Non-canonical TGF-β Signaling Pathways Research Articles

Complex and variable regulation of ΔNp63 and TAp63 by TGFβ has implications for the dynamics of squamous cell epithelial to mesenchymal transition

TGFβ has roles in inflammation, wound healing, epithelial to mesenchymal transition (EMT), and cancer stem cell states, and acts as a tumor suppressor gene for squamous cell carcinoma (SCC). SCCs are also characterized by high levels of ΔNp63, which induces epithelial cell phenotypes and maintains squamous stem cells. Previous studies indicate a complex interplay between ΔNp63 and TGFβ signaling, with contradictory effects reported. We investigated the effects of TGFβ on p63 isoform proteins and mRNAs in non-malignant squamous and SCC cells, and the role of either canonical or non-canonical TGFβ signaling pathways. TGFβ selectively increased ΔNp63 protein levels in non-malignant keratinocytes in association with SMAD3 activation and was prevented by TGFβ receptor inhibition, indicating activation of canonical TGFβ pathway signaling. TP63 isoform mRNAs showed discordance from protein levels, with an initial increase in both TAP63 and ΔNP63 mRNAs followed by a decrease at later times. These data demonstrate complex and heterogeneous effects of TGFβ in squamous cells that depend on the extent of canonical TGFβ pathway aberrations. The interplay between TGFβ and p63 is likely to influence the magnitude of EMT states in SCC, with clinical implications for tumor progression and response to therapy.

Copper chelation suppresses epithelial-mesenchymal transition by inhibition of canonical and non-canonical TGF-β signaling pathways in cancer

BackgroundMetastatic cancer cells exploit Epithelial-mesenchymal-transition (EMT) to enhance their migration, invasion, and resistance to treatments. Recent studies highlight that elevated levels of copper are implicated in cancer progression and metastasis. Clinical trials using copper chelators are associated with improved patient survival; however, the molecular mechanisms by which copper depletion inhibits tumor progression and metastasis are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Here, we propose that copper chelation inhibits metastasis by reducing TGF-β levels and EMT signaling. Given that many drugs targeting TGF-β have failed in clinical trials, partly because of severe side effects arising in patients, we hypothesized that copper chelation therapy might be a less toxic alternative to target the TGF-β/EMT axis.ResultsOur cytokine array and RNA-seq data suggested a link between copper homeostasis, TGF-β and EMT process. To validate this hypothesis, we performed single-cell imaging, protein assays, and in vivo studies. Here, we used the copper chelating agent TEPA to block copper trafficking. Our in vivo study showed a reduction of TGF-β levels and metastasis to the lung in the TNBC mouse model. Mechanistically, TEPA significantly downregulated canonical (TGF-β/SMAD2&3) and non-canonical (TGF-β/PI3K/AKT, TGF-β/RAS/RAF/MEK/ERK, and TGF-β/WNT/β-catenin) TGF-β signaling pathways. Additionally, EMT markers of MMP-9, MMP-14, Vimentin, β-catenin, ZEB1, and p-SMAD2 were downregulated, and EMT transcription factors of SNAI1, ZEB1, and p-SMAD2 accumulated in the cytoplasm after treatment.ConclusionsOur study suggests that copper chelation therapy represents a potentially effective therapeutic approach for targeting TGF-β and inhibiting EMT in a diverse range of cancers.

Tetraspanin CD82 Regulates Hematopoietic Stem and Progenitor Cell Quiescence & Regeneration

