TGF-beta 1 induces human alveolar epithelial to mesenchymal cell transition (EMT)

Background: Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF). They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-β1 could induce epithelial mesenchymal transition (EMT) in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-β1-mediated EMT. Methods: A549 cells were examined for evidence of EMT after treatment with TGF-β1. EMT was assessed by: morphology under phase-contrast microscopy; Western analysis of cell lysates for expression of mesenchymal phenotypic markers including fibronectin EDA (Fn-EDA), and expression of epithelial phenotypic markers including E-cadherin (E-cad). Markers of fibrogenesis, including collagens and connective tissue growth factor (CTGF) were also evaluated by measuring mRNA level using RT-PCR, and protein by immunofluorescence or Western blotting. Signaling pathways for EMT were characterized by Western analysis of cell lysates using monoclonal antibodies to detect phosphorylated Erk1/2 and Smad2 after TGF-β1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-β1-mediated EMT was investigated using siRNA. Results: The data showed that TGF-β1, but not TNF-α or IL-1β, induced A549 cells with an alveolar epithelial type II cell phenotype to undergo EMT in a time-and concentration-dependent manner. The process of EMT was accompanied by morphological alteration and expression of the fibroblast phenotypic markers Fn-EDA and vimentin, concomitant with a downregulation of the epithelial phenotype marker E-cad. Furthermore, cells that had undergone EMT showed enhanced expression of markers of fibrogenesis including collagens type I and III and CTGF. MMP-2 expression was also evidenced. TGF-β1-induced EMT occurred through phosphorylation of Smad2 and was inhibited by Smad2 gene silencing; MEK inhibitors failed to attenuate either EMT-associated Smad2 phosphorylation or the observed phenotypic changes. Conclusion: Our study shows that TGF-β1 induces A549 alveolar epithelial cells to undergo EMT via Smad2 activation. Our data support the concept of EMT in lung epithelial cells, and suggest the need for further studies to investigate the phenomenon

Global gene expression patterns in the post-pneumonectomy lung of adult mice

<p>Abstract</p> <p>Background</p> <p>Adult mice have a remarkable capacity to regenerate functional alveoli following either lung resection or injury that exceeds the regenerative capacity observed in larger adult mammals. The molecular basis for this unique capability in mice is largely unknown. We examined the transcriptomic responses to single lung pneumonectomy in adult mice in order to elucidate prospective molecular signaling mechanisms used in this species during lung regeneration.</p> <p>Methods</p> <p>Unilateral left pneumonectomy or sham thoracotomy was performed under general anesthesia (n = 8 mice per group for each of the four time points). Total RNA was isolated from the remaining lung tissue at four time points post-surgery (6 hours, 1 day, 3 days, 7 days) and analyzed using microarray technology.</p> <p>Results</p> <p>The observed transcriptomic patterns revealed mesenchymal cell signaling, including up-regulation of genes previously associated with activated fibroblasts (Tnfrsf12a, Tnc, Eln, Col3A1), as well as modulation of Igf1-mediated signaling. The data set also revealed early down-regulation of pro-inflammatory cytokine transcripts and up-regulation of genes involved in T cell development/function, but few similarities to transcriptomic patterns observed during embryonic or post-natal lung development. Immunohistochemical analysis suggests that early fibroblast but not myofibroblast proliferation is important during lung regeneration and may explain the preponderance of mesenchymal-associated genes that are over-expressed in this model. This again appears to differ from embryonic alveologenesis.</p> <p>Conclusion</p> <p>These data suggest that modulation of mesenchymal cell transcriptome patterns and proliferation of S100A4 positive mesenchymal cells, as well as modulation of pro-inflammatory transcriptome patterns, are important during post-pneumonectomy lung regeneration in adult mice.</p

Identification of a Novel Signaling Pathway and Its Relevance for GluA1 Recycling

We previously showed that the serum- and glucocorticoid-inducible kinase 3 (SGK3) increases the AMPA-type glutamate receptor GluA1 protein in the plasma membrane. The activation of AMPA receptors by NMDA-type glutamate receptors eventually leads to postsynaptic neuronal plasticity. Here, we show that SGK3 mRNA is upregulated in the hippocampus of new-born wild type Wistar rats after NMDA receptor activation. We further demonstrate in the Xenopus oocyte expression system that delivery of GluA1 protein to the plasma membrane depends on the small GTPase RAB11. This RAB-dependent GluA1 trafficking requires phosphorylation and activation of phosphoinositol-3-phosphate-5-kinase (PIKfyve) and the generation of PI(3,5)P2. In line with this mechanism we could show PIKfyve mRNA expression in the hippocampus of wild type C57/BL6 mice and phosphorylation of PIKfyve by SGK3. Incubation of hippocampal slices with the PIKfyve inhibitor YM201636 revealed reduced CA1 basal synaptic activity. Furthermore, treatment of primary hippocampal neurons with YM201636 altered the GluA1 expression pattern towards reduced synaptic expression of GluA1. Our findings demonstrate for the first time an involvement of PIKfyve and PI(3,5)P2 in NMDA receptor-triggered synaptic GluA1 trafficking. This new regulatory pathway of GluA1 may contribute to synaptic plasticity and memory

