Induction of galectin-1 by TGF-β1 accelerates fibrosis through enhancing nuclear retention of Smad2

Fibrosis is one of the most serious side effects in cancer patients undergoing radio-/ chemo-therapy, especially of the lung, pancreas or kidney. Based on our previous finding that galectin-1 (Gal-1) was significantly increased during radiation-induced lung fibrosis in areas of pulmonary fibrosis, we herein clarified the roles and action mechanisms of Gal-1 during fibrosis. Our results revealed that treatment with TGF-β1 induced the differentiation of fibroblast cell lines (NIH3T3 and IMR-90) to myofibroblasts, as evidenced by increased expression of the fibrotic markers smooth muscle actin-alpha (α-SMA), fibronectin, and collagen (Col-1). We also observed marked and time-dependent increases in the expression level and nuclear accumulation of Gal-1. The TGF-β1-induced increases in Gal-1, α-SMA and Col-1 were decreased by inhibitors of PI3-kinase and p38 MAPK, but not ERK. Gal-1 knockdown using shRNA decreased the phosphorylation and nuclear retention of Smad2, preventing the differentiation of fibroblasts. Gal-1 interacted with Smad2 and phosphorylated Smad2, which may accelerate fibrotic processes. In addition, up-regulation of Gal-1 expression was demonstrated in a bleomycin (BLM)-induced mouse model of lung fibrosis in vivo. Together, our results indicate that Gal-1 may promote the TGF-β1-induced differentiation of fibroblasts by sustaining nuclear localization of Smad2, and could be a potential target for the treatment of pulmonary fibrotic diseases.     

Antifibrotic properties of caveolin-1 scaffolding domain in vitro and in vivo

Lung fibrosis involves the overexpression of ECM proteins, primarily collagen, by alpha-smooth muscle actin (ASMA)-positive cells. Caveolin-1 is a master regulator of collagen expression by cultured lung fibroblasts and of lung fibrosis in vivo. A peptide equivalent to the caveolin-1 scaffolding domain (CSD peptide) inhibits collagen and tenascin-C expression by normal lung fibroblasts (NLF) and fibroblasts from the fibrotic lungs of scleroderma patients (SLF). CSD peptide inhibits ASMA expression in SLF but not NLF. Similar inhibition of collagen, tenascin-C, and ASMA expression was also observed when caveolin-1 expression was upregulated using adenovirus. These observations suggest that the low caveolin-1 levels in SLF cause their overexpression of collagen, tenascin-C, and ASMA. In mechanistic studies, MEK, ERK, JNK, and Akt were hyperactivated in SLF, and CSD peptide inhibited their activation and altered their subcellular localization. These studies and experiments using kinase inhibitors suggest many differences between NLF and SLF in signaling cascades. To validate these data, we determined that the alterations in signaling molecule activation observed in SLF also occur in fibrotic lung tissue from scleroderma patients and in mice with bleomycin-induced lung fibrosis. Finally, we demonstrated that systemic administration of CSD peptide to bleomycin-treated mice blocks epithelial cell apoptosis, inflammatory cell infiltration, and changes in tissue morphology as well as signaling molecule activation and collagen, tenascin-C, and ASMA expression associated with lung fibrosis. CSD peptide may be a prototype for novel treatments for human lung fibrosis that act, in part, by inhibiting the expression of ASMA and ECM proteins.     

Aortic Carboxypeptidase-like Protein (ACLP) Enhances Lung Myofibroblast Differentiation through Transforming Growth Factor β Receptor-dependent and -independent Pathways

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFβ, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFβ signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFβ receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFβ receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFβ signaling and promoting collagen expression through a TGFβ receptor-independent pathway.     

