/mL) for a further 120 minutes before RNA isolation and qPCR analysis. *P < 0.05. The lower panel shows the results of Western analysis. C and D: Fibroblasts from Smad3^+/+ or Smad3^-/- mouse embryos were incubated in parallel with TGF-β2 (10 ng/mL) for up to 120 minutes before RNA isolation and qPCR analysis (C) or up to 28 hours before isolation of whole cell lysates for Western analysis (D). ***P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; cgn, collagen.

Figure 4

Egr-1 stimulates Egr-2 expression. A and B: Foreskin fibroblasts at confluence were infected with Ad–Egr-1 or Ad-GFP, followed by incubation in serum-free media for the indicated periods. RNA was isolated and subjected to qPCR analysis. A: Analysis using microarrays (Illumina). Egr-2 mRNA signal intensity for each point was determined. Results, indicating fold change in Egr-1–infected fibroblasts compared with Ad-GFP–infected fibroblasts, are expressed as mean ± SEM from triplicate determinations. B: qPCR was performed. The results represent the mean ± SEM of triplicate determinations from three independent experiments. **P < 0.01.

Figure 5

Overexpressed Egr-2 stimulates profibrotic gene expression. A: Confluent fibroblasts were infected with increasing concentrations of Ad–Egr-2 or control virus. After 48 hours' incubation, fibroblasts were harvested and whole cell lysates were subjected to Western analysis. Representative immunoblots are shown. B and C: Fibroblasts were transiently cotransfected with pCMV–Egr-2, along with 772COL1A2–chloramphenicol acetyltransferase (CAT) (B) or ASMA-luc (C). After 48 hours' incubation, whole cell lysates were assayed for their CAT or luc activities. The results represent the means of triplicate determinations from two independent experiments. **P < 0.01. cgn indicates; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; cgn, collagen; MOI, multiplicity of infection.

Figure 6

Egr-2 is required for maximal TGF-β responses. Fibroblasts were transiently transfected with Egr-2–specific siRNA or scrambled control siRNA. After incubation with 10 ng/mL TGF-β for 24 hours, RNA was isolated and analyzed by qPCR. The results are shown as fold change compared with control siRNA and represent the mean ± SEM of triplicate determinations from three independent experiments. *P <0.05; **P <0.01. CTGF, connective tissue growth factor.

Figure 7

Genomewide expression changes induced by Egr-2. Fibroblasts were transfected with Ad-GFP or Ad–Egr-2 or left untransfected (control) for 48 hours before RNA isolation and processing for microarray analysis. A: Heat map. The genes that showed greater than twofold up- or down-regulation by Ad–Egr-2 compared with Ad-GFP were further analyzed. B: The top gene ontology categories were regulated by Egr-2. C: qPCR validation for selected Egr-2–regulated genes. D: Overlap between genes regulated by Egr-1 (751) and Egr-2 (677). *P < 0.05; **P < 0.01. rRNA, ribosomal RNA; FC, fold change; Plod, procollagen-lysine 1, 2-oxoglutarate 5-dioxygenase; Itgb, integrin β.

Figure 8

Elevated Egr-2 expression in mouse scleroderma. Dermal fibrosis was induced by daily s.c. injections of bleomycin (Bleo) for 28 days, and lesional skin was harvested. A: Sections were stained with antibodies against Egr-2 and counterstained with hematoxylin. Representative photomicrographs are shown. Original magnification: ×100 (upper panels); ×400 (lower panels). Lower panels in A represent the areas delineated by the squares in the upper panels. Arrows indicate immunopositive fibroblastic cells in the dermis. Omission of primary antibodies resulted in no staining (negative control). The number of immunopositive fibroblastic cells was determined in five separate microscopic fields by a blinded observer. B: Total RNA was isolated from the skin and subjected to qPCR. The results represent the mean ± SEM of three determinations from five mice per group.

Figure 9

Elevated Egr-2 expression in SSc. A: Skin biopsy specimens from patients with SSc (n = 3) or healthy controls (n = 3) were stained with antibodies to Egr-2 and examined by immunofluorescence. Representative images are shown, with red lines delineating the epidermis. Original magnification: ×200 (upper panels); ×630 (lower panels). B: Quantification of Egr-2 immunofluorescence in the dermis (the Materials and Methods section provides details). Bars represent the mean ± SEM of three determinations. C: Lung tissues from nonfibrotic donors (n = 3) (normal) or patients with SSc who have pulmonary fibrosis (n = 3) (SSc) were examined. Representative images were captured by a microscope (Zeiss Axioskop) with a CRi Nuance spectral camera. Original magnification: ×100 (left); ×1000 (middle). Right: Panels showing the magnified images (from the boxes). Brown indicates Egr-2–positive cells. Nuclei are counterstained with hematoxylin (blue).

Figure 10

Proposed mechanistic model highlighting the role of Egr-2 in TGF-β–induced profibrotic responses. The expression of Egr-1 is rapidly and transiently induced by TGF-β via extracellular signal–regulated kinase (ERK) 1/2. In turn, Egr-1 induces Egr-2. The ERK1/2 and Smad2/3 pathways also stimulate Egr-2, leading to its sustained expression. Egr-1 and Egr-2 directly stimulate COL1A2 transcription. SBE, smad binding element.