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TGF-β signals through a receptor complex composed of 2 type I and 2 type II (TGF-βRII) subunits. We investigated the role of macrophage TGF-β signaling in fibrosis after AKI in mice with selective monocyte/macrophage TGF-βRII deletion (macrophage TGF-βRII-/- mice). Four weeks after injury, renal TGF-β1 expression and fibrosis were higher in WT mice than macrophage TGF-βRII-/- mice, which had decreased renal macrophages. The in vitro chemotactic response to f-Met-Leu-Phe was comparable between bone marrow-derived monocytes (BMMs) from WT and macrophage TGF-βRII-/- mice, but TGF-βRII-/- BMMs did not respond to TGF-β. We then implanted Matrigel plugs suffused with either f-Met-Leu-Phe or TGF-β1 into WT or macrophage TGF-βRII-/- mice. After 6 days, f-Met-Leu-Phe induced similar macrophage infiltration into the Matrigel plugs of WT and macrophage TGF-βRII-/- mice, but TGF-β induced infiltration only in WT mice. We further determined the number of labeled WT or TGF-βRII-/- BMMs infiltrating into WT kidneys 20 days after ischemic injury. There were more labeled WT BMMs than TGF-βRII-/- BMMs. Therefore, macrophage TGF-βRII deletion protects against the development of tubulointerstitial fibrosis following severe ischemic renal injury. Chemoattraction of macrophages to the injured kidney through a TGF-β/TGF-βRII axis is a heretofore undescribed mechanism by which TGF-β can mediate renal fibrosis during progressive renal injury.
Pulmonary fibrosis is a progressive inflammatory disease with high mortality and limited therapeutic options. Previous genetic and immunologic investigations suggest common intersections between idiopathic pulmonary fibrosis (IPF), sarcoidosis, and murine models of pulmonary fibrosis. To identify immune responses that precede collagen deposition, we conducted molecular, immunohistochemical, and flow cytometric analysis of human and murine specimens. Immunohistochemistry revealed programmed cell death-1 (PD-1) up-regulation on IPF lymphocytes. PD-1CD4 T cells with reduced proliferative capacity and increased transforming growth factor-β (TGF-β)/interleukin-17A (IL-17A) expression were detected in IPF, sarcoidosis, and bleomycin CD4 T cells. PD-1 T helper 17 cells are the predominant CD4 T cell subset expressing TGF-β. Coculture of PD-1CD4 T cells with human lung fibroblasts induced collagen-1 production. Strikingly, ex vivo PD-1 pathway blockade resulted in reductions in TGF-β and IL-17A expression from CD4 T cells, with concomitant declines in collagen-1 production from fibroblasts. Molecular analysis demonstrated PD-1 regulation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Chemical blockade of STAT3, using the inhibitor STATTIC, inhibited collagen-1 production. Both bleomycin administration to PD-1 null mice or use of antibody against programmed cell death ligand 1 (PD-L1) demonstrated significantly reduced fibrosis compared to controls. This work identifies a critical, previously unrecognized role for PD-1CD4 T cells in pulmonary fibrosis, supporting the use of readily available therapeutics that directly address interstitial lung disease pathophysiology.
Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
Background & Aims - Chronic inflammation is a predisposing condition for colorectal cancer. Many studies to date have focused on proinflammatory signaling pathways in the colon. Understanding the mechanisms that suppress inflammation, particularly in epithelial cells, is critical for developing therapeutic interventions. Here, we explored the roles of transforming growth factor β (TGFβ) family signaling through SMAD4 in colonic epithelial cells.
Methods - The gene was deleted specifically in adult murine intestinal epithelium. Colitis was induced by 3 rounds of dextran sodium sulfate in drinking water, after which mice were observed for up to 3 months. Nontransformed mouse colonocyte cell lines and colonoid cultures and human colorectal cancer cell lines were analyzed for responses to TGFβ1 and bone morphogenetic protein 2.
Results - Dextran sodium sulfate treatment was sufficient to drive carcinogenesis in mice lacking colonic expression, with resulting tumors bearing striking resemblance to human colitis-associated carcinoma. Loss of SMAD4 protein was observed in 48% of human colitis-associated carcinoma samples as compared with 19% of sporadic colorectal carcinomas. Loss of increased the expression of inflammatory mediators within nontransformed mouse colon epithelial cells in vivo. In vitro analysis of mouse and human colonic epithelial cell lines and organoids indicated that much of this regulation was cell autonomous. Furthermore, TGFβ signaling inhibited the epithelial inflammatory response to proinflammatory cytokines.
