Abstract Objective The hallmark feature of systemic sclerosis (SSc) is excessive accumulation of extracellular matrix (ECM) in skin and other affected organs that leads to fibrosis. The accumulation of ECM is caused by pathologically activated fibroblast that releases collagen, fibronectin, and other ECM components. Strong evidence suggests transforming growth factor beta (TGF‐β) plays a pivotal role in fibroblast activation and induces excessive ECM production. In addition, heat shock protein (HSPs) have been implicated in SSc such that pharmacological inhibition of hsp90 blocks the pro‐fibrotic effect of TGF‐β. In this study we evaluate the role of hsp90 and hsp70 on TGF‐β signaling and ECM production in human dermal fibroblasts. Method Expression of hsp90 and hsp70 were quantified by immunoblot and immunofluorescence microscopy following TGF‐β induction of dermal fibroblasts. The effect of hsp90 and hsp70 inhibition on TGF‐β‐induced ECM expression was analyzed in dermal fibroblasts. Results Expression of hsp90 and hsp70 increased in a TGF‐β‐ dependent manner 48 hours after treatments following a marginal, proteasomal‐dependent decrease at 24 hours. Inhibition of hsp90 by 17‐allylamino‐17‐demethoxy‐geldanamycin (17‐AAG) blocked the effects of TGF‐β on fibronectin and alpha‐smooth muscle actin and myofibroblast differentiation. In turn, there was an increase in antifibrotic matrix decorin. Conclusion Our data suggest a critical role for hsp90 in TGF‐β signaling. We propose the use of hsp90 inhibitors as potential antifibrotic treatments. Our data suggest a critical role for hsp90 and hsp70 in TGF‐β signaling. Blockade of TGF‐β‐mediated myofibroblast differentiation by 17‐AAG suggests that hsp90 inhibitors merit additional study as novel antifibrotic agents.
We report a case of immunoglobulin(Ig)G4-related disease with the radiologic and histopathological manifestations resembling usual interstitial pneumonia (UIP). The patient was a 62-year-old man who presented with progressive dyspnea of insidious onset. High resolution computed tomography of the chest showed lower-lobe predominant peripheral reticulation and traction bronchiectasis but no honeycomb change. Microscopic examination of the surgical lung biopsy showed characteristic features of UIP including architectural distortion by fibrosis with peripheral and paraseptal accentuation, scattered fibroblast foci and microscopic honeycomb change. In addition there were prominent multifocal lymphoplasmacytic infiltrates with a marked increase of IgG4-positive plasma cells (79 per high power field in hot spots) and high IgG4/IgG ratio (up to 67%). The serum IgG4 level was elevated at 760 mg/dl (reference range 9-89), with normal levels for the other IgG subclasses and negative serologic markers for autoimmune diseases. The patient’s symptoms improved significantly with oral corticosteroid treatment.
Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued for trans-acting modifiers of the cleavage-polyadenylation reaction operating differentially during B-cell developmental stages. Using four complementary approaches, we demonstrate that a change in the level of hnRNP F is an important determinant in the regulated use of alternative polyadenylation sites between memory and plasma stage B cells. First, by Western analyses of cellular proteins, the ratio of hnRNP F to H or H' was found to be higher in memory B cells than in plasma cells. In memory B cells the activity of CstF-64 binding to pre-mRNA, but not its amount, was reduced. Second, examination of the complexes formed on input pre-mRNA in nuclear extracts revealed large assemblages containing hnRNP H, H', and F but deficient in CstF-64 in memory B-cell extracts but not in plasma cells. Formation of these large complexes is dependent on the region downstream of the AAUAAA in pre-mRNA, suggesting that CstF-64 and the hnRNPs compete for a similar region. Third, using a recombinant protein we showed that hnRNP F could bind to the region downstream of a poly(A) site, block CstF-64 association with RNA, and inhibit the cleavage reaction. Fourth, overexpression of recombinant hnRNP F in plasma cells resulted in a decrease in the endogenous Ig heavy-chain mRNA secretory form-to-membrane ratio. These results demonstrate that mammalian hnRNP F can act as a negative regulator in the pre-mRNA cleavage reaction and that increased expression of F in memory B cells contributes to the suppression of the Ig heavy-chain secretory poly(A) site.
To determine the role of insulin-like growth factor binding protein 3 (IGFBP-3) in mediating the effects of transforming growth factor β (TGFβ) on tenascin-C (TN-C) production and to assess the levels of TN-C in vivo in patients with systemic sclerosis (SSc)-associated pulmonary fibrosis.Human primary lung fibroblasts were stimulated with TGFβ or IGFBP-3 in the presence or absence of specific small interfering RNAs and chemical inhibitors of the signaling cascade. TN-C levels in lung tissue specimens obtained from patients with SSc-associated pulmonary fibrosis were assessed using immunohistochemical analysis and were compared with the levels in specimens obtained from normal donors. TN-C levels were quantified in sera from normal donors and patients with SSc with or without pulmonary fibrosis, using an enzyme-linked immunosorbent assay.IGFBP-3 mediated the induction of TN-C by TGFβ. Direct induction of TN-C by IGFBP-3 occurred in a p38 MAP kinase-dependent manner. TN-C levels were abundant in lung tissues from patients with SSc and were localized to subepithelial layers of the distal airways. No TN-C was detectable around the proximal airways. Patients with SSc-associated pulmonary fibrosis had significantly higher levels of circulating TN-C compared with SSc patients without pulmonary fibrosis. Longitudinal samples obtained from patients with SSc before and after the onset of pulmonary fibrosis showed increased levels of TN-C after the onset of pulmonary fibrosis.IGFBP-3, which is overexpressed in fibrotic lungs, induces production of TN-C by subepithelial fibroblasts. The increased lung tissue levels of TN-C parallel the levels detected in the sera of SSc patients with pulmonary fibrosis, suggesting that TN-C may be a useful biomarker for SSc-related pulmonary fibrosis.
Idiopathic Pulmonary Fibrosis (IPF) is a disease that may have pathophysiological similarities to some of the mechanisms involved in aging. Loss of proteostasis is one of the current hallmarks of aging and heat shock proteins (Hsp) are currently considered chaperones that regulate proteostasis. Preliminary data have demonstrated that Hsp-70 might be associated with IPF. We sought to investigate the potential association between the deficiency of Hsp70 with aging and IPF. Hsp70 expression was assessed using immunofluorescence in human lungs, and found that Hsp70 was not expressed in older compared to younger IPF tissues. . We also observed decreased Hsp70 mRNA and protein in primary fibroblasts from IPF versus normal donors. Treatment of primary human lung fibroblasts in vitro with TGF-β1 decreased Hsp70 in parallel with increased extracellular matrix proteins, collagen and fibronectin. Young Hsp70 knock-out mice (8-10 weeks) were subjected to an inhalational bleomycin model of pulmonary fibrosis and demonstrated accelerated fibrosis versus wild-type controls. No spontaneous fibrosis was observed in older knock-out mice (> 20 weeks). We therefore conclude that reduced Hsp70 protein is associated with pulmonary fibrosis. Interventions aimed at restoring normal expression of Hsp70 represent a novel therapeutic strategy for pulmonary fibrosis.
Idiopathic pulmonary fibrosis (IPF) pathogenesis has been postulated to involve a variety of mechanisms associated with the aging process, including loss of protein homeostasis (proteostasis). Heat shock proteins are cellular chaperones that serve a number of vital maintenance and repair functions, including the regulation of proteostasis. Previously published data have implicated heat shock protein 70 (Hsp70) in the development of pulmonary fibrosis in animal models. We sought to identify alterations in Hsp70 expression in IPF lung. Hsp70 mRNA and protein were decreased in primary fibroblasts cultured from IPF versus normal donor lung tissue. In addition to cultured fibroblasts, Hsp70 expression was decreased in intact IPF lung, a stressed environment in which upregulation of protective heat shock proteins would be anticipated. In support of a mechanistic association between decreased Hsp70 and fibrosis, cultured primary lung fibroblasts deficient in Hsp70 secreted increased extracellular matrix proteins. Treatment of primary normal human lung fibroblasts in vitro with either of the profibrotic molecules IGFBP5 (insulin-like growth factor-binding protein 5) or transforming growth factor-β1 downregulated Hsp70, suggesting Hsp70 is a downstream target in the fibrotic cascade. Hsp70-knockout mice subjected to an inhalational bleomycin model of pulmonary fibrosis demonstrated accelerated fibrosis versus wild-type control animals. We therefore conclude that reduced Hsp70 protein contributes to fibrosis and that interventions aimed at restoring normal expression of Hsp70 represent a novel therapeutic strategy for pulmonary fibrosis.
We have determined that the DNA sequence downstream of the well-characterized gonococcal fbp gene contains two open reading frames: one designated fbpB, which encodes a protein proposed to function as a cytoplasmic permease, and one designated fbpC, which encodes a protein proposed to function as a nucleotide-binding protein. The fpbABC operon composes an iron transport system that is homologous to the sfu and hit operons previously reported for Serratia marcescens and Haemophilus influenzae, respectively, and displays elements characteristic of ATP binding cassette transporters. The fpbABC operon differs from these loci in that it is lethal when overexpressed in Escherichia coli.
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