The significant cellular demand of the hematopoietic system is maintained by a rare pool of tissue-specific, hematopoietic stem and progenitor cells (HSPCs) that are primarily found in a quiescent state. Upon hematopoietic stress, HSPCs undergo rapid cell cycle activation, but ultimately must return to quiescence to prevent hematopoietic exhaustion. While the diverse mechanisms used by HSPCs to regulate quiescence are still being defined, it is clear that a combination of cell-intrinsic and bone marrow niche-mediated signaling pathways are involved. Tetraspanins are a unique family of membrane scaffold proteins with the capacity to modulate intrinsic and extrinsic signaling, through the formation of tetraspanin-enriched microdomains. Collectively, tetraspanins, and the microdomains they assemble, act as molecular facilitators that spatiotemporally compartmentalize membrane-associated signaling and adhesion proteins, providing a means to regulate the cell-cell communication and signal transduction associated with a breadth of cellular processes. More specifically, studies from our laboratory identified a critical role for the tetraspanin CD82 in the regulation of HSPC maintenance where we find a reduction in long term-HSPCs (LT-HSPCs) in CD82 knockout (CD82KO) mice, resulting from increased HSPC activation and a reduction of quiescent G0 cells. To further delineate how the CD82 scaffold modulates HSPC quiescence, we transplanted CD82 overexpressing (CD82OE) leukemia cell model into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Despite the fact that CD82OE cells display similar proliferation rates to control cells in culture, once localized to the bone marrow, CD82OE cells demonstrate reduced proliferation and increased quiescence, as indicated by diminished BrdU incorporation and IVIS signal. Additional mechanistic insight is provided by analysis of cells expressing a mutant form of CD82, where three N-linked glycosylation sites are inhibited (Ngly-CD82). Ngly-CD82 cells do not exhibit the same level of quiescence as CD82OE cells when injected into NSG mice, demonstrating an important role for CD82 glycosylation in the regulation of HSPC quiescence. Mechanistically, the cytokine TGF-β plays an essential role in supporting HSPC quiescence and activation, as one of the most potent inhibitors of HSPC growth. To determine if CD82 plays a role in TGF-β signaling, we stimulated WT and CD82KO HSPCs with TGF-β and measured nuclear translocation of SMAD2/3, a downstream indicator of TGF-β activation. Upon TGF-β activation, CD82KO HSPCs display decreased canonical TGF-β signaling, although our data indicate that the non-canonical TGF-β signaling pathway is not impacted. Additionally, we analyzed TGF-β signaling in primary human CD34+ HSPCs, finding that CD34+ human HSPCs with low CD82 expression also have decreased SMAD2/3 nuclear translocation suggestive of a reduced response to TGF-β stimulation. Analysis of the cell line models with differential CD82 expression suggests that cells with higher CD82 expression have increased SMAD2/3 activation following TGFβ stimulation when compared to lower expressing CD82 cells or Ngly-CD82 cells, but only when cells are engaged with fibronectin. Together, these data indicate that the CD82 scaffold has the capacity to modulate TGFb signaling through niche-mediated interactions and implicate integrins in the modulation of signal transduction.

Tumor-Induced T Cell Polarization by Schwann Cells.

Nerve-cancer crosstalk resulting in either tumor neurogenesis or intratumoral neurodegeneration is critically controlled by Schwann cells, the principal glial cells of the peripheral nervous system. Though the direct stimulating effect of Schwann cells on malignant cell proliferation, motility, epithelial-mesenchymal transition, and the formation of metastases have been intensively investigated, the ability of Schwann cells to affect the effector and regulatory immune cells in the tumor environment is significantly less studied. Here, we demonstrated that tumor cells could stimulate Schwann cells to produce high levels of prostaglandin E, which could be blocked by COX-2 inhibitors. This effect was mediated by tumor-derived TGF-β as neutralization of this cytokine in the tumor-conditioned medium completely blocked the inducible prostaglandin E production by Schwann cells. Similar protective effects were also induced by the Schwann cell pretreatment with TGF-βR1/ALK4/5/7 and MAPK/ERK kinase inhibitors of the canonical and non-canonical TGF-β signaling pathways, respectively. Furthermore, prostaglandin E derived from tumor-activated Schwann cells blocked the proliferation of CD3/CD28-activated T cells and upregulated the expression of CD73 and PD-1 on both CD4+ and CD8+ T cells, suggesting T cell polarization to the exhausted phenotype. This new pathway of tumor-induced T cell inhibition via the activation of neuroglial cells represents new evidence of the importance of nerve-cancer crosstalk in controlling tumor development and progression. A better understanding of the tumor-neuro-immune axis supports the development of efficient targets for harnessing this axis and improving the efficacy of cancer therapy.

Tumor-produced and aging-associated oncometabolite methylmalonic acid promotes cancer-associated fibroblast activation to drive metastatic progression

The systemic metabolic shifts that occur during aging and the local metabolic alterations of a tumor, its stroma and their communication cooperate to establish a unique tumor microenvironment (TME) fostering cancer progression. Here, we show that methylmalonic acid (MMA), an aging-increased oncometabolite also produced by aggressive cancer cells, activates fibroblasts in the TME, which reciprocally secrete IL-6 loaded extracellular vesicles (EVs) that drive cancer progression, drug resistance and metastasis. The cancer-associated fibroblast (CAF)-released EV cargo is modified as a result of reactive oxygen species (ROS) generation and activation of the canonical and noncanonical TGFβ signaling pathways. EV-associated IL-6 functions as a stroma-tumor messenger, activating the JAK/STAT3 and TGFβ signaling pathways in tumor cells and promoting pro-aggressive behaviors. Our findings define the role of MMA in CAF activation to drive metastatic reprogramming, unveiling potential therapeutic avenues to target MMA at the nexus of aging, the tumor microenvironment and metastasis.

Antitumor Effect of Demethylzeylasteral (T-96) on Triple-Negative Breast Cancer via LSD1-Mediate Epigenetic Mechanisms.