A new FGFR inhibitor disrupts the TGF‐β1‐induced fibrotic process

Pulmonary fibrosis (PF) is chronic and irreversible damage to the lung characterized by fibroblast activation and matrix deposition. Although recently approved novel anti‐fibrotic agents can improve the lung function and survival of patients with PF, the overall outcomes remain poor. In this study, a novel imidazopurine compound, 3‐(2‐chloro‐6‐fluorobenzyl)‐1,6,7‐trimethyl‐1H‐imidazo[2,1‐f]purine‐2,4(3H,8H)‐dione (IM‐1918), markedly inhibited transforming growth factor (TGF)‐β‐stimulated reporter activity and reduced the expression of representative fibrotic markers, such as connective tissue growth factor, fibronectin, collagen and α‐smooth muscle actin, on human lung fibroblasts. However, IM‐1918 neither decreased Smad‐2 and Smad‐3 nor affected p38MAPK and JNK. Instead, IM‐1918 reduced Akt and extracellular signal‐regulated kinase 1/2 phosphorylation increased by TGF‐β. Additionally, IM‐1918 inhibited the phosphorylation of fibroblast growth factor receptors 1 and 3. In a bleomycin‐induced murine lung fibrosis model, IM‐1918 profoundly reduced fibrotic areas and decreased collagen and α‐smooth muscle actin accumulation. These results suggest that IM‐1918 can be applied to treat lung fibrosis.     

MicroRNA-411-3p inhibits bleomycin-induced skin fibrosis by regulating transforming growth factor-β/Smad ubiquitin regulatory factor-2 signalling

Skin fibrosis, which is characterized by fibroblast proliferation and increased extracellular matrix, has no effective treatment. An increasing number of studies have shown that microRNAs (miRNAs/miRs) participate in the mechanism of skin fibrosis, such as in limited cutaneous systemic sclerosis and pathological scarring. The objective of the present study was to determine the role of miR-411-3p in bleomycin (BLM)-induced skin fibrosis and skin fibroblast transformation. Using Western blot analysis and real-time quantitative polymerase chain reaction assess the expression levels of miR-411-3p, collagen (COLI) and transforming growth factor (TGF)-β/Smad ubiquitin regulatory factor (Smurf)-2/Smad signalling factors both in vitro and in vivo with or without BLM. To explore the regulatory relationship between miR-411-3p and Smurf2, we used the luciferase reporter assay. Furthermore, miR-411-3p overexpression was identified in vitro and in vivo via transfection with Lipofectamine 2000 reagent and injection. Finally, we tested the dermal layer of the skin using haematoxylin and eosin and Van Gieson's staining. We found that miR-411-3p expression was decreased in bleomycin (BLM)-induced skin fibrosis and fibroblasts. However, BLM accelerated transforming growth factor (TGF)-β signalling and collagen production. Overexpression of miR-411-3p inhibited the expression of collagen, F-actin and the TGF-β/Smad signalling pathway factors in BLM-induced skin fibrosis and fibroblasts. In addition, miR-411-3p inhibited the target Smad ubiquitin regulatory factor (Smurf)-2. Furthermore, Smurf2 was silenced, which attenuated the expression of collagen via suppression of the TGF-β/Smad signalling pathway. We demonstrated that miR-411-3p exerts antifibrotic effects by inhibiting the TGF-β/Smad signalling pathway via targeting of Smurf2 in skin fibrosis.     

Smad3 mediates TGF-beta1-induced collagen gel contraction by human lung fibroblasts

Transforming growth factor-beta1 (TGF-beta1) is a key mediator in tissue repair and fibrosis. Using small interference RNA (siRNA), the role of Smad2 and Smad3 in TGF-beta stimulation of human lung fibroblast contraction of collagenous matrix and induction of alpha-SMA and the role of alpha-SMA in contraction were assessed. HFL-1 cells were transfected with Smad2, Smad3 or control-siRNA, and cultured in floating Type I collagen gels +/- -TGF-beta1. TGF-beta1 augmented gel contraction in Smad2-siRNA- and control-siRNA-treated cells, but had no effect in Smad3-siRNA-treated cells. Similarly, TGF-beta1 upregulated alpha-SMA in Smad2-siRNA- and control-siRNA-treated cells, but had no effect on Smad3-siRNA-treated cells. Alpha-SMA-siRNA-treated cells did not contact the collagen gels with or without TGF-beta1, suggesting alpha-SMA is required for gel contraction. Thus, Smad3 mediates TGF-beta1-induced contraction and alpha-SMA induction in human lung fibroblasts. Smad3, therefore, could be a target for blocking contraction of human fibrotic tissue induced by TGF-beta1.     

