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

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

Comparison of oral versus parenteral methotrexate in the treatment of rheumatoid arthritis: A meta-analysis.

OBJECTIVE:Studies suggest that parenteral MTX may be more efficacious than the oral form at equivalent doses for the treatment of rheumatoid arthritis. We carried out a meta-analysis to compare the efficacy of oral versus parenteral MTX in RA. METHODS:PubMed, Web of Science and Embase were systematically searched from inception to June 8th 2017 and reviewed following PRISMA 2009 guidelines, by two independent reviewers. To be included, trials had to study adults with RA randomized to the same dose of either oral or parenteral MTX. The primary endpoint was ACR20 at 6 months. Intention-to-treat analysis results were used when possible. Data from direct comparisons between oral and parenteral methotrexate quantitatively analyzed using maximum likelihood random effects meta-analysis. Relative treatment effects were generated as an odds ratio [OR] (OR>1 indicated a benefit for parenteral therapy). RESULTS:The search yielded 357 papers or abstracts. After review of titles or abstracts and full text papers, we found 4 that met inclusion criteria with 703 patients randomized. Dose of MTX started at 15mg/week and increased up to 25mg/week. The summary OR for achieving ACR20 using parenteral vs. oral MTX was 3.02 (95% CI 1.41, 6.46), with no significant difference in the risk for all adverse events. CONCLUSION:Parenteral MTX therapy had significantly higher odds than oral MTX of achieving reduction in disease activity. We propose that parenteral MTX is more effective than weekly oral MTX; its widespread use may lead to better control of disease and a decrease in demand for biologic agents

Ciprofloxacin has antifibrotic effects in scleroderma fibroblasts via downregulation of Dnmt1 and upregulation of Fli1

Systemic sclerosis (SSc) is characterized by fibrosis of the skin and internal organs. The present study was undertaken to examine the effects of ciprofloxacin, a fluoroquinolone antibiotic implicated in matrix remodeling, on dermal and lung fibroblasts obtained from SSc patients. Dermal and lung fibroblasts from SSc patients and healthy subjects were treated with ciprofloxacin. Western blotting was used to analyze protein levels and RT-PCR was used to measure in RNA expression. The pharmacologic inhibitor UO126 was used to block Erk1/2 signaling. SSc dermal fibroblasts demonstrated a significant decrease in collagen type I mRNA and protein levels after antibiotic treatment, while healthy dermal fibroblasts were less sensitive to ciprofloxacin, downregulating collagen only at the protein levels. Connective tissue growth factor (CCN2) gene expression was significantly reduced and matrix metalloproteinase (MMPI) levels were enhanced after ciprofloxacin treatment to a similar extent in healthy and SSc fibroblasts. Ciprofloxacin induced Erk1/2 phosphorylation, and Erk1/2 blockade completely prevented MMP1 upregulation. However. Smad1 and Smad3 activation in response to TGF beta was not affected. The expression of friend leukemia integration factor 1 (Fli1). a transcriptional repressor of collagen, was increased after treatment with ciprofloxacin only in SSc fibroblasts, and this was accompanied by a decrease in the levels of DNA methyltransferase 1 (Dnmt1). Similar effects were observed in SSc-interstitial lung disease (ILD) lung fibroblasts. In summary, our study demonstrates that ciprofloxacin has antifibrotic actions in SSc dermal and lung fibroblasts via the downregulation of Dnmt1, the upregulation of Fli1 and induction of MMPI gene expression via an Erk1/2-dependent mechanism. Thus, our data suggest that ciprofloxacin may he an attractive therapy for SSc skin and lung fibrosis

Clinical characteristics and outcomes in pulmonary manifestations of systemic sclerosis: Contribution from pulmonary hypertension and interstitial lung disease severity

Abstract Patients with systemic sclerosis complicated by both pulmonary hypertension (SSc‐PH) and interstitial lung disease (SSc‐PH‐ILD) have poor prognosis compared to those with SSc‐PH or SSc‐ILD alone. Little is known of how ILD severity affects outcomes in those with SSc‐PH, or how PH severity affects outcomes in those with SSc‐ILD. Herein, we aimed to delineate clinical features of patients with SSc‐PH and SSc‐ILD and determine to what degree PH and ILD severity contribute to mortality in patients with SSc. We conducted parallel retrospective studies in cohorts of patients with SSc‐PH and SSc‐ILD. We categorized ILD severity by pulmonary function testing and PH severity by cardiopulmonary hemodynamics. Our primary outcome was all‐cause mortality from time of PH or ILD diagnosis for the SSc‐PH and SSc‐ILD cohorts, respectively. We calculated adjusted risks of time to all‐cause mortality using Cox proportional hazards models. In patients with SSc‐PH, severe ILD (HR: 3.54; 95% CI: 1.05, 11.99) was associated with increased hazards for all‐cause mortality. By contrast, mild and moderate ILD were not associated with increased mortality risk. In patients with SSc‐ILD, both moderate (HR: 2.65; 95% CI: 1.12, 6.31) and severe PH (HR: 6.60; 95% CI: 2.98, 14.61) were associated with increased hazards for all‐cause mortality, while mild PH was not. Through our parallel study design, the risk of all‐cause mortality increases as severity of concomitant ILD or PH worsens. Therapies that target slowing disease progression earlier in the disease course may be beneficial