Background and Purpose. Breast cancer ranks first in the incidence of female tumors. Triple-negative breast cancer (TNBC), one type of breast cancer, is more aggressive and has a worse prognosis. Demethylzeylasteral (T-96) is isolated from Tripterygium wilfordii Hook F. Our previous study found that T96 could inhibit TNBC invasion via suppressing the canonical and noncanonical TGF-β signaling pathways. However, the antitumor effects and mechanisms of T-96 on TNBC have not been studied. This study is aimed at investigating the antitumor effect and mechanism of T-96 on breast cancer. Experimental approach. MTT assay, Live and Dead cell assay, and TUNEL were used to observe the antitumor effect of breast cancer cells treated with T-96. siRNA of LSD1, Co-IP, and molecular docking were used to explore the direct target and mechanism of T-96. Subcutaneous murine xenograft models were used to detect the efficacy of T-96 antitumor activity in vivo. Key Results. T-96 was more susceptible to inducing the apoptosis of highly metastatic TNBC cell lines (SUM-1315). An abnormal level of histone methylation is a crucial characteristic of metastatic cancer cells. LSD1 is a histone demethylase. We found that T-96 could significantly decrease the protein expression of LSD1, increase its target protein PTEN expression and enhance histone methylation. T-96 could also down-regulate the PI3K/AKT signaling pathway, which could be blocked by PTEN. Knockdown of LSD1 by siRNA blocked the pharmacological activity of T-96. And the molecular docking predicted T-96 processed affinity toward LSD1 through hydrogen bonding. Finally, T-96 was evaluated in a murine xenograft model of SUM-1315 cells. And T-96 could significantly inhibit tumor growth without showing marked toxicity. Conclusions & Implications. The results illustrated that T-96 exerted antitumor activity in highly metastatic TNBC by inactivating the LSD1 function.

Asthmatic Eosinophils Alter the Gene Expression of Extracellular Matrix Proteins in Airway Smooth Muscle Cells and Pulmonary Fibroblasts.

The impaired production of extracellular matrix (ECM) proteins by airway smooth muscle cells (ASMC) and pulmonary fibroblasts (PF) is a part of airway remodeling in asthma. This process might be influenced by eosinophils that migrate to the airway and abundantly secrete various cytokines, including TGF-β. We aimed to investigate the effect of asthmatic eosinophils on the gene expression of ECM proteins in ASMC and PF. A total of 34 study subjects were recruited: 14 with allergic asthma (AA), 9 with severe non-allergic eosinophilic asthma (SNEA), and 11 healthy subjects (HS). All AA patients underwent bronchial allergen challenge with D. pteronyssinus. The peripheral blood eosinophils were isolated using high-density centrifugation and magnetic separation. The individual cell cultures were made using hTERT ASMC and MRC-5 cell lines and the subjects’ eosinophils. The gene expression of ECM and the TGF-β signaling pathway was analyzed using qRT-PCR. We found that asthmatic eosinophils significantly promoted collagen I, fibronectin, versican, tenascin C, decorin, vitronectin, periostin, vimentin, MMP-9, ADAM33, TIMP-1, and TIMP-2 gene expression in ASMC and collagen I, collagen III, fibronectin, elastin, decorin, MMP-2, and TIMP-2 gene expression in PF compared with the HS eosinophil effect. The asthmatic eosinophils significantly increased the gene expression of several canonical and non-canonical TGF-β signaling pathway components in ASMC and PF compared with the HS eosinophil effect. The allergen-activated AA and SNEA eosinophils had a greater effect on these changes. In conclusion, asthmatic eosinophils, especially SNEA and allergen-activated eosinophils, imbalanced the gene expression of ECM proteins and their degradation-regulating proteins. These changes were associated with increased gene expression of TGF-β signaling pathway molecules in ASMC and PF.

Expanding the mutation and phenotype spectrum of MYH3-associated skeletal disorders

Pathogenic variants in MYH3 cause distal arthrogryposis type 2A and type 2B3 as well as contractures, pterygia and spondylocarpotarsal fusion syndromes types 1A and 1B. These disorders are ultra-rare and their natural course and phenotypic variability are not well described. In this study, we summarize the clinical features and genetic findings of 17 patients from 10 unrelated families with vertebral malformations caused by dominant or recessive pathogenic variants in MYH3. Twelve novel pathogenic variants in MYH3 (NM_002470.4) were identified: three of them were de novo or inherited in autosomal dominant way and nine were inherited in autosomal recessive way. The patients had vertebral segmentation anomalies accompanied with variable joint contractures, short stature and dysmorphic facial features. There was a significant phenotypic overlap between dominant and recessive MYH3-associated conditions regarding the degree of short stature as well as the number of vertebral fusions. All monoallelic variants caused significantly decreased SMAD3 phosphorylation, which is consistent with the previously proposed pathogenic mechanism of impaired canonical TGF-β signaling. Most of the biallelic variants were predicted to be protein-truncating, while one missense variant c.4244T>G,p.(Leu1415Arg), which was inherited in an autosomal recessive way, was found to alter the phosphorylation level of p38, suggesting an inhibition of the non-canonical pathway of TGF-β signaling. In conclusion, the identification of 12 novel pathogenic variants and overlapping phenotypes in 17 affected individuals from 10 unrelated families expands the mutation and phenotype spectrum of MYH3-associated skeletal disorders. We show that disturbances of canonical or non-canonical TGF-β signaling pathways are involved in pathogenesis of MYH3-associated skeletal fusion (MASF) syndrome.

