Regulator Of TGF-β Signaling Research Articles

Unraveling the Role of RSPRY1 in TGF-β Pathway Dysregulation: Insights into the Pathogenesis of Spondyloepimetaphyseal Dysplasia

Skeletal dysplasias, characterized by bone, cartilage, and connective tissue abnormalities, often arise due to disruptions in extracellular matrix (ECM) dynamics and growth factor-dependent signaling pathways. RSPRY1, a secreted protein with RING and SPRY domains, has been implicated in bone development, yet its exact role remains to be determined. RSPRY1 gene mutations are associated with spondyloepimetaphyseal dysplasia (SEMD), a rare skeletal disorder characterized by severe epiphyseal and metaphyseal deformities. This study aimed to determine the molecular and cellular mechanisms by which RSPRY1 deficiency affects skeletal homeostasis. Transcriptome analysis of fibroblasts from patients with homozygous RSPRY1 mutations showed there was significant enrichment of transforming growth factor beta (TGF-β) signaling and ECM-related pathways. Functional wound healing assays showed that RSPRY1 knockout fibroblasts exhibited enhanced motility, a phenotype that was abrogated in RSPRY1 + SMAD3 double knockout fibroblasts, highlighting the SMAD3-dependence of RSPRY1′s effects. The observed limited response to exogenous TGF-β in RSPRY1-deficient cells indicated that there was constitutive pathway activation. These findings show that RSPRY1 is a critical regulator of TGF-β signaling in ECM dynamics and cell motility, contributing to the pathophysiology of SEMD. An improvement in our understanding of the molecular roles of RSPRY1 might yield novel therapeutic strategies that target TGF-β signaling in patients with SEMD and other skeletal dysplasias.

A stepwise mode of TGFβ-SMAD signaling and DNA methylation regulates naïve-to-primed pluripotency and differentiation

The formation of transcription regulatory complexes by the association of Smad4 with Smad2 and Smad3 (Smad2/3) is crucial in the canonical TGFβ pathway. Although the central requirement of Smad4 as a common mediator is emphasized in regulating TGFβ signaling, it is not obligatory for all responses. The role of Smad2/3 independently of Smad4 remains understudied. Here, we introduce a stepwise paradigm in which Smad2/3 regulate the lineage priming and differentiation of mouse embryonic stem cells (mESCs) by collaboration with different effectors. During the naïve-to-primed transition, Smad2/3 upregulate DNA methyltransferase 3b (Dnmt3b), which establishes the proper DNA methylation patterns and, in turn, enables Smad2/3 binding to the hypomethylated centers of promoters and enhancers of epiblast marker genes. Consequently, in the absence of Smad2/3, Smad4 alone cannot initiate epiblast-specific gene transcription. When primed epiblast cells begin to differentiate, Dnmt3b becomes less actively engaged in global genome methylation, and Smad4 takes over the baton in this relay race, forming a complex with Smad2/3 to support mesendoderm induction. Thus, mESCs lacking Smad4 can undergo the priming process but struggle with the downstream differentiation. This work sheds light on the intricate mechanisms underlying TGFβ signaling and its role in cellular processes.

Suppression of Class Switch Recombination to IgA by RASA2 and RASA3 through Inhibition of TGF-β Signaling.

Abs play a pivotal role in adaptive immunity by binding to pathogens and initiating immune responses against infections. Processes such as somatic hypermutation and class switch recombination (CSR) enhance Ab affinity and effector functions. We previously carried out a CRISPR/Cas9 screen in the CH12F3-2 (CH12) lymphoma B cell line to identify novel factors involved in CSR. The screen showed that guide RNAs targeting both Rasa2 and Rasa3 genes were decreased in IgA-negative CH12 B cells, implying that these genes might suppress CSR. Indeed, CSR was increased when either Rasa2 or Rasa3 were knocked out in CH12 cells. Compared to controls, Rasa2-/- and Rasa3-/- CH12 cells had increased expression of activation-induced cytidine deaminase (AID) and Iα transcripts, providing an explanation for the increased CSR. The increased CSR, AID, and Iα expression in Rasa2-/- or Rasa3-/- CH12F3-2 is mediated through TGF-β stimulation. Indeed, we found that deletion of RASA2 or RASA3 promotes a shift from noncanonical to canonical TGF-β signaling through SMAD3. These results show that RASA2 and RASA3 are both novel regulators of TGF-β signaling in B cells, a pathway known to be essential for CSR to IgA.