Protection from fluorescein isothiocyanate-induced fibrosis in IL-13-deficient, but not IL-4-deficient, mice results from impaired collagen synthesis by fibroblasts

Intratracheal injection of FITC results in acute lung injury and progresses to fibrosis by day 21 postchallenge. In response to FITC, BALB/c mice produce IL-4 and IL-13 in the lung. To investigate whether IL-4 and/or IL-13 were important profibrotic mediators in this model, we examined the fibrotic response to FITC in mice that were genetically deficient in IL-4 (IL-4(-/-)), IL-13 (IL-13(-/-)), or IL-4 and IL-13 combined (IL-4/13(-/-)). Baseline levels of collagen were similar in all mice. In response to FITC, both BALB/c and IL-4(-/-) mice developed fibrosis, whereas the IL-13(-/-) and IL-4/13(-/-) mice were significantly protected, as measured by total lung collagen levels and histology. Total leukocyte recruitment to the lung was similar in all four strains of mice when measured on days 7, 14, and 21 post-FITC. BALB/c mice showed prominent eosinophilia on day 7 that was absent in IL-4(-/-), IL-13(-/-), and IL-4/13(-/-) mice, suggesting that eosinophilia is not necessary for development of a fibrotic response. There were no significant differences in the percentages of any other leukocytes analyzed between the genotypes. Similarly, protection in IL-13(-/-) mice was not associated with alterations in cytokine or eicosanoid profiles. Interestingly, TGF-beta1 production was not reduced in IL-13(-/-) mice. Analyses of fibroblasts isolated from the four genotypes demonstrated that although there were similar numbers of fibroblasts present in cultures of lung minces, fibroblasts from IL-13-deficient strains have reduced basal and stimulated levels of collagen production. IL-13Ralpha1 expression increases on fibroblasts during fibrotic responses in vivo, and IL-13 increases collagen synthesis in fibroblasts. Thus, IL-13 mediates its profibrotic actions through direct effects on fibroblast production of extracellular matrix.     

Angiotensin II and the fibroproliferative response to acute lung injury

Angiotensin II (ANG II), generated by activation of local renin-angiotensin systems, is believed to play an important role in tissue repair and remodeling, in part via transforming growth factor-beta (TGF-beta). Angiotensin-converting enzyme (ACE) inhibitors have been shown to abrogate experimental lung injury via a number of potential mechanisms; however, the potentially fibroproliferative role for ANG II in the lung has not been characterized. We hypothesized that, after lung injury, ANG II would stimulate fibroblast procollagen synthesis and promote lung collagen deposition in rats. In vitro, ANG II was a potent inducer of procollagen production in human lung fibroblasts via activation of the type 1 receptor and, at least in part, via the autocrine action of TGF-beta. After bleomycin-induced lung injury, an increase in lung ANG II concentration was observed by day 3 that preceded increases in lung collagen and was maintained until death at day 21. Administration of an ACE inhibitor (ramipril) reduced ACE activity, ANG II concentration, TGF-beta expression, and collagen deposition. Losartan (an ANG II type 1 receptor antagonist) also attenuated the increase in TGF-beta expression and lung collagen deposition. These observations suggest that ANG II, possibly generated locally within the lung, may play an important role in the fibrotic response to acute lung injury, at least in part via the action of TGF-beta. ACE inhibitors and receptor antagonists, already widely used clinically, should be assessed as potential new therapies for fibrotic lung disease.     

A novel reciprocal loop between microRNA-21 and TGFβRIII is involved in cardiac fibrosis

Cardiac fibrosis is characterized by aberrant proliferation of cardiac fibroblasts and exaggerated deposition of extracellular matrix (ECM) in the myocardial interstitial, and ultimately impairs cardiac function. It is still controversial whether microRNA-21 (miR-21) participates in the process of cardiac fibrosis. Our previous study confirmed that transforming growth factor beta receptor III (TGFβRIII) is a negative regulator of TGF-β pathway. Here, we aimed to decipher the relationship between miR-21 and TGFβRIII in the pathogenic process of myocardial fibrosis. We found that TGF-β1 and miR-21 were up-regulated, whereas TGFβRIII was down-regulated in the border zone of mouse hearts in response to myocardial infarction. After transfection of miR-21 into cardiac fibroblasts, TGFβRIII expression was markedly reduced and collagen content was increased. And, luciferase results confirmed that TGFβRIII was a target of miR-21. It suggests that up-regulation of miR-21 could increase the collagen content and at least in part through inhibiting TGFβRIII. Conversely, we also confirmed that overexpression of TGFβRIII could inhibit the expression of miR-21 and reduce collagen production in fibroblasts. Further studies showed that overexpression of TGFβRIII could also deactivate TGF-β1 pathway by decreasing the expression of TGF-β1 and phosphorylated-Smad3 (p-Smad3). TGF-β1 has been proven as a positive regulator of miR-21. Taken together, we found a novel reciprocal loop between miR-21 and TGFβRIII in cardiac fibrosis caused by myocardial infarction in mice, and targeting this pathway could be a new strategy for the prevention and treatment of myocardial remodeling.     