The Role of Toll-Like Receptor 2 in Inflammation and Fibrosis during Progressive Renal Injury

Tissue fibrosis and chronic inflammation are common causes of progressive organ damage, including progressive renal disease, leading to loss of physiological functions. Recently, it was shown that Toll-like receptor 2 (TLR2) is expressed in the kidney and activated by endogenous danger signals. The expression and function of TLR2 during renal fibrosis and chronic inflammation has however not yet been elucidated. Therefore, we studied TLR2 expression in human and murine progressive renal diseases and explored its role by inducing obstructive nephropathy in TLR2−/− or TLR2+/+ mice. We found that TLR2 is markedly upregulated on tubular and tubulointerstitial cells in patients with chronic renal injury. In mice with obstructive nephropathy, renal injury was associated with a marked upregulation and change in distribution of TLR2 and upregulation of murine TLR2 danger ligands Gp96, biglycan, and HMGB1. Notably, TLR2 enhanced inflammation as reflected by a significantly reduced influx of neutrophils and production of chemokines and TGF-β in kidneys of TLR2−/− mice compared with TLR2+/+ animals. Although, the obstructed kidneys of TLR2−/− mice had less interstitial myofibroblasts in the later phase of obstructive nephropathy, tubular injury and renal matrix accumulation was similar in both mouse strains. Together, these data demonstrate that TLR2 can initiate renal inflammation during progressive renal injury and that the absence of TLR2 does not affect the development of chronic renal injury and fibrosis

Rapamycin Ameliorates Kidney Fibrosis by Inhibiting the Activation of mTOR Signaling in Interstitial Macrophages and Myofibroblasts

Interstitial fibrosis is an inevitable outcome of all kinds of progressive chronic kidney disease (CKD). Emerging data indicate that rapamycin can ameliorate kidney fibrosis by reducing the interstitial infiltrates and accumulation of extra cellular matrix (ECM). However, the cellular mechanism that regulates those changes has not been well understood yet. In this study, we revealed the persistent activation of mammalian target of rapamycin (mTOR) signaling in the interstitial macrophages and myofibroblasts, but rarely in injured proximal epithelial cells, CD4+ T cells, neutrophils, or endothelial cells, during the development of kidney fibrosis. Administration of rapamycin to unilateral ureteral obstruction (UUO) mice significantly suppressed the immunoreactivity of mTOR signaling, which decreased the inflammatory responses and ECM accumulation in the obstructed kidneys. Isolated macrophages from rapamycin-treated obstructed kidneys presented less inflammatory activity than vehicle groups. In vitro study confirmed that rapamycin significantly inhibited the fibrogenic activation of cultured fibroblasts (NIH3T3 cells), which was induced by the stimulation of TGF-β1. Further experiment revealed that rapamycin did not directly inhibit the fibrogenesis of HK2 cells with aristolochic acid treatment. Our findings clarified that rapamycin can ameliorate kidney fibrosis by blocking the mTOR signaling in interstitial macrophages and myofibroblasts

Cancer-associated fibroblasts are positively correlated with metastatic potential of human gastric cancers

<p>Abstract</p> <p>Background</p> <p>The prognosis of gastric cancer patients is difficult to predict because of defects in establishing the surgical-pathological features. Cancer-associated fibroblasts (CAFs) have been found to play prominent role in promoting tumor growth, invasion and metastasis. Thus raises the hypothesis that the extent of CAFs prevalence may help to establish the prognosis of gastric cancer patients.</p> <p>Methods</p> <p>Immunochemistry and realtime-PCR experiments were carried out to compare the expression of proteins which are specific markers of CAFs or secreted by CAFs in the tumor and normal tissue specimens. The extent of CAFs' prevalence was graded according to immunochemical staining, and correlation was further analyzed between CAFs' prevalence and other tumor characteristics which may influence the prognosis of gastric cancer patients.</p> <p>Results</p> <p>Nearly 80 percent of normal gastric tissues were negative or weak positive for CAFs staining, while more than 60 percent of gastric cancer tissues were moderate or strong positive for CAFs staining. Realtime-PCR results also showed significant elevated expression of FAP, SDF-1 and TGF-β1 in gastric cancer tissues compared to normal gastric tissues. Further analysis showed that CAFs' prevalence was correlated with tumor size, depth of the tumor, lymph node metastasis, liver metastasis or peritoneum metastasis.</p> <p>Conclusions</p> <p>Reactive cancer associated fibroblasts (CAFs) were frequently accumulated in gastric cancer tissues, and the prevalence of CAFs was correlated with tumor size, depth of the tumor and tumor metastasis, thus give some supports for establishing the prognosis of the gastric cancer patients.</p

Transgenic Expression of Nonclassically Secreted FGF Suppresses Kidney Repair

FGF1 is a signal peptide-less nonclassically released growth factor that is involved in angiogenesis, tissue repair, inflammation, and carcinogenesis. The effects of nonclassical FGF export in vivo are not sufficiently studied. We produced transgenic mice expressing FGF1 in endothelial cells (EC), which allowed the detection of FGF1 export to the vasculature, and studied the efficiency of postischemic kidney repair in these animals. Although FGF1 transgenic mice had a normal phenotype with unperturbed kidney structure, they showed a severely inhibited kidney repair after unilateral ischemia/reperfusion. This was manifested by a strong decrease of postischemic kidney size and weight, whereas the undamaged contralateral kidney exhibited an enhanced compensatory size increase. In addition, the postischemic kidneys of transgenic mice were characterized by hyperplasia of interstitial cells, paucity of epithelial tubular structures, increase of the areas occupied by connective tissue, and neutrophil and macrophage infiltration. The continuous treatment of transgenic mice with the cell membrane stabilizer, taurine, inhibited nonclassical FGF1 export and significantly rescued postischemic kidney repair. It was also found that similar to EC, the transgenic expression of FGF1 in monocytes and macrophages suppresses kidney repair. We suggest that nonclassical export may be used as a target for the treatment of pathologies involving signal peptide-less FGFs

Sitagliptin reduces cardiac apoptosis, hypertrophy and fibrosis primarily by insulin-dependent mechanisms in experimental type-II diabetes. Potential roles of GLP-1 isoforms

Background:Myocardial fibrosis is a key process in diabetic cardiomyopathy. However, their underlying mechanisms have not been elucidated, leading to a lack of therapy. The glucagon-like peptide-1 (GLP-1) enhancer, sitagliptin, reduces hyperglycemia but may also trigger direct effects on the heart.Methods:Goto-Kakizaki (GK) rats developed type-II diabetes and received sitagliptin, an anti-hyperglycemic drug (metformin) or vehicle (n=10, each). After cardiac structure and function assessment, plasma and left ventricles were isolated for biochemical studies. Cultured cardiomyocytes and fibroblasts were used for in vitro assays.Results:Untreated GK rats exhibited hyperglycemia, hyperlipidemia, plasma GLP-1 decrease, and cardiac cell-death, hypertrophy, fibrosis and prolonged deceleration time. Moreover, cardiac pro-apoptotic/necrotic, hypertrophic and fibrotic factors were up-regulated. Importantly, both sitagliptin and metformin lessened all these parameters. In cultured cardiomyocytes and cardiac fibroblasts, high-concentration of palmitate or glucose induced cell-death, hypertrophy and fibrosis. Interestingly, GLP-1 and its insulinotropic-inactive metabolite, GLP-1(9-36), alleviated these responses. In addition, despite a specific GLP-1 receptor was only detected in cardiomyocytes, GLP-1 isoforms attenuated the pro-fibrotic expression in cardiomyocytes and fibroblasts. In addition, GLP-1 receptor signalling may be linked to PPARδ activation, and metformin may also exhibit anti-apoptotic/necrotic and anti-fibrotic direct effects in cardiac cells.Conclusions:Sitagliptin, via GLP-1 stabilization, promoted cardioprotection in type-II diabetic hearts primarily by limiting hyperglycemia e hyperlipidemia. However, GLP-1 and GLP-1(9-36) promoted survival and anti-hypertrophic/fibrotic effects on cultured cardiac cells, suggesting cell-autonomous cardioprotective actionsThis work was supported by national funding from Ministerio de Educación y Ciencia (SAF2009-08367), Comunidad de Madrid (CCG10-UAM/ BIO-5289), and a unrestricted grant from by Merck/MS