Autocrine Transforming Growth Factor β Signaling Regulates Extracellular Signal-regulated Kinase 1/2 Phosphorylation via Modulation of Protein Phosphatase 2A Expression in Scleroderma Fibroblasts

BACKGROUND. During scleroderma (SSc) pathogenesis, fibroblasts acquire an activated phenotype characterized by enhanced production of extracellular matrix (ECM) and constitutive activation of several major signaling pathways including extracellular signal-related kinase (ERK1/2). Several studies have addressed the role of ERK1/2 in SSc fibrosis however the mechanism of its prolonged activation in SSc fibroblasts is still unknown. Protein phosphatase 2A (PP2A) is a key serine threonine phosphatase responsible for dephosphorylation of a wide array of signaling molecules. Recently published microarray data from cultured SSc fibroblasts suggests that the catalytic subunit (C-subunit) of PP2A is downregulated in SSc. In this study we examined the role and regulation of PP2A in SSc fibroblasts in the context of ERK1/2 phosphorylation and matrix production. RESULTS. We show for the first time that PP2A mRNA and protein expression are significantly reduced in SSc fibroblasts and correlate with an increase in ERK1/2 phosphorylation and collagen expression. Furthermore, transforming growth factor β (TGFβ), a major profibrotic cytokine implicated in SSc fibrosis, downregulates PP2A expression in healthy fibroblasts. PP2A-specific small interfering RNA (siRNA) was utilized to confirm the role of PP2A in ERK1/2 dephosphorylation in dermal fibroblasts. Accordingly, blockade of autocrine TGFβ signaling in SSc fibroblasts using soluble recombinant TGFβ receptor II (SRII) restored PP2A levels and decreased ERK1/2 phosphorylation and collagen expression. In addition, we observed that inhibition of ERK1/2 in SSc fibroblasts increased PP2A expression suggesting that ERK1/2 phosphorylation also contributes to maintaining low levels of PP2A, leading to an even further amplification of ERK1/2 phosphorylation. CONCLUSIONS. Taken together, these studies suggest that decreased PP2A levels in SSc is a result of constitutively activated autocrine TGFβ signaling and could contribute to enhanced phosphorylation of ERK1/2 and matrix production in SSc fibroblasts.National Institutes of Health (AR-44883

Fresolimumab Treatment Decreases Biomarkers and Improves Clinical Symptoms in Systemic Sclerosis Patients

BACKGROUND. TGF-β has potent profibrotic activity in vitro and has long been implicated in systemic sclerosis (SSc), as expression of TGF-β–regulated genes is increased in the skin and lungs of patients with SSc. Therefore, inhibition of TGF-β may benefit these patients. METHODS. Patients with early, diffuse cutaneous SSc were enrolled in an open-label trial of fresolimumab, a high-affinity neutralizing antibody that targets all 3 TGF-β isoforms. Seven patients received two 1 mg/kg doses of fresolimumab, and eight patients received one 5 mg/kg dose of fresolimumab. Serial mid-forearm skin biopsies, performed before and after treatment, were analyzed for expression of the TGF-β–regulated biomarker genes thrombospondin-1 (THBS1) and cartilage oligomeric protein (COMP) and stained for myofibroblasts. Clinical skin disease was assessed using the modified Rodnan skin score (MRSS). RESULTS. In patient skin, THBS1 expression rapidly declined after fresolimumab treatment in both groups (P = 0.0313 at 7 weeks and P = 0.0156 at 3 weeks), and skin expression of COMP exhibited a strong downward trend in both groups. Clinical skin disease dramatically and rapidly decreased (P \u3c 0.001 at all time points). Expression levels of other TGF-β–regulated genes, including SERPINE1 and CTGF, declined (P = 0.049 and P = 0.012, respectively), and a 2-gene, longitudinal pharmacodynamic biomarker of SSc skin disease decreased after fresolimumab treatment (P = 0.0067). Dermal myofibroblast infiltration also declined in patient skin after fresolimumab (P \u3c 0.05). Baseline levels of THBS1 were predictive of reduced THBS1 expression and improved MRSS after fresolimumab treatment. CONCLUSION. The rapid inhibition of TGF-β–regulated gene expression in response to fresolimumab strongly implicates TGF-β in the pathogenesis of fibrosis in SSc. Parallel improvement in the MRSS indicates that fresolimumab rapidly reverses markers of skin fibrosis

CCN2 Is Required for the TGF-β Induced Activation of Smad1 - Erk1/2 Signaling Network

Connective tissue growth factor (CCN2) is a multifunctional matricellular protein, which is frequently overexpressed during organ fibrosis. CCN2 is a mediator of the pro-fibrotic effects of TGF-β in cultured cells, but the specific function of CCN2 in the fibrotic process has not been elucidated. In this study we characterized the CCN2-dependent signaling pathways that are required for the TGF-β induced fibrogenic response. By depleting endogenous CCN2 we show that CCN2 is indispensable for the TGF-β-induced phosphorylation of Smad1 and Erk1/2, but it is unnecessary for the activation of Smad3. TGF-β stimulation triggered formation of the CCN2/β3 integrin protein complexes and activation of Src signaling. Furthermore, we demonstrated that signaling through the αvβ3 integrin receptor and Src was required for the TGF-β induced Smad1 phosphorylation. Recombinant CCN2 activated Src and Erk1/2 signaling, and induced phosphorylation of Fli1, but was unable to stimulate Smad1 or Smad3 phosphorylation. Additional experiments were performed to investigate the role of CCN2 in collagen production. Consistent with the previous studies, blockade of CCN2 abrogated TGF-β-induced collagen mRNA and protein levels. Recombinant CCN2 potently stimulated collagen mRNA levels and upregulated activity of the COL1A2 promoter, however CCN2 was a weak inducer of collagen protein levels. CCN2 stimulation of collagen was dose-dependent with the lower doses (<50 ng/ml) having a stimulatory effect and higher doses having an inhibitory effect on collagen gene expression. In conclusion, our study defines a novel CCN2/αvβ3 integrin/Src/Smad1 axis that contributes to the pro-fibrotic TGF-β signaling and suggests that blockade of this pathway may be beneficial for the treatment of fibrosis

Fli1 Is a Negative Regulator of Estrogen Receptor α in Dermal Fibroblasts

Estrogen is an important regulator of dermal fibroblast functions, including extracellular matrix (ECM) synthesis. Estrogen mediates its effects through estrogen receptors (ERs), ERα and ERβ; however, regulation of ERs in dermal fibroblasts remains poorly understood. Friend leukemia integration factor 1 (Fli1), a member of the Ets transcription factor family, has been shown to play a pivotal role in regulation of the ECM genes in dermal fibroblasts. The aim of this study was to examine a possible interaction between Fli1 and estrogen pathways, focusing on ERα. We show that treatment of human dermal fibroblasts with transforming growth factor-β (TGF-β) increases ERα protein and mRNA levels. Similarly, ERα expression was increased in response to small interfering RNA (siRNA)-mediated depletion of Fli1, suggesting that Fli1 is a mediator of the TGF-β effects on ERα expression. Accordingly, we showed that Fli1 binds to the most proximal region of the ERα promoter, and dissociates from the promoter upon TGF-β treatment. An inverse correlation between Fli1 and ERα expression levels was confirmed in cultured skin fibroblasts obtained from Fli1+/− mice and in the skin of Fli1+/− mice in vivo. This study supports a role of Fli1 as a negative regulator of the ERα gene in dermal fibroblasts