A linkage between effectual genes in progression of CRC through canonical and non-canonical TGF-β signaling pathways.

Different molecular signaling pathways have been involved in the incidence and progression of CRC. We aimed to examine the correlation between eight candidate genes, including TFGβ, SMAD2, SMAD4, RhoA, EGFR, MAP2K1, MTA1, and LEF1 in the progression of colorectal cancer (CRC) and their association with clinicopathological variables and CRC patients prognosis. Immunohistochemistry and quantitative real-time polymerase chain reaction (qRT-PCR) analysis 2-ΔΔct, were performed to assess the expression of eight genes in 64 and 122 patients with CRC, respectively and 20 normal samples were added for verification. We showed a positive correlation between SMAD2 and MAP2K1 (r = 0.337, P < 0.001), MAP2K1 and LEF1 (r = 0.187, P = 0.03), SMAD4 and RhoA (r = 0.214, P = 0.01) and as well, a negative correlation between SMAD2 and TGFβ (r = - 0.197, P = 0.02), and RhoA and LEF1 (r = - 0.180, P = 0.04) in tumor tissues. A decrease in RhoA mRNA expression was associated with the advanced TNM stage (P = 0.01), while the EGFR and SMAD2 mRNA expression upregulated in advanced stages (P = 0.03, P = 0.03), respectively. Also, an increase in EGFR and SMAD4 protein expression was significantly associated with the advanced TNM stage (P = 0.000) (P = .002), respectively. Perceiving the connections between canonical and non-canonical Transforming growth factor (TGF-β) signaling pathway along with the epidermal growth factor receptor (EGFR) and WNT cascades may trigger the development of novel approaches for CRC prediction.

Butyrate Protects against Diet-Induced NASH and Liver Fibrosis and Suppresses Specific Non-Canonical TGF-β Signaling Pathways in Human Hepatic Stellate Cells.

In obesity-associated non-alcoholic steatohepatitis (NASH), persistent hepatocellular damage and inflammation are key drivers of fibrosis, which is the main determinant of NASH-associated mortality. The short-chain fatty acid butyrate can exert metabolic improvements and anti-inflammatory activities in NASH. However, its effects on NASH-associated liver fibrosis remain unclear. Putative antifibrotic effects of butyrate were studied in Ldlr-/-.Leiden mice fed an obesogenic diet (HFD) containing 2.5% (w/w) butyrate for 38 weeks and compared with a HFD-control group. Antifibrotic mechanisms of butyrate were further investigated in TGF-β-stimulated primary human hepatic stellate cells (HSC). HFD-fed mice developed obesity, insulin resistance, increased plasma leptin levels, adipose tissue inflammation, gut permeability, dysbiosis, and NASH-associated fibrosis. Butyrate corrected hyperinsulinemia, lowered plasma leptin levels, and attenuated adipose tissue inflammation, without affecting gut permeability or microbiota composition. Butyrate lowered plasma ALT and CK-18M30 levels and attenuated hepatic steatosis and inflammation. Butyrate inhibited fibrosis development as demonstrated by decreased hepatic collagen content and Sirius-red-positive area. In TGF-β-stimulated HSC, butyrate dose-dependently reduced collagen deposition and decreased procollagen1α1 and PAI1 protein expression. Transcriptomic analysis and subsequent pathway and upstream regulator analysis revealed deactivation of specific non-canonical TGF-β signaling pathways Rho-like GTPases and PI3K/AKT and other important pro-fibrotic regulators (e.g., YAP/TAZ, MYC) by butyrate, providing a potential rationale for its antifibrotic effects. In conclusion, butyrate protects against obesity development, insulin resistance-associated NASH, and liver fibrosis. These antifibrotic effects are at least partly attributable to a direct effect of butyrate on collagen production in hepatic stellate cells, involving inhibition of non-canonical TGF-β signaling pathways.

MicroRNAs in Transforming Growth Factor-Beta Signaling Pathway Associated With Fibrosis Involving Different Systems of the Human Body.