Deciphering the prognostic and therapeutic significance of BAG1 and BAG2 for predicting distinct survival outcome and effects on liposarcoma

Liposarcoma (LPS) is the second most common kind of soft tissue sarcoma, and a heterogeneous malignant tumor derived from adipose tissue. Up to now, the prognostic value of BAG1 or BAG2 in LPS has not been defined yet. Expression profiling data of LPS patients were collected from TCGA and GEO database. Survival curves were plotted to verify the outcome differences of patients based on BAG1 or BAG2 expression. Univariate and multivariate Cox regression models were used to analyze the prognostic ability of BAG1 or BAG2. Chaperone’s regulators BAG1 and BAG2 were identified as prognostic biomarkers for LPS patients, which exhibited distinct expression patterns and survival outcome prediction performances. Patients with high BAG2 expression and/or low BAG1 expression had worse prognosis. Enrichment analysis showed that BAG1 was involved in negative regulation of TGF-β signaling. Low expression of BAG1 was associated with high abundance of regulatory T cells (Tregs). The 2-gene signature model further confirmed the improved risk assessment performance of BAG1 and BAG2: high risk patients displayed poor prognosis. BAG1 and BAG2 are supposed to be potential prognostic biomarkers for LPS and have impacts on liposarcomagenesis and immune infiltration in distinctive manners, which may function as potential therapy targets (BAG1 agonists/BAG2 inhibitors) for LPS.

Targeting the liver clock improves fibrosis by restoring TGF-β signaling

Liver fibrosis is the major driver of hepatocellular carcinoma and liver disease-related death. Approved antifibrotic therapies are absent and compounds in development have limited efficacy. Increased TGF-β signaling drives collagen deposition by hepatic stellate cells (HSCs)/myofibroblasts. Here, we aimed to dissect the role of the circadian clock (CC) in controlling TGF-β signaling and liver fibrosis. Using CC-mutant mice, enriched HSCs and myofibroblasts obtained from healthy and fibrotic mice in different CC phases and loss-of-function studies in human hepatocytes and myofibroblasts, we investigated the relationship between CC and TGF-β signaling. We explored hepatocyte-myofibroblast communication through bioinformatic analyses of single-nuclei transcriptomes and performed validation in cell-based models. Using mouse models for MASH (metabolic dysfunction-associated steatohepatitis)-related fibrosis and spheroids from patients with liver disease, we performed proof-of-concept studies to validate pharmacological targetability and clinical translatability. We discovered that the CC oscillator temporally gates TGF-β signaling and this regulation is broken in fibrosis. We demonstrate that HSCs and myofibroblasts contain a functional CC with rhythmic expression of numerous genes, including fibrogenic genes. Perturbation studies in hepatocytes and myofibroblasts revealed a reciprocal relationship between TGF-β activation and CC perturbation, which was confirmed in patient-derived exvivo and invivo models. Pharmacological modulation of CC-TGF-β signaling inhibited fibrosis in mouse models invivo as well as in patient-derived liver spheroids. The CC regulates TGF-β signaling, and the breakdown of this control is associated with liver fibrosis in patients. Pharmacological proof-of-concept studies across different models have uncovered the CC as a novel therapeutic target for liver fibrosis - a growing unmet medical need. Liver fibrosis due to metabolic diseases is a global health challenge. Many liver functions are rhythmic throughout the day, being controlled by the circadian clock (CC). Here we demonstrate that regulation of the CC is perturbed upon chronic liver injury and this perturbation contributes to fibrotic disease. By showing that a compound targeting the CC improves liver fibrosis in patient-derived models, this study provides a novel therapeutic candidate strategy to treat fibrosis in patients. Additional studies will be needed for clinical translation. Since the findings uncover a previously undiscovered profibrotic mechanism and therapeutic target, the study is of interest for scientists investigating liver disease, clinical hepatologists and drug developers.

LncRNA ERICD interacts with TROAP to regulate TGF-β signaling in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most prevalent and common malignant tumors worldwide, accounting for 85–90 % of primary liver cancer cases. Accumulating evidence shows that long non-coding RNAs (LncRNAs) play regulatory roles in HCC occurrence and progression. However, little is known about the biological role of the LncRNA “E2F1-regulated inhibitor of cell death” (ERICD) in HCC. Our study revealed that ERICD is highly expressed in HCC and correlates with TNM staging; high ERICD levels were associated with poor patient prognoses. We revealed the targeting relationship between ERICD and miR-142-5p for the first time by bioinformatics prediction and further verified the targeting relationship between ERICD and miR-142-5p using a luciferase reporting experiment. In summary, our results showed that ERICD promotes the occurrence and metastasis of HCC by downregulating miR-142-5p expression. Our study provides a target for new potential therapeutic strategies for HCC.

LRG1 loss effectively restrains glomerular TGF-β signaling to attenuate diabetic kidney disease

Transforming growth factor (TGF)-β signaling is a well-established pathogenic mediator of diabetic kidney disease (DKD). However, owing to its pleiotropic actions, its systemic blockade is not therapeutically optimal. The expression of TGF-β signaling regulators can substantially influence TGF-β's effects in a cell- or context-specific manner. Among these, leucine-rich α2-glycoprotein 1 (LRG1) is significantly increased in glomerular endothelial cells (GECs) in DKD. As LRG1 is a secreted molecule that can exert autocrine and paracrine effects, we examined the effects of LRG1 loss in kidney cells in diabetic OVE26 mice by single-cell transcriptomic analysis. Gene expression analysis confirmed a predominant expression of Lrg1 in GECs, which further increased in diabetic kidneys. Loss of Lrg1 led to the reversal of angiogenic and TGF-β-induced gene expression in GECs, which were associated with DKD attenuation. Notably, Lrg1 loss also mitigated the increased TGF-β-mediated gene expression in both podocytes and mesangial cells in diabetic mice, indicating that GEC-derived LRG1 potentiates TGF-β signaling in glomerular cells in an autocrine and paracrine manner. Indeed, a significant reduction in phospho-Smad proteins was observed in the glomerular cells of OVE26 mice with LRG1 loss. These results indicate that specific antagonisms of LRG1 may be an effective approach to curb the hyperactive glomerular TGF-β signaling to attenuate DKD.

TSPYL1 as a Critical Regulator of TGFβ Signaling through Repression of TGFBR1 and TSPYL2.

Nucleosome assembly proteins (NAPs) have been identified as histone chaperons. Testis-Specific Protein, Y-Encoded-Like (TSPYL) is a newly arisen NAP family in mammals. TSPYL2 can be transcriptionally induced by DNA damage and TGFβ causing proliferation arrest. TSPYL1, another TSPYL family member, has been poorly characterized and is the only TSPYL family member known to be causal of a lethal recessive disease in humans. This study shows that TSPYL1 and TSPYL2 play an opposite role in TGFβ signaling. TSPYL1 partners with the transcription factor FOXA1 and histone methyltransferase EZH2, and at the same time represses TGFBR1 and epithelial-mesenchymal transition (EMT). Depletion of TSPYL1 increases TGFBR1 expression, upregulates TGFβ signaling, and elevates the protein stability of TSPYL2. Intriguingly, TSPYL2 forms part of the SMAD2/3/4 signal transduction complex upon stimulation by TGFβ to execute the transcriptional responses. Depletion of TSPYL2 rescues the EMT phenotype of TSPYL1 knockdown in A549 lung carcinoma cells. The data demonstrates the prime role of TSPYL2 in causing the dramatic defects in TSPYL1 deficiency. An intricate counter-balancing role of TSPYL1 and TSPYL2 in regulating TGFβ signaling is also unraveled.

Unboxing the network among long non-coding RNAs and TGF-β signaling in cancer.

Deeper analysis of molecular mechanisms arising in tumor cells is an unmet need to provide new diagnostic and therapeutic strategies to prevent and treat tumors. The transforming growth factor β (TGF-β) signaling has been steadily featured in tumor biology and linked to poor prognosis of cancer patients. One pro-tumorigenic mechanism induced by TGF-β is the epithelial-to-mesenchymal transition (EMT), which can initiate cancer dissemination, enrich the tumor stem cell population, and increase chemoresistance. TGF-β signals via SMAD proteins, ubiquitin ligases, and protein kinases and modulates the expression of protein-coding and non-coding RNA genes, including those encoding larger than 500 nt transcripts, defined as long non-coding RNAs (lncRNAs). Several reports have shown lncRNAs regulating malignant phenotypes by directly affecting epigenetic processes, transcription, and post-transcriptional regulation. Thus, this review aims to update and summarize the impact of TGF-β signaling on the expression of lncRNAs and the function of such lncRNAs as regulators of TGF-β signaling, and how these networks might impact specific hallmarks of cancer.

Abstract 6964: Regulation of TGF-β signaling via a novel TβRIII sheddase

Abstract In a normal cell, TGF-β signaling serves as a tumor suppressor by reducing proliferation and promoting apoptosis. Paradoxically, the function of TGF-β signaling reverses in a cancer cell and begins to promote oncogenic pathways such as proliferation, survival, evasion of the immune system, and epithelial to mesenchymal transition (EMT). This has made TGF-β signaling a very attractive therapeutic target for a wide variety of human carcinomas. As a result, many small molecule inhibitors of TGF-β signaling have been developed, but none have achieved FDA approval for use in human cancers due to the deleterious effects of inhibiting TGF-β responsiveness in normal cells. In effort to overcome this obstacle, we aim to restore the normal biological mechanisms that regulate TGF-β signaling. Specifically, the membrane bound co-receptor TβRIII can be proteolytically cleaved and shed from the membrane, resulting in an extracellular protein capable of binding and sequestering TGF-β ligand. This decoupling of ligand binding from the receptor allows TβRIII to suppress signaling. TβRIII shedding decreases TGF-β mediated oncogenic phenotypes such as migration and metastasis in multiple cancer types. However, this shedding is often lost in cancer. Therefore, restoration of TβRIII shedding could be a viable therapeutic approach for multiple cancer types. One potential mechanism for the loss of shedding is the negative regulation of the protease responsible for shedding (sheddase). However, the identity of the sheddase still remains unknown. Here, our goal is to identify the sheddase and uncover regulatory mechanisms that could be leveraged to restore TβRIII shedding in cancer. To achieve this goal, a library of protease CRISPR knockout viruses was screened to determine which proteases have an effect on shedding. The quantity of TβRIII shed into the cell culture media was measured via ELISA for each of these CRISPR knockout cell lines, and the candidates with the largest reduction of shedding were retained for further investigation. The list of candidates was further screened in silico via gene set enrichment analysis of human patient data against a TGF-β signaling gene set signature. Only the candidates demonstrating a negative correlation between sheddase candidate expression and TGF-β signaling activity were studied further. To further validate these candidate sheddases, knockdown and overexpression cell lines were created and characterized. Overexpression of the most promising candidate reduced TGF-β mediated phenotypes such as migration and invasion, while knockdown of this candidate increased the same phenotypes. Knockdown also increased expression of TGF-β and EMT markers. Importantly, these effects of sheddase knockdown were neutralized in the presence of the TGF-β inhibitor galunisertib or when the cells were rescued with exposure to purified soluble TβRIII, indicating the effects are mediated through TβRIII shedding. Citation Format: Benjamin M. Greulich, John Pawlak, John Post, Elena Karas, Gerard Blobe. Regulation of TGF-β signaling via a novel TβRIII sheddase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6964.

CD109 Attenuates Bleomycin-induced Pulmonary Fibrosis by Inhibiting TGF-β Signaling.

Pulmonary fibrosis is a fatal condition characterized by fibroblast and myofibroblast proliferation and collagen deposition. TGF-β plays a pivotal role in the development of pulmonary fibrosis. Therefore, modulation of TGF-β signaling is a promising therapeutic strategy for treating pulmonary fibrosis. To date, however, interventions targeting TGF-β have not shown consistent efficacy. CD109 is a GPI-anchored glycoprotein that binds to TGF-β receptor I and negatively regulates TGF-β signaling. However, no studies have examined the role and therapeutic potential of CD109 in pulmonary fibrosis. The purpose of this study was to determine the role and therapeutic value of CD109 in bleomycin-induced pulmonary fibrosis. CD109-transgenic mice overexpressing CD109 exhibited significantly attenuated pulmonary fibrosis, preserved lung function, and reduced lung fibroblasts and myofibroblasts compared with wild-type (WT) mice. CD109-/- mice exhibited pulmonary fibrosis comparable to WT mice. CD109 expression was induced in variety types of cells, including lung fibroblasts and macrophages, upon bleomycin exposure. Recombinant CD109 protein inhibited TGF-β signaling and significantly decreased ACTA2 expression in human fetal lung fibroblast cells invitro. Administration of recombinant CD109 protein markedly reduced pulmonary fibrosis in bleomycin-treated WT mice invivo. Our results suggest that CD109 is not essential for the development of pulmonary fibrosis, but excess CD109 protein can inhibit pulmonary fibrosis development, possibly through suppression of TGF-β signaling. CD109 is a novel therapeutic candidate for treating pulmonary fibrosis.

TSP50 Attenuates DSS-Induced Colitis by Regulating TGF-β Signaling Mediated Maintenance of Intestinal Mucosal Barrier Integrity.

The integrity of the intestinal mucosal barrier is crucial for protecting the intestinal epithelium against invasion by commensal bacteria and pathogens, thereby combating colitis. The investigation revealed that the absence of TSP50 compromised the integrity of the intestinal mucosal barrier in murine subjects. This disruption facilitated direct contact between intestinal bacteria and the intestinal epithelium, thereby increasing susceptibility to colitis. Mechanistic analysis indicated that TSP50 deficiency in intestinal stem cells (ISCs) triggered aberrant activation of the TGF-β signaling pathway and impeded the differentiation of goblet cells in mice, leading to impairment of mucosal permeability. By inhibiting the TGF-β pathway, the functionality of the intestinal mucosal barrier is successfully restored and mitigated colitis in TSP50-deficient mice. In conclusion, TSP50 played a crucial role in maintaining the intestinal mucosal barrier function and exhibited the preventive effect against the development of colitis by regulating the TGF-β signaling pathway.

SET8 is a novel negative regulator of TGF-β signaling in a methylation-independent manner

Transforming growth factor β (TGF-β) is a multifunctional cytokine that induces a diverse set of cellular processes principally through Smad-dependent transcription. Transcriptional responses induced by Smads are tightly regulated by Smad cofactors and histone modifications; however, the underlying mechanisms have not yet been elucidated in detail. We herein report lysine methyltransferase SET8 as a negative regulator of TGF-β signaling. SET8 physically associates with Smad2/3 and negatively affects transcriptional activation by TGF-β in a catalytic activity-independent manner. The depletion of SET8 results in an increase in TGF-β-induced plasminogen activator inhibitor-1 (PAI-1) and p21 expression and enhances the antiproliferative effects of TGF-β. Mechanistically, SET8 occupies the PAI-1 and p21 promoters, and a treatment with TGF-β triggers the replacement of the suppressive binding of SET8 with p300 on these promoters, possibly to promote gene transcription. Collectively, the present results reveal a novel role for SET8 in the negative regulation of TGF-β signaling.

Toll-interacting protein inhibits transforming growth factor beta signaling in mouse lung fibroblasts.

Variations in the Toll-interacting protein (TOLLIP) gene have been identified in genome-wide association studies to correlate with risk of disease, mortality, and response to N-acetylcysteine therapy in idiopathic pulmonary fibrosis. Although TOLLIP is known to modulate innate immune responses, its relevance in organ fibrogenesis remains unknown. Prior work in the literature suggests TOLLIP dampens transforming growth factor beta (TGFβ) signaling in human cell lines. In this study, we examined the role of TOLLIP in mouse lung fibroblast (MLF) responses to TGFβ and in the bleomycin model of experimental lung fibrosis using Tollip-/- mice. We hypothesize that if TOLLIP negatively regulates TGFβ signaling, then Tollip-/- mouse lung fibroblasts (MLFs) would have enhanced response to TGFβ treatment, and Tollip-/- mice would develop increased fibrosis following bleomycin challenge. Primary MLFs were stimulated with TGFβ (1 ng/mL) for 24 h. RNA was obtained to assess global transcriptional responses by RNA-seq and markers of myofibroblast transition by qPCR. Functional assessment of TGFβ-stimulated MLFs included cell migration by scratch assay, cell proliferation, and matrix invasion through Matrigel. In the invivo model of lung fibrosis, Tollip-/- mice and wild-type (WT) littermates were administered bleomycin intratracheally and assessed for fibrosis. We further examined TGFβ signaling invivo after bleomycin injury by SMAD2, ERK1/2, and TGFβR1 Western blot. In response to TGFβ treatment, both WT and Tollip-/- MLFs exhibited global transcriptional changes consistent with myofibroblast differentiation. However, Tollip-/- MLFs showed greater number of differentially expressed genes compared to WT MLFs and greater upregulation of Acta2 by qPCR. Functionally, Tollip-/- MLFs also exhibited increased migration and Matrigel invasiveness compared to WT. We found evidence of enhanced TGFβ signaling in Tollip-/- through SMAD2 invitro and invivo. Tollip-/- mice experienced lower survival using a standard weight-adjusted dosing without evidence of differences in fibrosis at Day 21. With adjustment of dosing for sex, no differences were observed in fibrosis at Day 21. However, Tollip-/- mice had greater weight loss and increased bronchoalveolar lavage fluid total protein during early resolution at Day 14 compared to WT without evidence of differences in acute lung injury at Day 7, suggesting impaired resolution of lung injury.

Cavin-2 promotes fibroblast-to-myofibroblast trans-differentiation and aggravates cardiac fibrosis.

Transforming growth factor β (TGF-β) signalling is one of the critical pathways in fibroblast activation, and several drugs targeting the TGF-β/Smad signalling pathway in heart failure with cardiac fibrosis are being tested in clinical trials. Some caveolins and cavins, which are components of caveolae on the plasma membrane, are known for their association with the regulation of TGF-β signalling. Cavin-2 is particularly abundant in fibroblasts; however, the detailed association between Cavin-2 and cardiac fibrosis is still unclear. We tried to clarify the involvement and role of Cavin-2 in fibroblasts and cardiac fibrosis. To clarify the role of Cavin-2 in cardiac fibrosis, we performed transverse aortic constriction (TAC) operations on four types of mice: wild-type (WT), Cavin-2 null (Cavin-2 KO), Cavin-2flox/flox , and activated fibroblast-specific Cavin-2 conditional knockout (Postn-Cre/Cavin-2flox/flox , Cavin-2 cKO) mice. We collected mouse embryonic fibroblasts (MEFs) from WT and Cavin-2 KO mice and investigated the effect of Cavin-2 in fibroblast trans-differentiation into myofibroblasts and associated TGF-β signalling. Four weeks after TAC, cardiac fibrotic areas in both the Cavin-2 KO and the Cavin-2 cKO mice were significantly decreased compared with each control group (WT 8.04±1.58% vs. Cavin-2 KO 0.40±0.03%, P<0.01; Cavin-2flox/flox , 7.19±0.50% vs. Cavin-2 cKO 0.88±0.44%, P<0.01). Fibrosis-associated mRNA expression (Col1a1, Ctgf, and Col3) was significantly attenuated in the Cavin-2 KO mice after TAC. α1 type I collagen deposition and non-vascular αSMA-positive cells (WT 43.5±2.4% vs. Cavin-2 KO 25.4±3.2%, P<0.01) were reduced in the heart of the Cavin-2 cKO mice after TAC operation. The levels of αSMA protein (0.36-fold, P<0.05) and fibrosis-associated mRNA expression (Col1a1, 0.69-fold, P<0.01; Ctgf, 0.27-fold, P<0.01; Col3, 0.60-fold, P<0.01) were decreased in the Cavin-2 KO MEFs compared with the WT MEFs. On the other hand, αSMA protein levels were higher in the Cavin-2 overexpressed MEFs compared with the control MEFs (2.40-fold, P<0.01). TGF-β1-induced Smad2 phosphorylation was attenuated in the Cavin-2 KO MEFs compared with WT MEFs (0.60-fold, P<0.01). Heat shock protein 90 protein levels were significantly reduced in the Cavin-2 KO MEFs compared with the WT MEFs (0.69-fold, P<0.01). Cavin-2 loss suppressed fibroblast trans-differentiation into myofibroblasts through the TGF-β/Smad signalling. The loss of Cavin-2 in cardiac fibroblasts suppresses cardiac fibrosis and may maintain cardiac function.

The long non-coding RNA LINC00707 interacts with Smad proteins to regulate TGFβ signaling and cancer cell invasion

BackgroundLong non-coding RNAs (lncRNAs) regulate cellular processes by interacting with RNAs or proteins. Transforming growth factor β (TGFβ) signaling via Smad proteins regulates gene networks that control diverse biological processes, including cancer cell migration. LncRNAs have emerged as TGFβ targets, yet, their mechanism of action and biological role in cancer remain poorly understood.MethodsWhole-genome transcriptomics identified lncRNA genes regulated by TGFβ. Protein kinase inhibitors and RNA-silencing, in combination with cDNA cloning, provided loss- and gain-of-function analyses. Cancer cell-based assays coupled to RNA-immunoprecipitation, chromatin isolation by RNA purification and protein screening sought mechanistic evidence. Functional validation of TGFβ-regulated lncRNAs was based on new transcriptomics and by combining RNAscope with immunohistochemical analysis in tumor tissue.ResultsTranscriptomics of TGFβ signaling responses revealed down-regulation of the predominantly cytoplasmic long intergenic non-protein coding RNA 707 (LINC00707). Expression of LINC00707 required Smad and mitogen-activated protein kinase inputs. By limiting the binding of Krüppel-like factor 6 to the LINC00707 promoter, TGFβ led to LINC00707 repression. Functionally, LINC00707 suppressed cancer cell invasion, as well as key fibrogenic and pro-mesenchymal responses to TGFβ, as also attested by RNA-sequencing analysis. LINC00707 also suppressed Smad-dependent signaling. Mechanistically, LINC00707 interacted with and retained Smad proteins in the cytoplasm. Upon TGFβ stimulation, LINC00707 dissociated from the Smad complex, which allowed Smad accumulation in the nucleus. In vivo, LINC00707 expression was negatively correlated with Smad2 activation in tumor tissues.ConclusionsLINC00707 interacts with Smad proteins and limits the output of TGFβ signaling, which decreases LINC00707 expression, thus favoring cancer cell invasion.EZy4eavcmbb38C5oFYKmkjVideo

Putting transcriptional brakes on fibrosis: a negative regulator of TGFβ signaling.

Due to the pleiotropic effect of transforming growth factor β (TGFβ) signaling inhibition, function-specific targeted inhibition of TGFβ signaling is required. In a recent study, Yang et al. found that Krüppel-like factor (KLF)-13 acts as a negative regulator of TGFβ. Thus, activating KLF13 in fibrotic tissues may protect them from fibrosis by decreasing TGFβ signaling.

Cardiac Pericytes Acquire a Fibrogenic Phenotype and Contribute to Vascular Maturation After Myocardial Infarction.

Pericytes have been implicated in tissue repair, remodeling, and fibrosis. Although the mammalian heart contains abundant pericytes, their fate and involvement in myocardial disease remains unknown. We used NG2Dsred;PDGFRαEGFP pericyte:fibroblast dual reporter mice and inducible NG2CreER mice to study the fate and phenotypic modulation of pericytes in myocardial infarction. The transcriptomic profile of pericyte-derived cells was studied using polymerase chain reaction arrays and single-cell RNA sequencing. The role of transforming growth factor-β (TGF-β) signaling in regulation of pericyte phenotype was investigated in vivo using pericyte-specific TGF-β receptor 2 knockout mice and in vitro using cultured human placental pericytes. In normal hearts, neuron/glial antigen 2 (NG2) and platelet-derived growth factor receptor α (PDGFRα) identified distinct nonoverlapping populations of pericytes and fibroblasts, respectively. After infarction, a population of cells expressing both pericyte and fibroblast markers emerged. Lineage tracing demonstrated that in the infarcted region, a subpopulation of pericytes exhibited transient expression of fibroblast markers. Pericyte-derived cells accounted for ~4% of PDGFRα+ infarct fibroblasts during the proliferative phase of repair. Pericyte-derived fibroblasts were overactive, expressing higher levels of extracellular matrix genes, integrins, matricellular proteins, and growth factors, when compared with fibroblasts from other cellular sources. Another subset of pericytes contributed to infarct angiogenesis by forming a mural cell coat, stabilizing infarct neovessels. Single-cell RNA sequencing showed that NG2 lineage cells diversify after infarction and exhibit increased expression of matrix genes, and a cluster with high expression of fibroblast identity markers emerges. Trajectory analysis suggested that diversification of infarct pericytes may be driven by proliferating cells. In vitro and in vivo studies identified TGF-β as a potentially causative mediator in fibrogenic activation of infarct pericytes. However, pericyte-specific TGF-β receptor 2 disruption had no significant effects on infarct myofibroblast infiltration and collagen deposition. Pericyte-specific TGF-β signaling was involved in vascular maturation, mediating formation of a mural cell coat investing infarct neovessels and protecting from dilative remodeling. In the healing infarct, cardiac pericytes upregulate expression of fibrosis-associated genes, exhibiting matrix-synthetic and matrix-remodeling profiles. A fraction of infarct pericytes exhibits expression of fibroblast identity markers. Pericyte-specific TGF-β signaling plays a central role in maturation of the infarct vasculature and protects from adverse dilative remodeling, but it does not modulate fibrotic remodeling.

Secreted ADAMTS-like 2 promotes myoblast differentiation by potentiating WNT signaling

Myogenesis is the process that generates multinucleated contractile myofibers from muscle stem cells during skeletal muscle development and regeneration. Myogenesis is governed by myogenic regulatory transcription factors, including MYOD1. Here, we identified the secreted matricellular protein ADAMTS-like 2 (ADAMTSL2) as part of a Wnt-dependent positive feedback loop, which augmented or sustained MYOD1 expression and thus promoted myoblast differentiation. ADAMTSL2 depletion resulted in severe retardation of myoblast differentiation in vitro and its ablation in myogenic precursor cells resulted in aberrant skeletal muscle architecture. Mechanistically, ADAMTSL2 potentiated WNT signaling by binding to WNT ligands and WNT receptors. We identified the WNT-binding ADAMTSL2 peptide, which was sufficient to promote myogenesis in vitro. Since ADAMTSL2 was previously described as a negative regulator of TGFβ signaling in fibroblasts, ADAMTSL2 now emerges as a signaling hub that could integrate WNT, TGFβ and potentially other signaling pathways within the dynamic microenvironment of differentiating myoblasts during skeletal muscle development and regeneration.

PROTEIN PHOSPHATASE PPM1A INHIBITION ATTENUATES OSTEOARTHRITIS VIA REGULATION OF TGF-Β/SMAD2 SIGNALLING IN CHONDROCYTES

TGF-β/Smad2 signaling is considered to be one of the important pathways involved in osteoarthritis (OA) and protein phosphatase magnesium-dependent 1A (PPM1A) functions as an exclusive phosphatase of Smad2 and regulates TGF-β signaling, here, we investigated the functional role of PPM1A in OA pathogenesis.PPM1A expressions in both human OA cartilage and experimental OA mice chondrocytes were analyzed immunohistochemically. Besides, the mRNA and protein expression of PPM1A induced by IL-1β treatment were also detected by q-PCR and immunofluorescence in vitro. OA was induced in PPM1A knockout (KO) mice by destabilization of the medial meniscus (DMM), and histopathological examination was performed. OA was also induced in wild-type (WT) mice, which were then treated with an intra-articular injection of a selective PPM1A inhibitor for 8 weeks.PPM1A protein expressions were increased in both human OA cartilage and experimental OA mice chondrocytes. We also found that treatment with IL-1β in mouse primary chondrocytes significantly increased both mRNA and protein expression of PPM1A in vitro. Importantly, our data showed that PPM1A deletion could substantially protect against surgically induced OA. Concretely, the average OARSI score and quantification of BV/TV of subchondral bone in KO mice were significantly lower than that in WT mice 8 weeks after DMM surgery. Besides, TUNEL staining revealed a significant decrease in apoptotic chondrocytes in PPM1A-KO mice with DMM operation. With OA induction, the rates of chondrocytes positive for Mmp-13 and Adamts-5 in KO mice were also significantly lower than those in WT mice. Moreover, compared with WT mice, the phosphorylation of Smad2 in chondrocytes was increased in KO mice underwent DMM surgery. However, articular-injection with SD-208, a selective inhibitor of TGF-β/Smad2 signaling could significantly abolish the chondroprotective phenotypes in PPM1A-KO mice. Additionally, both cartilage degeneration and subchondral bone subchondral bone sclerosis in DMM model were blunted following intra-articular injection with BC-21, a small-molecule inhibitor for PPM1A.Our study demonstrated that PPM1A inhibition attenuates OA by regulating TGF-β/Smad2 signaling. Furthermore, PPM1A is a potential target for OA treatment and BC-21 may be employed as alternative therapeutic agents for the management of OA.