Endothelin-1 mediated glycosaminoglycan synthesizing gene expression involves NOX-dependent transactivation of the transforming growth factor-ß receptor.

G protein-coupled receptor (GPCR) agonist endothelin-1 (ET-1) through transactivation of the transforming growth factor (TGF) ß receptor (TGFBR1) stimulates glycosaminoglycan (GAG) elongation on proteoglycans. GPCR agonists thrombin and lysophosphatidic acid (LPA) via respective receptors transactivate the TGFBR1 via Rho/ROCK dependent pathways however mechanistic insight for ET-1 transactivation of the TGFBR1 remains unknown. NADPH oxidase (NOX) generates reactive oxygen species (ROS) and is a signalling entity implicated in the pathogenesis of many diseases including atherosclerosis. If implicated in this pathway, NOX/ROS would be a potential therapeutic target. In this study, we investigated the involvement of NOX in ET-1/ET receptor-mediated transactivation of TGFBR1 to stimulate mRNA expression of GAG chain synthesizing enzymes chondroitin 4-O-sulfotransferase 1 (C4ST-1) and chondroitin sulfate synthase 1 (ChSy-1). The invitro model used vascular smooth muscle cells that were treated with pharmacological antagonists in the presence and absence of ET-1 or TGF-ß. Proteins and phosphoproteins isolated from treated cells were quantified by western blotting and quantitative real-time PCR was used to assess mRNA expression of GAG synthesizing enzymes. In the presence of diphenyliodonium (DPI) (NOX inhibitor), ET-1 stimulated phospho-Smad2C levels were inhibited. ET-1 mediated mRNA expression of GAG synthesizing enzymes C4ST-1 and ChSy-1 was also blocked by TGBFR1 antagonists, SB431542, broad spectrum ET receptor antagonist bosentan, DPI and ROS scavenger N-acetyl-L-cysteine. This work shows that NOX and ROS play an important role in ET-1 mediated transactivation of the TGFBR1 and downstream gene targets associated with GAG chain elongation. As ROS is involved in GPCR to protein tyrosine kinase receptor transactivation, the NOX/ROS axis presents as the first common biochemical target in all GPCR to kinase receptor transactivation signalling.

MicroRNA-665 Regulates Cell Proliferation and Apoptosis of Vascular Smooth Muscle Cells by Targeting TGFBR1.

Coronary artery disease (CAD) is one of the heavy health burdens worldwide. Aberrant proliferation of vascular smooth muscle cells (VSMCs) contributes to the occurrence and development of CAD. This study aimed at exploring differentially expressed microRNAs (miRNAs) and their regulatory mechanisms in the development of CAD.The miRNA expression profile of GSE28858 was obtained from the Gene Expression Omnibus database. Differentially expressed miRNAs (DEmiRNAs) between CAD and healthy control samples were analyzed using limma package in R. Target genes of DEmiRNAs were predicted, and a miRNA-target gene network was constructed. The relationship between miR-665 and transforming growth factor beta receptor 1 (TGFBR1) was selected for further analysis. The interaction between miR-665 and TGFBR1 was confirmed by dual luciferase reporter assay. Effects of miR-665 on cell viability and apoptosis of VSMCs were evaluated by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Besides, western blot assays for BCL2L11 and caspase 3 were also conducted.A total of 38 upregulated miRNAs and 28 downregulated miRNAs were identified. The expression level of miR-665 was significantly downregulated in patients with CAD. TGFBR1 was proved to be a target gene of miR-665. Besides, ectopic expression of miR-665 obviously inhibited VSMC growth and promoted VSMC apoptosis. TGFBR1 overexpression in VSMCs transfected with miR-665 mimic could restore the effect of miR-665 on the proliferation and apoptosis of VSMCs.MiR-665 might participate in the proliferation and apoptosis of VSMCs by targeting TGFBR1.

Distinct effects of pharmacological inhibition of stromelysin1 on endothelial-to-mesenchymal transition and myofibroblast differentiation.

