The inhibitory effect of ginsan on TGF-β mediated fibrotic process

Transforming growth factor-beta (TGF-β) plays a central role in the development of fibrosis by stimulating extracellular matrix accumulation, and signals either directly or indirectly through types I, II, and III (TβRI, II, and III) TGF-β receptor complexes. Ginsan, a polysaccharide extracted from Panax ginseng, has multiple immunomodulatory effects. Here, we examine whether ginsan regulates the fibrogenic process by interfering with TGF-β signaling pathways. TGF-β treatment of murine or human normal lung fibroblasts enhanced the levels of several fibrotic markers, including smooth muscle alpha actin (α-SMA), collagen-1, and fibronectin. Interestingly, ginsan treatment either before or after TGF-β administration led to significant reductions in all of α-SMA, collagen-1, and fibronectin expression levels. Ginsan not only inhibited phosphorylation of Smad2 and Smad3, but also attenuated pERK and pAKT signaling induced by TGF-β. Moreover, ginsan restored TβRIII protein expression, which was significantly downregulated by TGF-β, but reduced TβRI and TβRII protein levels. In a murine model of bleomycin (BLM)-induced pulmonary fibrosis, ginsan significantly suppressed accumulation of collagen, α-SMA, and TGF-β. These data collectively suggest that ginsan acts as an effective anti-fibrotic agent in the treatment of pulmonary fibrosis by blocking multiple TGF-β signaling pathways.     

CD109-mediated degradation of TGF-β receptors and inhibition of TGF-β responses involve regulation of SMAD7 and Smurf2 localization and function

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates a wide variety of cellular processes including proliferation, differentiation, and extracellular matrix deposition. Dysregulation of TGF-β signaling is associated with several diseases such as cancer and tissue fibrosis. TGF-β signals through two transmembrane proteins known as the type I (TGFBR1) and type II (TGFBR2) receptors. The levels of these receptors at the cell surface are tightly regulated by several mechanisms, including degradation following recruitment of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor (Smurf) 2 by SMAD7. In addition, TGF-β co-receptors can modulate TGF-β signaling receptor activity in a cell-specific manner. We have previously identified a novel TGF-β co-receptor, CD109, a glycosyl phosphatidylinositol (GPI)-anchored protein that negatively regulates TGF-β signaling. Despite CD109's potential relevance as a regulator of TGF-β action in vivo, the mechanisms by which CD109 regulates TGF-β signaling are still incompletely understood. Previously, we have shown that CD109 downregulates TGF-β signaling by promoting TGF-β receptor localization into the lipid raft/caveolae compartment and by enhancing TGF-β receptor degradation. Here, we demonstrate that CD109 enhances SMAD7/Smurf2-mediated degradation of TGFBR1 in a ligand-dependent manner. Moreover, we show that CD109 regulates the localization and the association of SMAD7/Smurf2 with TGFBR1. Finally, we demonstrate that CD109's inhibitory effect on TGF-β signaling and responses require SMAD7 expression and Smurf2 ubiquitin ligase activity. Taken together, these results suggest that CD109 is an important regulator of SMAD7/Smurf2-mediated degradation of TGFBR1.     

Hsp70 and Hsp90 oppositely regulate TGF-β signaling through CHIP/Stub1

Transforming growth factor-β (TGF-β) signaling plays an important role in regulation of a wide variety of cellular processes. Canonical TGF-β signaling is mediated by Smads which were further regulated by several factors. We previously reported that E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein, also named Stub1) controlled the sensitivity of TGF-β signaling by modulating the basal level of Smad3 through ubiquitin-mediated degradation. Here, we present evidence that Hsp70 and Hsp90 regulate the complex formation of Smad3/CHIP. Furthermore, we observed that over-expressed Hsp70 or inhibition of Hsp90 by geldanamycin (GA) leads to facilitated CHIP-induced ubiquitination and degradation of Smad3, which finally enhances TGF-β signaling. In contrast, over-expressed Hsp90 antagonizes CHIP mediated Smad3 ubiquitination and degradation and desensitizes cells in response to TGF-β signaling. Taken together, our data reveal an opposite role of Hsp70 and Hsp90 in regulating TGF-β signaling by implicating CHIP-mediated Smad3 ubiquitination and degradation. This study provides a new insight into understanding the regulation of the TGF-β signaling by chaperones.     