Conclusions - TGFβ suppresses the expression of proinflammatory genes in the colon epithelium, and loss of its downstream mediator, SMAD4, is sufficient to initiate inflammation-driven colon cancer. Transcript profiling: GSE100082.
The TGF- and Wnt/-catenin pathways have important roles in modulating CKD, but how these growth factors affect the epithelial response to CKD is not well studied. TGF- has strong profibrotic effects, but this pleiotropic factor has many different cellular effects depending on the target cell type. To investigate how TGF- signaling in the proximal tubule, a key target and mediator of CKD, alters the response to CKD, we injured mice lacking the TGF- type 2 receptor specifically in this epithelial segment. Compared with littermate controls, mice lacking the proximal tubular TGF- receptor had significantly increased tubular injury and tubulointerstitial fibrosis in two different models of CKD. RNA sequencing indicated that deleting the TGF- receptor in proximal tubule cells modulated many growth factor pathways, but Wnt/-catenin signaling was the pathway most affected. We validated that deleting the proximal tubular TGF- receptor impaired -catenin activity and Genetically restoring -catenin activity in proximal tubules lacking the TGF- receptor dramatically improved the tubular response to CKD in mice. Deleting the TGF- receptor alters many growth factors, and therefore, this ameliorated response may be a direct effect of -catenin activity or an indirect effect of -catenin interacting with other growth factors. In conclusion, blocking TGF- and -catenin crosstalk in proximal tubules exacerbates tubular injury in two models of CKD.
Copyright © 2017 by the American Society of Nephrology.
Pulmonary vascular endothelial function may be impaired by oxidative stress in endotoxemia-derived acute lung injury. Growing evidence suggests that endothelial-to-mesenchymal transition (EndMT) could play a pivotal role in various respiratory diseases; however, it remains unclear whether EndMT participates in the injury/repair process of septic acute lung injury. Here, we analyzed lipopolysaccharide (LPS)-treated mice whose total number of pulmonary vascular endothelial cells (PVECs) transiently decreased after production of reactive oxygen species (ROS), while the population of EndMT-PVECs significantly increased. NAD(P)H oxidase inhibition suppressed EndMT of PVECs. Most EndMT-PVECs derived from tissue-resident cells, not from bone marrow, as assessed by mice with chimeric bone marrow. Bromodeoxyuridine-incorporation assays revealed higher proliferation of capillary EndMT-PVECs. In addition, EndMT-PVECs strongly expressed c-kit and CD133. LPS loading to human lung microvascular endothelial cells (HMVEC-Ls) induced reversible EndMT, as evidenced by phenotypic recovery observed after removal of LPS. LPS-induced EndMT-HMVEC-Ls had increased vasculogenic ability, aldehyde dehydrogenase activity, and expression of drug resistance genes, which are also fundamental properties of progenitor cells. Taken together, our results demonstrate that LPS induces EndMT of tissue-resident PVECs during the early phase of acute lung injury, partly mediated by ROS, contributing to increased proliferation of PVECs.
Copyright © 2016 the American Physiological Society.
BACKGROUND - The recurrent BRAF driver mutation V600E (BRAF (V600E)) is currently one of the most clinically relevant mutations in melanoma. However, the genome-wide transcriptional and epigenetic dysregulations induced by BRAF (V600E) are still unclear. The investigation of this driver mutation's functional consequences is critical to the understanding of tumorigenesis and the development of therapeutic strategies.
METHODS AND RESULTS - We performed an integrative analysis of transcriptomic and epigenomic changes disturbed by BRAF (V600E) by comparing the gene expression and methylation profiles of 34 primary cutaneous melanoma tumors harboring BRAF (V600E) with those of 27 BRAF (WT) samples available from The Cancer Genome Atlas (TCGA). A total of 711 significantly differentially expressed genes were identified as putative BRAF (V600E) target genes. Functional enrichment analyses revealed the transcription factor MITF (p < 3.6 × 10(-16)) and growth factor TGFB1 (p < 3.1 × 10(-9)) were the most significantly enriched up-regulators, with MITF being significantly up-regulated, whereas TGFB1 was significantly down-regulated in BRAF (V600E), suggesting that they may mediate tumorigenesis driven by BRAF (V600E). Further investigation using the MITF ChIP-Seq data confirmed that BRAF (V600E) led to an overall increased level of gene expression for the MITF targets. Furthermore, DNA methylation analysis revealed a global DNA methylation loss in BRAF (V600E) relative to BRAF (WT). This might be due to BRAF dysregulation of DNMT3A, which was identified as a potential target with significant down-regulation in BRAF (V600E). Finally, we demonstrated that BRAF (V600E) targets may play essential functional roles in cell growth and proliferation, measured by their effects on melanoma tumor growth using a short hairpin RNA silencing experimental dataset.
CONCLUSIONS - Our integrative analysis identified a set of BRAF (V600E) target genes. Further analyses suggested a complex mechanism driven by mutation BRAF (V600E) on melanoma tumorigenesis that disturbs specific cancer-related genes, pathways, and methylation modifications.
Deregulation of the tumour suppressor PTEN occurs in lung and skin fibrosis and diabetic and ischaemic renal injury. However, the potential role of PTEN and associated mechanisms in the progression of kidney fibrosis is unknown. Tubular and interstitial PTEN expression was dramatically decreased in several models of renal injury, including aristolochic acid nephropathy (AAN), streptozotocin (STZ)-mediated injury and ureteral unilateral obstruction (UUO), correlating with Akt, p53 and SMAD3 activation and fibrosis. Stable silencing of PTEN in HK-2 human tubular epithelial cells induced dedifferentiation and CTGF, PAI-1, vimentin, α-SMA and fibronectin expression, compared to HK-2 cells expressing control shRNA. Furthermore, PTEN knockdown stimulated Akt, SMAD3 and p53(Ser15) phosphorylation, with an accompanying decrease in population density and an increase in epithelial G1 cell cycle arrest. SMAD3 or p53 gene silencing or pharmacological blockade partially suppressed fibrotic gene expression and relieved growth inhibition orchestrated by deficiency or inhibition of PTEN. Similarly, shRNA suppression of PAI-1 rescued the PTEN loss-associated epithelial proliferative arrest. Moreover, TGFβ1-initiated fibrotic gene expression is further enhanced by PTEN depletion. Combined TGFβ1 treatment and PTEN silencing potentiated epithelial cell death via p53-dependent pathways. Thus, PTEN loss initiates tubular dysfunction via SMAD3- and p53-mediated fibrotic gene induction, with accompanying PAI-1-dependent proliferative arrest, and cooperates with TGFβ1 to induce the expression of profibrotic genes and tubular apoptosis.
Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Pulmonary arterial hypertension (PAH) is commonly associated with chronic hypoxemia in disorders such as chronic obstructive pulmonary disease (COPD). Prostacyclin analogs are widely used in the management of PAH patients; however, clinical efficacy and long-term tolerability of some prostacyclin analogs may be compromised by concomitant activation of the E-prostanoid 3 (EP3) receptor. Here, we found that EP3 expression is upregulated in pulmonary arterial smooth muscle cells (PASMCs) and human distal pulmonary arteries (PAs) in response to hypoxia. Either pharmacological inhibition of EP3 or Ep3 deletion attenuated both hypoxia and monocrotaline-induced pulmonary hypertension and restrained extracellular matrix accumulation in PAs in rodent models. In a murine PAH model, Ep3 deletion in SMCs, but not endothelial cells, retarded PA medial thickness. Knockdown of EP3α and EP3β, but not EP3γ, isoforms diminished hypoxia-induced TGF-β1 activation. Expression of either EP3α or EP3β in EP3-deficient PASMCs restored TGF-β1 activation in response to hypoxia. EP3α/β activation in PASMCs increased RhoA-dependent membrane type 1 extracellular matrix metalloproteinase (MMP) translocation to the cell surface, subsequently activating pro-MMP-2 and promoting TGF-β1 signaling. Activation or disruption of EP3 did not influence PASMC proliferation. Together, our results indicate that EP3 activation facilitates hypoxia-induced vascular remodeling and pulmonary hypertension in mice and suggest EP3 inhibition as a potential therapeutic strategy for pulmonary hypertension.