Endothelial-to-mesenchymal transition (EndMT) and fibroblast-to-myofibroblast (FibroMF) differentiation are frequently reported in organ fibrosis. Stromelysin1, a matrix metalloprotease-3 (MMP3) has been indicated in vascular pathologies and organ injuries that often lead to fibrosis. In the current study, we investigated the role of stromelysin1 in EndMT and FibroMF differentiation, which is currently unknown. In our results, whereas TGFß2 treatment of endothelial cells (ECs) induced EndMT associated with increased expression of stromelysin1 and mesenchymal markers such as α-smooth muscle actin (αSMA), N-cadherin, and activin linked kinase-5 (ALK5), inhibition of stromelysin1 blunted TGFß2-induced EndMT. In contrast, treatment of NIH-3T3 fibroblasts with TGFß1 promoted FibroMF differentiation accompanied by increased expression of αSMA, N-cadherin, and ALK5. Intriguingly, stromelysin1 inhibition in TGFß1-stimulated myofibroblasts further exacerbated fibroproliferation with increased FibroMF marker expression. Gene Expression Omnibus (GEO) data analysis indicated increased stromelysin1 expression associated with EndMT and decreased stromelysin1 expression in human pulmonary fibrosis fibroblasts. In conclusion, our study has identified that EndMT and FibroMF differentiation are reciprocally regulated by stromelysin1.

The Effects of the Transforming Growth Factor-ß1 (TGF-ß1) Signaling Pathway on Cell Proliferation and Cell Migration are Mediated by Ubiquitin Specific Protease 4 (USP4) in Hypertrophic Scar Tissue and Primary Fibroblast Cultures.

BACKGROUND Hypertrophic scar results from an abnormal repair response to trauma in the skin and involves fibroblasts proliferation with increased collagen deposition. Transforming growth factor-ß1 (TGF-ß1) and TGF-ß receptor type I (TGF-ßR1) are involved in tissue repair and are increased by ubiquitin-specific protease 4 (USP4). This study aimed to investigate the effects of TGF-ßR1 and USP4 in human tissue samples of hypertrophic scar and on cell proliferation and cell migration in primary fibroblast cultures in vitro. MATERIAL AND METHODS Skin excision tissue samples with adjacent normal skin were obtained from 15 patients with hypertrophic scar, which provided tissue sections and primary fibroblast culture for analysis. Immunohistochemistry detected the expression of USP4 and TGF-ßR1 in tissue sections. MicroRNA (miRNAs) expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was performed to measure protein expression levels. Cultured skin fibroblasts were investigated using immunofluorescence staining. Fibroblast proliferation, apoptosis, and migration were measured with the Cell Counting Kit-8 (CCK-8) assay, flow cytometry, and a wound-healing assay, respectively. RESULTS The expression of USP4 and TGF-ßR1 in hypertrophic scar were increased compared with normal skin. Fibroblasts cultured from hypertrophic scar tissue showed increased expression of of USP4 and TGF-ßR1. Fibroblast transfection with USP4 short-interfering RNA (siRNA) resulted in reduced fibroblast proliferation and migration, and increased apoptosis. Downregulation of USP4 inhibited the expression of TGF-ßR1 protein and increased the expression levels of Smad7 protein. CONCLUSIONS USP4 regulated the proliferation, migration, and apoptosis of hypertrophic scar fibroblasts by regulating the TGF-ß1 signaling pathway.

TGFßRI antagonist inhibits HIV-1 Nef-induced CC chemokine family ligand 2 (CCL2) in the brain and prevents spatial learning impairment.

BACKGROUND: HIV-1-associated neurocognitive disorders (HAND) progression is related to continued inflammation despite undetectable viral loads and may be caused by early viral proteins expressed by latently infected cells. Astrocytes represent an HIV reservoir in the brain where the early viral neurotoxin negative factor (Nef) is produced. We previously demonstrated that astrocytic expression of Nef in the hippocampus of rats causes inflammation, macrophage infiltration, and memory impairment. Since these processes are affected by TGFß signaling pathways, and TGFß-1 is found at higher levels in the central nervous system of HIV-1+ individuals and is released by astrocytes, we hypothesized a role for TGFß-1 in our model of Nef neurotoxicity. METHODS: To test this hypothesis, we compared cytokine gene expression by cultured astrocytes expressing Nef or green fluorescent protein. To determine the role of Nef and a TGFßRI inhibitor on memory and learning, we infused astrocytes expressing Nef into the hippocampus of rats and then treated them daily with an oral dose of SD208 (10 mg/kg) or placebo for 7 days. During this time, locomotor activity was recorded in an open field and spatial learning tested in the novel location recognition paradigm. Postmortem tissue analyses of inflammatory and signaling molecules were conducted using immunohistochemistry and immunofluorescence. RESULTS: TGFß-1 was induced in cultures expressing Nef at 24 h followed by CCL2 induction which was prevented by blocking TGFßRI with SD208 (competitive inhibitor). Interestingly, Nef seems to change the TGFßRI localization as suggested by the distribution of the immunoreactivity. Nef caused a deficit in spatial learning that was recovered upon co-administration of SD208. Brain tissue from Nef-treated rats given SD208 showed reduced CCL2, phospho-SMAD2, cluster of differentiation 163 (CD163), and GFAP immunoreactivity compared to the placebo group. CONCLUSIONS: Consistent with our previous findings, rats treated with Nef showed deficits in spatial learning and memory in the novel location recognition task. In contrast, rats treated with Nef + SD208 showed better spatial learning suggesting that Nef disrupts memory formation in a TGFß-1-dependent manner. The TGFßRI inhibitor further reduced the induction of inflammation by Nef which was concomitant with decreased TGFß signaling. Our findings suggest that TGFß-1 signaling is an intriguing target to reduce neuroHIV.