RB1CC1 protein positively regulates transforming growth factor-beta signaling through the modulation of Arkadia E3 ubiquitin ligase activity

Transforming growth factor-β (TGF-β) signaling is controlled by a variety of regulators, of which Smad7, c-Ski, and SnoN play a pivotal role in its negative regulation. Arkadia is a RING-type E3 ubiquitin ligase that targets these negative regulators for degradation to enhance TGF-β signaling. In the present study we identified a candidate human tumor suppressor gene product RB1CC1/FIP200 as a novel positive regulator of TGF-β signaling that functions as a substrate-selective cofactor of Arkadia. Overexpression of RB1CC1 enhanced TGF-β signaling, and knockdown of endogenous RB1CC1 attenuated TGF-β-induced expression of target genes as well as TGF-β-induced cytostasis. RB1CC1 down-regulated the protein levels of c-Ski but not SnoN by enhancing the activity of Arkadia E3 ligase toward c-Ski. Substrate selectivity is primarily attributable to the physical interaction of RB1CC1 with substrates, suggesting its role as a scaffold protein. RB1CC1 thus appears to play a unique role as a modulator of TGF-β signaling by restricting substrate specificity of Arkadia.     

A novel small compound that promotes nuclear translocation of YB-1 ameliorates experimental hepatic fibrosis in mice

Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-β/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-β-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.     

TMEPAI inhibits TGF-β signaling by promoting lysosome degradation of TGF-β receptor and contributes to lung cancer development

Transforming growth factor-β (TGF-β) signaling plays important roles in embryogenesis and tumorigenesis by controlling cell growth, differentiation and migration. The transmembrane prostate androgen-induced protein (TMEPAI) is elevated in several cancers. TMEPAI expression is induced by TGF-β signaling, and in turn, expression of TMEPAI negatively regulates TGF-β signaling, but the molecular mechanisms of TMEPAI induced TGF-β signaling inhibition are not well understood. Here we report that TMEPAI is localized to the lysosome and late endosome, and that association of TMEPAI with the E3 ubiquitin ligase Nedd4 is required for its transport to the lysosome. TMEPAI associates with the TGF-β type I receptor (TβRI) and promotes its degradation in the lysosome. Depletion of TMEPAI in A549 lung cancer cells inhibits cell proliferation, migration and invasion, while TMEPAI expression in nude mice promotes tumorigenesis. These results reveal a novel function for TMEPAI in regulating TGF-β signaling through the modulation of TβRI levels, which has important implications for cancer development in vivo.     

Mechanisms of pulmonary fibrosis induced by core fucosylation in pericytes

Pulmonary fibrosis is a common outcome of a variety of pulmonary interstitial diseases, and myofibroblasts are the main culprit for this process. Recent studies have found that pericytes are one of the major sources of myofibroblasts; the transformation of which involves a complex process of activation of TGF-β/Smad2/3 and PDGFβ/Erk signaling pathways. We have reported that the transforming growth factor-β receptor and platelet-derived growth factor-β receptor (TGF-βR I and PDGFβR, respectively) are modified by glycosylation. Thus, we hope to regulate the above-mentioned signal pathways through core fucosylation (CF) catalyzed by α-1,6-fucosyltransferase (FUT8). Previous work has confirmed that TGF-β1 can induce the transformation of pericytes into myofibroblasts, while FUT8siRNA can inhibit such transformation. In the present study, we used an adenovirus packaging FUT8 shRNA to infect a bleomycin-induced pulmonary fibrosis mouse model and determined the effect of CF on pulmonary fibrosis by analyzing the mechanism of CF-mediated pericyte transformation. Our findings may shed new light on the mechanism of pulmonary interstitial fibrosis and provide a novel therapeutic target for clinical applications.     