Effective therapy to prevent organ fibrosis, which is associated with more than half of all mortalities, remains elusive. Involvement of tumor suppressor ataxia telangiectasia mutated (ATM) in the TGF-β1 pathway related to renal fibrosis is largely unknown. ATM activation (pATM(Ser1981)) increased 4-fold in the tubulointerstitial region of the unilateral ureteral obstruction-injured kidney in mice correlating with SMAD3 and p53(Ser15) phosphorylation and elevated levels of p22(phox) subunit of the NADPH oxidases (NOXs), and fibrotic markers, plasminogen activator inhibitor-1 (PAI-1), and fibronectin, when compared to contralateral (contra) or sham controls. In fact, ATM is rapidly phosphorylated at Ser(1981) by TGF-β1 stimulation. Stable silencing and pharmacologic inhibition of ATM ablated TGF-β1-induced p53 activation (>95%) and subsequent PAI-1, fibronectin, connective tissue growth factor, and p21 expression in human kidney 2 (HK-2) tubular epithelial cells and normal rat kidney-49 fibroblasts (NRK-49F). ATM or p53 depletion in HK-2 cells, moreover, bypassed TGF-β1-mediated cytostasis evident in control short hairpin RNA-expressing HK-2 cells. Interestingly, stable silencing of NOX subunits, p22(phox) and p47(phox), in HK-2 cells blocked TGF-β1-induced pATM(Ser1981) (>90%) and target gene induction via p53-dependent mechanisms. Furthermore, NRK-49F fibroblast proliferation triggered by conditioned media from TGF-β1-stimulated, control vector-transfected HK-2 cells decreased (∼ 50%) when exposed to conditioned media from ATM-deficient, TGF-β1-treated HK-2 cells. Thus, TGF-β1 promotes NOX-dependent ATM activation leading to p53-mediated fibrotic gene reprogramming and growth arrest in HK-2 cells. Furthermore, TGF-β1/ATM-initiated paracrine factor secretion by dysfunctional renal epithelium promotes interstitial fibroblast growth, suggesting a role of tubular ATM in mediating epithelial-mesenchymal cross-talk highlighting the translational benefit of targeting the NOX/ATM/p53 axis in renal fibrosis.
Breast cancer survival rates decrease from 99% for patients with local disease to 25% for those with distant metastases. Matrix metalloproteinases (MMPs), including MMP2, are associated with metastatic progression. We found that loss of host MMP2 reduces the proliferation of experimental metastases in the lungs and identified fibroblasts in tumour-bearing lungs as the major source of MMP2. In vitro, spheroidal mammary tumour growth was increased by co-culture with control fibroblasts isolated from tumour-bearing lungs, but not when fibroblasts with stable Mmp2 knockdown were used. This result prompted us to assess whether MMP2 was responsible for a tumour-proliferative, activated fibroblast phenotype. To test this, we evaluated: (a) fibroblasts from wild-type tumour-bearing lungs, with or without shRNA-mediated MMP2 knockdown; and (b) normal, quiescent fibroblasts isolated from either WT or Mmp2(-/-) mice. Quantitative PCR revealed that Mmp2 knockdown attenuated expression of two markers of activation (α-smooth muscle actin and vimentin), but there was minimal expression in quiescent WT or Mmp2(-/-) fibroblasts, as expected. Placing quiescent fibroblasts under activating conditions led to increases in activation-associated transcripts in WT but not Mmp2(-/-) fibroblasts. Additionally, Mmp2 knockdown fibroblasts showed significantly decreased expression of the matrix transcripts collagen I, collagen IV and fibronectin. Addition of active TGFβ was sufficient to rescue the MMP2-dependent collagen I and IV expression, while MMP2-induced collagen expression was blocked by the addition of TGFβ1-neutralizing antibody. Gene expression data in stromal cells of human breast cancers reveal that MMP2 expression is also positively correlated with activation and matrix transcripts. Thus, we present a model whereby MMP2 production in tumour fibroblasts is important for TGFβ1 activity and subsequent activation of fibroblasts to a matrix-producing, proliferation-supportive phenotype. Overall, our results reveal a previously undefined role for MMP2 in metastatic outgrowth mediated by fibroblasts, and extend the mechanisms by which MMPs contribute to tumour progression.
Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.