Fibrosis, a major cause of morbidity and mortality, is a histopathological manifestation of many chronic inflammatory diseases affecting different systems of the human body. Two types of transforming growth factor beta (TGF-β) signaling pathways regulate fibrosis: the canonical TGF-β signaling pathway, represented by SMAD-2 and SMAD-3, and the noncanonical pathway, which functions without SMAD-2/3 participation and currently includes TGF-β/mitogen-activated protein kinases, TGF-β/SMAD-1/5, TGF-β/phosphatidylinositol-3-kinase/Akt, TGF-β/Janus kinase/signal transducer and activator of transcription protein-3, and TGF-β/rho-associated coiled-coil containing kinase signaling pathways. MicroRNA (miRNA), a type of non-coding single-stranded small RNA, comprises approximately 22 nucleotides encoded by endogenous genes, which can regulate physiological and pathological processes in fibrotic diseases, particularly affecting organs such as the liver, the kidney, the lungs, and the heart. The aim of this review is to introduce the characteristics of the canonical and non-canonical TGF-β signaling pathways and to classify miRNAs with regulatory effects on these two pathways based on the influenced organ. Further, we aim to summarize the limitations of the current research of the mechanisms of fibrosis, provide insights into possible future research directions, and propose therapeutic options for fibrosis.

MO011PODOCYTE NEPHRONECTIN IS REGULATED BY TRANSFORMING GROWTH FACTOR BETA VIA THE CANONICAL AND NON-CANONICAL PATHWAYS

Abstract Background and Aims The glomerular basement membrane (GBM), podocytes and glomerular endothelial cells (GEC) are composing the glomerular filtration barrier (GFB) within the glomerulus. Both podocytes and GEC are essential components for the synthesis of the extracellular matrix (ECM) of the GBM. One ECM protein of the GBM, which is mainly produced by podocytes is nephronectin (NPNT). An altered expression pattern of NPNT has been observed in different kidney diseases. While NPNT was shown to be down regulated in membranous glomerulonephropathy, its expression was elevated in diabetic glomerulopathy, compared to healthy controls. Using a morpholino-induced knockdown of npnt in zebrafish larvae, proteinuria, podocyte foot process effacement and thickening of the lamia rara interna of the GBM were observed. Mice that were intra peritoneally injected with a microRNA 378a (miR-378a) mimic showed a decrease in NPNT expression in the kidneys on both mRNA and protein level, suggesting a regulatory effect of miR-378a on NPNT. In addition, in cultured human podocytes treatment with transforming growth factor beta (TGFβ), as well as transfection with miR-378a mimic down regulated NPNT mRNA and protein expression. By blocking different parts of the TGFβ pathway, we want to further investigate the mechanisms by which TGFβ mediates the regulation of NPNT in podocytes. Method Our main model for this study are immortalized human podocytes, which are proliferating at 33°C and differentiating at 37°C due to a temperature sensitive SV40 large T antigen. Ten to 12 days differentiated podocytes were used for experiments. Differentiated cells were either transfected with a miR-192 or control miR mimic or pre-incubated with different inhibitors for components of both the canonical and the non-canonical TGFβ signaling pathways, followed by culture with or without additional TGFβ. Cell lysates were prepared to be used for Western Blot or qPCR analyses. Results Treatment of immortalized human podocytes with TGFβ decreased NPNT expression on mRNA and protein level. After transfecting immortalized human podocytes with a miR-192 mimic, a GEC-derived miR up regulated by TGFβ stimulation, we observed reduced NPNT expression, compared to control miR transfection. Blocking TGFβ receptor I signaling with the specific inhibitor SD208, caused higher NPNT protein abundance, while NPNT mRNA expression remained unchanged. Targeting downstream parts of both the canonical and non-canonical TGFβ pathways by using inhibitors for single molecules of the respective arms of the intricate TGFβ pathway showed ambiguous results. Suppression of either Smad2 and/or Smad3 tended to enhance NPNT protein levels, accompanied by mostly unaltered mRNA expression. Blockade of different parts of the non-canonical TGFβ pathway also up regulated protein expression of NPNT. However, NPNT mRNA expression was more variable. Therefore, regulation of podocyte NPNT might not be due to changes in mRNA transcription, but to modifications on posttranscriptional level. Conclusion Treating immortalized human podocytes with TGFβ or TGFβ-induced miR-192 reduced NPNT expression on both the mRNA and protein level. More detailed analysis with inhibition of different parts of the canonical and non-canonical TGFβ pathways hint that both pathways are involved in NPNT expression. Therefore, we suggest that the regulation of podocyte NPNT by TGFβ is fine-tuned via both the canonical and non-canonical pathways with additional modulation through TGFβ dependent miRs.

Effects of emodin, a plant-derived anthraquinone, on TGF-β1-induced cardiac fibroblast activation and function.

Cardiac fibrosis accompanies a number of pathological conditions and results in altered myocardial structure, biomechanical properties and function. The signaling networks leading to fibrosis are complex, contributing to the general lack of progress in identifying effective therapeutic approaches to prevent or reverse this condition. Several studies have shown protective effects of emodin, a plant-derived anthraquinone, in animal models of fibrosis. A number of questions remain regarding the mechanisms whereby emodin impacts fibrosis. Transforming growth factor beta 1 (TGF-β1) is a potent stimulus of fibrosis and fibroblast activation. In the present study, experiments were performed to evaluate the effects of emodin on activation and function of cardiac fibroblasts following treatment with TGF-β1. We demonstrate that emodin attenuates TGF-β1-induced fibroblast activation and collagen accumulation in vitro. Emodin also inhibits activation of several canonical (SMAD2/3) and noncanonical (Erk1/2) TGF-β signaling pathways, while activating the p38 pathway. These results suggest that emodin may provide an effective therapeutic agent for fibrosis that functions via specific TGF-β signaling pathways.

Integrin αVβ1 regulates procollagen I production through a non-canonical transforming growth factor β signaling pathway in human hepatic stellate cells.

Hepatic stellate cells (HSCs) are thought to play key roles in the development of liver fibrosis. Extensive evidence has established the concept that αV integrins are involved in the activation of latent transforming growth factor β (TGF-β), a master regulator of the fibrotic signaling cascade. Based on mRNA and protein expression profiling data, we found that αVβ1 integrin is the most abundant member of the αV integrin family in either quiescent or TGF-β1-activated primary human HSCs. Unexpectedly, either a selective αVβ1 inhibitor, Compound 8 (C8), or a pan-αV integrin inhibitor, GSK3008348, decreased TGF-β1-activated procollagen I production in primary human HSCs, in which the role of β1 integrin was confirmed by ITGB1 siRNA. In contrast with an Activin receptor-like kinase 5 (Alk5) inhibitor, C8 and GSK3008348 failed to inhibit TGF-β1 induced SMAD3 and SMAD2 phosphorylation, but inhibited TGF-β-induced phosphorylation of ERK1/2 and STAT3, suggesting that αVβ1 integrin is involved in non-canonical TGF-β signaling pathways. Consistently, ITGB1 siRNA significantly decreased phosphorylation of ERK1/2. Furthermore, a selective inhibitor of MEK1/2 blocked TGF-β1 induced phosphorylation of ERK1/2 and decreased TGF-β1 induced procollagen I production, while a specific inhibitor of STAT3 had no effect on TGF-β1 induced procollagen I production. Taken together, current data indicate that αVβ1 integrin can regulate TGF-β signaling independent of its reported role in activating latent TGF-β. Our data further support that αVβ1 inhibition is a promising therapeutic target for the treatment of liver fibrosis.

Physical Training Inhibits the Fibrosis Formation in Alzheimer’s Disease Kidney Influencing the TGFβ Signaling Pathways

Background:Alzheimer’s disease (AD) is a neurodegenerative illness, with several peripheral pathological signs such as accumulation of amyloid-β (Aβ) plaques in the kidney. Alterations of transforming growth factor β (TGFβ) signaling in the kidney can induce fibrosis, thus disturbing the elimination of Aβ.Objective:A protective role of increased physical activity has been proven in AD and in kidney fibrosis, but it is not clear whether TGFβ signalization is involved in this effect.Methods:The effects of long-term training on fibrosis were investigated in the kidneys of mice representing a model of AD (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J) by comparing wild type and AD organs. Alterations of canonical and non-canonical TGFβ signaling pathways were followed with PCR, western blot, and immunohistochemistry.Results:Accumulation of collagen type I and interstitial fibrosis were reduced in kidneys of AD mice after long-term training. AD induced the activation of canonical and non-canonical TGFβ pathways in non-trained mice, while expression levels of signal molecules of both TGFβ pathways became normalized in trained AD mice. Decreased amounts of phosphoproteins with molecular weight corresponding to that of tau and the cleaved C-terminal of AβPP were detected upon exercising, along with a significant increase of PP2A catalytic subunit expression.Conclusion:Our data suggest that physical training has beneficial effects on fibrosis formation in kidneys of AD mice and TGFβ signaling plays a role in this phenomenon.

Metastatic colorectal cancer cells maintain the TGFβ program and use TGFBI to fuel angiogenesis.

Colorectal cancer (CRC) cells are traditionally considered unresponsive to TGFβ due to mutations in the receptors and/or downstream signaling molecules. TGFβ influences CRC cells only indirectly via stromal cells, such as cancer-associated fibroblasts. However, CRC cell ability to directly respond to TGFβ currently remains unexplored. This represents a missed opportunity for diagnostic and therapeutic interventions.Methods: We examined whether cancer cells from primary CRC and liver metastases respond to TGFβ by inducing TGFβ-induced protein ig-h3 (TGFBI) expression, and the contribution of canonical and non-canonical TGFβ signaling pathways to this effect. We then investigated in vitro and in vivo TGFBI impact on metastasis formation and angiogenesis. Using patient serum samples and an orthotopic mouse model of CRC liver metastases we assessed the diagnostic/tumor targeting value of novel antibodies against TGFBI.Results: Metastatic CRC cells, such as circulating tumor cells, directly respond to TGFβ. These cells were characterized by the absence of TGFβ receptor mutations and the frequent presence of p53 mutations. The pro-tumorigenic program orchestrated by TGFβ in CRC cells was mediated through TGFBI, the expression of which was positively regulated by non-canonical TGFβ signaling cascades. TGFBI inhibition was sufficient to significantly reduce liver metastasis formation in vivo. Moreover, TGFBI pro-tumorigenic function was linked to its ability to stimulate angiogenesis. TGFBI levels were higher in serum samples from untreated patients with CRC than in patients who were receiving chemotherapy. A radiolabeled anti-TGFBI antibody selectively targeted metastatic lesions in vivo, underscoring its diagnostic and therapeutic potential.Conclusions: TGFβ signaling in CRC cells directly contributes to their metastatic potential and stromal cell-independence. Proteins downstream of activated TGFβ, such as TGFBI, represent novel diagnostic and therapeutic targets for more specific anti-metastatic therapies.

Anti‑proliferative effect of honokiol on SW620cells through upregulating BMP7 expression via the TGF‑β1/p53 signaling pathway.

Honokiol (HNK), a natural pharmaceutically active component extracted from magnolia bark, has been used for clinical treatments and has anti-inflammatory, antiviral and antioxidative effects. In recent years, anticancer research has become a major hotspot. However, the underlying molecular mechanisms of how HNK inhibits colorectal cancer have remained elusive. The present study focused on elucidating the effects of HNK on the expression of bone morphogenetic protein (BMP)7 and its downstream interaction with transforming growth factor (TGF)-β1 and p53 in colon cancer. In in vitro assays, cell viability, cell cycle distribution and apoptosis were examined using Cell Counting Kit-8, flow cytometry and reverse transcription-quantitative PCR, respectively. In addition, the expression of BMP7, TGF-β1 and relevant signaling proteins was determined by western blot analysis. In vivo, the anticancer effect of HNK was assessed in xenografts in nude mice. Furthermore, immunohistochemistry was performed to evaluate the association between BMP7 and TGF-β1 expression in colon cancer. The results indicated that HNK inhibited the proliferation of colon cancer cell lines, with SW620 cells being more sensitive than other colon cancer cell lines. Furthermore, HNK markedly promoted the expression of BMP7 at the mRNA and protein level. Exogenous BMP7 potentiated the effect of HNK on SW620 cells, while knocking down BMP7 inhibited it. As a downstream mechanism, HNK increased the expression of TGF-β1 and p53, which was enhanced by exogenous BMP7 in SW620 cells. In addition, immunohistochemical analysis indicated a positive association between BMP7 and TGF-β1 expression. Hence, the present results suggested that HNK is a promising agent for the treatment of colon cancer and enhanced the expression TGF-β1 and p53 through stimulating BMP7 activity via the non-canonical TGF-β signaling pathway.

Huangkui Capsule Ameliorates Renal Fibrosis in a Unilateral Ureteral Obstruction Mouse Model Through TRPC6 Dependent Signaling Pathways.

Renal fibrosis is the final common pathological manifestation of almost all progressive chronic kidney diseases (CKD). Transient receptor potential canonical (TRPC) channels, especially TRPC3/6, were proposed to be essential therapeutic targets for kidney injury. Huangkui capsule (HKC), an important adjuvant therapy for CKD, showed superior efficacy for CKD at stages 1–2 in clinical practice. However, its anti-fibrotic effect and the underlying mechanisms remain to be investigated. In the present study, we evaluated the efficacy of HKC on renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) and explored the potential underlying mechanism. Administration of HKC by intragastric gavage dose-dependently suppressed UUO-induced kidney injury and tubulointerstitial fibrosis. Similarly, HKC suppressed the expression level of α-smooth muscle actin (α-SMA), increased the expression of E-cadherin, and suppressed the mRNA expression of a plethora of proinflammatory mediators that are necessary for the progression of renal fibrosis. Mechanistically, HKC suppressed both canonical and non-canonical TGF-β signaling pathways in UUO mice as well as the TRPC6/calcineurin A (CnA)/nuclear factor of activated T cells (NFAT) signaling axis. In addition, TRPC6 knockout mice and HKC treated wild type mice displayed comparable protection on UUO-triggered kidney tubulointerstitial injury, interstitial fibrosis, and α-SMA expression. More importantly, HKC had no additional protective effect on UUO-triggered kidney tubulointerstitial injury and interstitial fibrosis in TRPC6 knockout mouse. Further investigation demonstrated that HKC could directly suppress TRPC3/6 channel activities. Considered together, these data demonstrated that the protective effect of HKC on renal injury and interstitial fibrosis is dependent on TRPC6, possibly through direct inhibition of TRPC6 channel activity and indirect suppression of TRPC6 expression.