Activation of Wnt11 by transforming growth factor-β drives mesenchymal gene expression through non-canonical Wnt protein signaling in renal epithelial cells

Transforming growth factor β1 (TGF-β) promotes renal interstitial fibrosis in vivo and the expression of mesenchymal genes in vitro; however, most of its direct targets in epithelial cells are still elusive. In a screen for genes directly activated by TGF-β, we found that components of the Wnt signaling pathway, especially Wnt11, were targets of activation by TGF-β and Smad3 in primary renal epithelial cells. In gain and loss of function experiments, Wnt11 mediated the actions of TGF-β through enhanced activation of mesenchymal marker genes, such as Zeb1, Snail1, Pai1, and αSMA, without affecting Smad3 phosphorylation. Inhibition of Wnt11 by receptor knockdown or treatment with Wnt inhibitors limited the effects of TGF-β on gene expression. We found no evidence that Wnt11 activated the canonical Wnt signaling pathway in renal epithelial cells; rather, the function of Wnt11 was mediated by the c-Jun N-terminal kinase (JNK) pathway. Consistent with the in vitro results, all the TGF-β, Wnt11, and JNK targets were activated in a unilateral ureteral obstruction (UUO) model of renal fibrosis in vivo. Our findings demonstrated cooperativity among the TGF-β, Wnt11, and JNK signaling pathways and suggest new targets for anti-fibrotic therapy in renal tissue.     

Transforming growth factor β induces expression of connective tissue growth factor in hepatic progenitor cells through Smad independent signaling

Hepatic progenitor cells (HPCs) are activated in the chronic liver injury and are found to participate in the progression of liver fibrosis, while the precise role of HPCs in liver fibrosis remains largely elusive. In this study, by immunostaining of human liver sections, we confirmed that HPCs were activated in the cirrhotic liver and secreted transforming growth factor β (TGF-β) and connective tissue growth factor (CTGF), both of which were important inducers of liver fibrosis. Besides, we used HPC cell lines LE/6 and WB-F344 as in vitro models and found that TGF-β induced secretion of CTGF in HPCs. Moreover, TGF-β signaling was intracrine activated and contributed to autonomous secretion of CTGF in HPCs. Furthermore, we found that TGF-β induced expression of CTGF was not mediated by TGF-β activated Smad signaling but mediated by TGF-β activated Erk, JNK and p38 MAPK signaling. Taken together, our results provide evidence for the role of HPCs in liver fibrosis and suggest that the production of CTGF by TGF-β activated MAPK signaling in HPCs may be a therapeutic target of liver fibrosis.     

TGF-β1-PML SUMOylation-peptidyl-prolyl cis–trans isomerase NIMA-interacting 1 (Pin1) form a positive feedback loop to regulate cardiac fibrosis

Transforming growth factor-β (TGF-β) signaling pathway is involved in fibrosis in most, if not all forms of cardiac diseases. Here, we evaluate a positive feedback signaling the loop of TGF-β1/promyelocytic leukemia (PML) SUMOylation/Pin1 promoting the cardiac fibrosis. To test this hypothesis, the mice underwent transverse aortic constriction (3 weeks) were developed and the morphological evidence showed obvious interstitial fibrosis with TGF-β1, Pin1 upregulation, and increase in PML SUMOylation. In neonatal mouse cardiac fibroblasts (NMCFs), we found that exogenous TGF-β1 induced the upregulation of TGF-β1 itself in a time- and dose-dependent manner, and also triggered the PML SUMOylation and the formation of PML nuclear bodies (PML-NBs), and consequently recruited Pin1 into nuclear to colocalize with PML. Pharmacological inhibition of TGF-β signal or Pin1 with LY364947 (3 μM) or Juglone (3 μM), the TGF-β1-induced PML SUMOylation was reduced significantly with downregulation of the messenger RNA and protein for TGF-β1 and Pin1. To verify the cellular function of PML by means of gain- or loss-of-function, the positive feedback signaling loop was enhanced or declined, meanwhile, TGF-β-Smad signaling pathway was activated or weakened, respectively. In summary, we uncovered a novel reciprocal loop of TGF-β1/PML SUMOylation/Pin1 leading to myocardial fibrosis.     

Regulation of TGF-β storage and activation in the human idiopathic pulmonary fibrosis lung