MicroRNA-26a modulates transforming growth factor beta-1-induced proliferation in human fetal lung fibroblasts

MicroRNA-26a is a newly discovered microRNA that has a strong anti-tumorigenic capacity and is capable of suppressing cell proliferation and activating tumor-specific apoptosis. However, whether miR-26a can inhibit the over-growth of lung fibroblasts remains unclear. The relationship between miR-26a and lung fibrosis was explored in the current study. We first investigated the effect of miR-26a on the proliferative activity of human lung fibroblasts with or without TGF-beta1 treatment. We found that the inhibition of endogenous miR-26a promoted proliferation and restoration of mature miR-26a inhibited the proliferation of human lung fibroblasts. We also examined that miR-26a can block the G1/S phase transition via directly targeting 3′-UTR of CCND2, degrading mRNA and decreasing protein expression of Cyclin D2. Furthermore, we showed that miR-26a mediated a TGF-beta 2-TGF-beta 1 feedback loop and inhibited TGF-beta R I activation. In addition, the overexpression of miR-26a also significantly suppressed the TGF-beta 1-interacting-CTGF–collagen fibrotic pathway. In summary, our studies indicated an essential role of miR-26a in the anti-fibrotic mechanism in TGF-beta1-induced proliferation in human lung fibroblasts, by directly targeting Cyclin D2, regulating TGF-beta R I as well as TGF-beta 2, and suggested the therapeutic potential of miR-26a in ameliorating lung fibrosis.     

Fibroblast-specific expression of a kinase-deficient type II transforming growth factor beta (TGFbeta) receptor leads to paradoxical activation of TGFbeta signaling pathways with fibrosis in transgenic mice

To better understand the role of disrupted transforming growth factor beta (TGFbeta) signaling in fibrosis, we have selectively expressed a kinase-deficient human type II TGFbeta receptor (TbetaRIIDeltak) in fibroblasts of transgenic mice, using a lineage-specific expression cassette subcloned from the pro-alpha2(I) collagen gene. Surprisingly, despite previous studies that characterized TbetaRIIDeltak as a dominant negative inhibitor of TGFbeta signaling, adult mice expressing this construct demonstrated TGFbeta overactivity and developed dermal and pulmonary fibrosis. Compared with wild type cells, transgenic fibroblasts proliferated more rapidly, produced more extracellular matrix, and showed increased expression of key markers of TGFbeta activation, including plasminogen activator inhibitor-1, connective tissue growth factor, Smad3, Smad4, and Smad7. Smad2/3 phosphorylation was increased in transgenic fibroblasts. Overall, the gene expression profile of explanted transgenic fibroblasts using cDNA microarrays was very similar to that of littermate wild type cells treated with recombinant TGFbeta1. Despite basal up-regulation of TGFbeta signaling pathways, transgenic fibroblasts were relatively refractory to further stimulation with TGFbeta1. Thus, responsiveness of endogenous genes to TGFbeta was reduced, and TGFbeta-regulated promoter-reporter constructs transiently transfected into transgenic fibroblasts showed little activation by recombinant TGFbeta1. Responsiveness was partially restored by overexpression of wild type type II TGFbeta receptors. Activation of MAPK pathways by recombinant TGFbeta1 appeared to be less perturbed than Smad-dependent signaling. Our results show that expression of TbetaRIIDeltak selectively in fibroblasts leads to paradoxical ligand-dependent activation of downstream signaling pathways and causes skin and lung fibrosis. As well as confirming the potential for nonsignaling receptors to regulate TGFbeta activity, these findings support a direct role for perturbed TGFbeta signaling in fibrosis and provide a novel genetically determined animal model of fibrotic disease.