Glial cell line-derived neurotrophic factor (GDNF) mediates hepatic stellate cell activation via ALK5/Smad signalling.

OBJECTIVE: Although glial cell line-derived neurotrophic factor (GDNF) is a member of the transforming growth factor-ß superfamily, its function in liver fibrosis has rarely been studied. Here, we investigated the role of GDNF in hepatic stellate cell (HSC) activation and liver fibrosis in humans and mice. DESIGN: GDNF expression was examined in liver biopsies and sera from patients with liver fibrosis. The functional role of GDNF in liver fibrosis was examined in mice with adenoviral delivery of the GDNF gene, GDNF sgRNA CRISPR/Cas9 and the administration of GDNF-blocking antibodies. GDNF was examined on HSC activation using human and mouse primary HSCs. The binding of activin receptor-like kinase 5 (ALK5) to GDNF was determined using surface plasmon resonance (SPR), molecular docking, mutagenesis and co-immunoprecipitation. RESULTS: GDNF mRNA and protein levels are significantly upregulated in patients with stage F4 fibrosis. Serum GDNF content correlates positively with α-smooth muscle actin (α-SMA) and Col1A1 mRNA in human fibrotic livers. Mice with overexpressed GDNF display aggravated liver fibrosis, while mice with silenced GDNF expression or signalling inhibition by GDNF-blocking antibodies have reduced fibrosis and HSC activation. GDNF is confined mainly to HSCs and contributes to HSC activation through ALK5 at His39 and Asp76 and through downstream signalling via Smad2/3, but not through GDNF family receptor alpha-1 (GFRα1). GDNF, ALK5 and α-SMA colocalise in human and mouse HSCs, as demonstrated by confocal microscopy. CONCLUSIONS: GDNF promotes HSC activation and liver fibrosis through ALK5/Smad signalling. Inhibition of GDNF could be a novel therapeutic strategy to combat liver fibrosis.

Transforming growth factor-ß (TGF-ß)-induced up-regulation of TGF-ß receptors at the cell surface amplifies the TGF-ß response.

Functional activation of the transforming growth factor-ß (TGF-ß) receptors (TGFBRs) is carefully regulated through integration of post-translational modifications, spatial regulation at the cellular level, and TGFBR availability at the cell surface. Although the bulk of TGFBRs resides inside the cells, AKT Ser/Thr kinase (AKT) activation in response to insulin or other growth factors rapidly induces transport of TGFBRs to the cell surface, thereby increasing the cell's responsiveness to TGF-ß. We now demonstrate that TGF-ß itself induces a rapid translocation of its own receptors to the cell surface and thus amplifies its own response. This mechanism of response amplification, which hitherto has not been reported for other cell-surface receptors, depended on AKT activation and TGF-ß type I receptor kinase. In addition to an increase in cell-surface TGFBR levels, TGF-ß treatment promoted TGFBR internalization, suggesting an overall amplification of TGFBR cycling. The TGF-ß-induced increase in receptor presentation at the cell surface amplified TGF-ß-induced SMAD family member (SMAD) activation and gene expression. Furthermore, bone morphogenetic protein 4 (BMP-4), which also induces AKT activation, increased TGFBR levels at the cell surface, leading to enhanced autocrine activation of TGF-ß-responsive SMADs and gene expression, providing context for the activation of TGF-ß signaling in response to BMP during development. In summary, our results indicate that TGF-ß- and BMP-induced activation of low levels of cell surface-associated TGFBRs rapidly mobilizes additional TGFBRs from intracellular stores to the cell surface, increasing the abundance of cell-surface TGFBRs and cells' responsiveness to TGF-ß signaling.

Upregulation of transforming growth factor-beta type I receptor by interferon consensus sequence-binding protein in osteosarcoma cells.

Transforming growth factor-beta (TGF-ß) is a known tumor suppressor, which also exerts a tumor promoting activity at an advanced stage of cancer. Previously, we reported that expression of interferon consensus sequence-binding protein (ICSBP), also known as interferon regulatory factor-8, is positively correlated with TGF-ß type I receptor (TGF-ß RI) expression in osteosarcoma patient tissues. In this study, we demonstrated that ICSBP upregulated TGF-ß RI and induced epithelial-to-mesenchymal transition-like phenomena in human osteosarcoma cell lines. As determined by soft agar growth of osteosarcoma cells and xenografted mouse models, ICSBP increased tumorigenicity, which was reversed by ICSBP knock-down or a TGF-ß RI inhibitor. To test whether ICSBP directly regulates the promoter activity of TGF-ß RI, we performed a TGF-ß RI promoter assay, an electro mobility shift assay, and a chromatin immunoprecipitation assay. We observed that TGF-ß RI promoter was activated in ICSBP-overexpressing osteosarcoma cells. Exploiting serial deletions and mutations of the TGF-ß RI promoter, we found a putative ICSBP-binding site at nucleotides -216/-211 (GGXXTC) in the TGF-ß RI promoter. Our data suggest that ICSBP upregulates TGF-ß RI expression by binding to this site, causing ICSBP-mediated tumor progression in osteosarcoma cells. In addition, we found a positive correlation between ICSBP and TGF-ß RI expression in several types of tumors using the cBioportal database. SUMMARY: We demonstrated that interferon consensus sequence-binding protein upregulates transforming growth factor-beta type I receptor (TGF-ß RI) expression by binding to nucleotides -216/-211 (GGXXTC) in the TGF-ß RI promoter, which resulted in increased tumorigenicity and tumor progression in human osteosarcoma cells.

Autoimmune Retinopathy: An Immunologic Cellular-Driven Disorder.

Autoimmune retinopathy (AIR) was often mistaken for retinitis pigmentosa (RP), due to an overlap of clinical findings, but increasingly has been recognized as a unique entity in the last decade. AIR has distinctive features: sudden onset of photopsias and scotomata in patients with no family history of RP, followed by visual field and central vision loss. Initially, retina exams are normal with no sign of pigment deposits or retinal degeneration. A family history of autoimmune diseases (all types) occurs in 60% of patients. One hallmark of AIR has been the presence of anti-retinal autoimmune antibodies (ARAs) in patients' sera, but patients can continue to have ARAs even when the disease has been quiescent for years. The accumulation of ARAs represents a breakdown of retinal immune tolerance with many different immunoreactive bands found at different reference weights in AIR patients. We began investigating cellular immunity using flow cytometry and found abnormal distributions (>2 StDev) of increased memory lymphocytes and NK cells and decreased regulatory B cell subsets in many AIR patients compared to normal controls. Culture of patient lymphocytes with small amounts (25 µg) of recoverin protein for 6 days led to significant elevations of interferon gamma (IFNγ) and in some cases tumor necrosis factor alpha (TNFα) production. We found the IFNγ/IL-10 ratio in response to recoverin was elevated in patients with more active disease (defined by visual field contraction between visits), but in some patients, there also appeared to be independent factors influencing severity, suggesting other autoimmune mechanisms were at play. These cellular immune parameters may provide improved markers for active AIR.

T Cell Receptor-Regulated TGF-ß Type I Receptor Expression Determines T Cell Quiescence and Activation.

It is unclear how quiescence is enforced in naive T cells, but activation by foreign antigens and self-antigens is allowed, despite the presence of inhibitory signals. We showed that active transforming growth factor ß (TGF-ß) signaling was present in naive T cells, and T cell receptor (TCR) engagement reduced TGF-ß signaling during T cell activation by downregulating TGF-ß type 1 receptor (TßRI) through activation of caspase recruitment domain-containing protein 11 (CARD11) and nuclear factor κB (NF-κB). TGF-ß prevented TCR-mediated TßRI downregulation, but this was abrogated by interleukin-6 (IL-6). Mitigation of TCR-mediated TßRI downregulation through overexpression of TßRI in naive and activated T cells rendered T cells less responsive and suppressed autoimmunity. Naive T cells in autoimmune patients exhibited reduced TßRI expression and increased TCR-driven proliferation compared to healthy subjects. Thus, TCR-mediated regulation of TßRI-TGF-ß signaling acts as a crucial criterion to determine T cell quiescence and activation.