Transforming Growth Factor Beta3 is Required for Cardiovascular Development.

Transforming growth factor beta3 (TGFB3) gene mutations in patients of arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD1) and Loeys-Dietz syndrome-5 (LDS5)/Rienhoff syndrome are associated with cardiomyopathy, cardiac arrhythmia, cardiac fibrosis, cleft palate, aortic aneurysms, and valvular heart disease. Although the developing heart of embryos express Tgfb3, its overarching role remains unclear in cardiovascular development and disease. We used histological, immunohistochemical, and molecular analyses of Tgfb3−/− fetuses and compared them to wildtype littermate controls. The cardiovascular phenotypes were diverse with approximately two thirds of the Tgfb3−/− fetuses having one or more cardiovascular malformations, including abnormal ventricular myocardium (particularly of the right ventricle), outflow tract septal and alignment defects, abnormal aortic and pulmonary trunk walls, and thickening of semilunar and/or atrioventricular valves. Ventricular septal defects (VSD) including the perimembranous VSDs were observed in Tgfb3−/− fetuses with myocardial defects often accompanied by the muscular type VSD. In vitro studies using TGFβ3-deficient fibroblasts in 3-D collagen lattice formation assays indicated that TGFβ3 was required for collagen matrix reorganization. Biochemical studies indicated the ‘paradoxically’ increased activation of canonical (SMAD-dependent) and noncanonical (MAP kinase-dependent) pathways. TGFβ3 is required for cardiovascular development to maintain a balance of canonical and noncanonical TGFβ signaling pathways.

Phenotypically Silent Bone Morphogenetic Protein Receptor 2 Mutations Predispose Rats to Inflammation-Induced Pulmonary Arterial Hypertension by Enhancing the Risk for Neointimal Transformation.

Bmpr2 (bone morphogenetic protein receptor 2) mutations are critical risk factors for hereditary pulmonary arterial hypertension (PAH) with approximately 20% of carriers developing disease. There is an unmet medical need to understand how environmental factors, such as inflammation, render Bmpr2 mutants susceptible to PAH. Overexpressing 5-LO (5-lipoxygenase) provokes lung inflammation and transient PAH in Bmpr2+/- mice. Accordingly, 5-LO and its metabolite, leukotriene B4, are candidates for the second hit. The purpose of this study was to determine how 5-LO-mediated pulmonary inflammation synergized with phenotypically silent Bmpr2 defects to elicit significant pulmonary vascular disease in rats. Monoallelic Bmpr2 mutant rats were generated and found phenotypically normal for up to 1 year of observation. To evaluate whether a second hit would elicit disease, animals were exposed to 5-LO-expressing adenovirus, monocrotaline, SU5416, SU5416 with chronic hypoxia, or chronic hypoxia alone. Bmpr2-mutant hereditary PAH patient samples were assessed for neointimal 5-LO expression. Pulmonary artery endothelial cells with impaired BMPR2 signaling were exposed to increased 5-LO-mediated inflammation and were assessed for phenotypic and transcriptomic changes. Lung inflammation, induced by intratracheal delivery of 5-LO-expressing adenovirus, elicited severe PAH with intimal remodeling in Bmpr2+/- rats but not in their wild-type littermates. Neointimal lesions in the diseased Bmpr2+/- rats gained endogenous 5-LO expression associated with elevated leukotriene B4 biosynthesis. Bmpr2-mutant hereditary PAH patients similarly expressed 5-LO in the neointimal cells. In vitro, BMPR2 deficiency, compounded by 5-LO-mediated inflammation, generated apoptosis-resistant and proliferative pulmonary artery endothelial cells with mesenchymal characteristics. These transformed cells expressed nuclear envelope-localized 5-LO consistent with induced leukotriene B4 production, as well as a transcriptomic signature similar to clinical disease, including upregulated nuclear factor Kappa B subunit (NF-κB), interleukin-6, and transforming growth factor beta (TGF-β) signaling pathways. The reversal of PAH and vasculopathy in Bmpr2 mutants by TGF-β antagonism suggests that TGF-β is critical for neointimal transformation. In a new 2-hit model of disease, lung inflammation induced severe PAH pathology in Bmpr2+/- rats. Endothelial transformation required the activation of canonical and noncanonical TGF-β signaling pathways and was characterized by 5-LO nuclear envelope translocation with enhanced leukotriene B4 production. This study offers an explanation of how an environmental injury unleashes the destructive potential of an otherwise silent genetic mutation.