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of unknown cause. The pathogenesis of the disease is characterized by fibroblast accumulation and excessive transforming growth factor-β (TGF-β) activation. Although TGF-β activation is a complex process involving various protein interactions, little is known of the specific routes of TGF-β storage and activation in human lung. Here, we have systematically analyzed the expression of specific proteins involved in extracellular matrix targeting and activation of TGF-β. Latent TGF-β-binding protein (LTBP)-1 was found to be significantly upregulated in IPF patient lungs. LTBP-1 expression was especially high in the fibroblastic foci, in which P-Smad2 immunoreactivity, indicative of TGF-β signaling activity, was less prominent. In cultured primary lung fibroblasts and epithelial cells, short-interfering-RNA-mediated downregulation of LTBP-1 resulted in either increased or decreased TGF-β signaling activity, respectively, suggesting that LTBP-1-mediated TGF-β activation is dependent on the cellular context in the lung. Furthermore, LTBP-1 was shown to colocalize with fibronectin, fibrillin-1 and fibrillin-2 proteins in the IPF lung. Fibrillin-2, a developmental gene expressed only in blood vessels in normal adult lung, was found specifically upregulated in IPF fibroblastic foci. The TGF-β-activating integrin β8 subunit was expressed at low levels in both control and IPF lungs. Alterations in extracellular matrix composition, such as high levels of the TGF-β storage protein LTBP-1 and the re-appearance of fibrillin-2, probably modulate TGF-β availability and activation in different pulmonary compartments in the fibrotic lung.     

TGF-β-mediated upregulation of Sox9 in fibroblast promotes renal fibrosis

TGF-β signaling plays a principal role in renal fibrosis, but the precise mechanisms and the downstream factors are still largely unknown. Sox9 exhibits diverse roles in regulating the production of extracellular matrix proteins. Here we found that Sox9 was induced by TGF-β in the kidney fibroblast and acted as an important downstream mediator of TGF-β signaling in promoting renal fibrosis. TGF-β/Smad signaling mediated the upregulation of Sox9 in kidney fibroblast by binding to a conserved enhancer. In different mouse models of renal fibrosis, as well as in the kidney biopsy tissue from patients with renal fibrosis, Sox9 expression significantly increased. Immunostaining confirmed the upregulation of Sox9 in the kidney fibroblast during renal fibrosis. Delivery of Sox9 knockdown plasmid to the kidney by ultrasound microbubble–mediated gene transfer suppressed the unilateral ureteral obstruction (UUO) or folic acid-induced mouse renal fibrosis, whereas ectopic expression of Sox9 aggravated renal fibrosis. In addition, we identified Sox9 as a direct target of miR-30. Notably, miR-30 expression was significantly inhibited by TGF-β1 in the kidney fibroblast and the downregulation of miR-30 was observed in renal fibrosis. Mechanistically, inhibition of miR-30 independently strengthened the effect of TGF-β/Smad signaling on Sox9 upregulation. Adenovirus-mediated ectopic expression of miR-30 in kidney fibroblast greatly reduced UUO-induced renal fibrosis by targeting Sox9. These findings link Sox9 to intrinsic mechanisms of TGF-β signaling in renal fibrosis and may have therapeutic potential for tissue fibrosis.     

Histopathologic and immunologic effects of the itraconazole treatment in a murine model of chronic pulmonary paracoccidioidomycosis

A comparative study, based on histopathologic findings (inflammation, cellularity, and fibrosis) and immunologic parameters (pro-inflammatory and anti-inflammatory cytokines), was carried out in order to evaluate the effects of itraconazole (ITC) treatment and its starting time in a BALB/c murine model of chronic pulmonary paracoccidioidomycosis (PCM), induced by intranasal inoculation of Paracoccidioides brasiliensis (Pb) conidia. Two different groups of mice were exposed to ITC therapy beginning at the 4th or 8th week after Pb infection, respectively. ITC was administered daily, via gavage, for a period of sixty days. At weeks 0, 4, 8, 12 and 16 the animals were sacrificed and their lungs removed for histology staining with hematoxylin and eosin (H&E), Masson’s trichromic and Gomori–Grocott; pulmonary levels of IL-1β, TNF-α, IFN-γ, IL-13 and TGF-β were also measured by ELISA. The development or absence of the principal pulmonary PCM sequela, lung fibrosis, was directly related to the therapy’s starting time. This and other histopathologic findings were related to the behavior of cytokine levels.     

Epithelial–mesenchymal transition of A549 cells is enhanced by co-cultured with THP-1 macrophages under hypoxic conditions

Epithelial–mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O₂) and hypoxic (2% O₂) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-β1 and IL-1β in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-β1 inhibitor or IL-1β neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-β1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-β1 in A549 cells and THP-1 macrophages and that of IL-1β in THP-1 macrophages when each cells were co-cultured. Anti-IL-1β neutralizing antibody attenuated TGF-β1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1β from THP-1 macrophages up-regulated the TGF-β1 from A549 cells and THP-1 macrophages, and then the TGF-β1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis.