Regulation Of TGF-beta Signaling Research Articles

Tumoral CD105 promotes immunosuppression, metastasis, and angiogenesis in renal cell carcinoma.

CD105 (endoglin) is a transmembrane protein that functions as a TGF-beta coreceptor and is highly expressed on endothelial cells. Unsurprisingly, preclinical and clinical evidence strongly suggests that CD105 is an important contributor to tumor angiogenesis and tumor progression. Emerging evidence suggests that CD105 is also expressed by tumor cells themselves in certain cancers such as renal cell carcinoma (RCC). In human RCC tumor cells, CD105 expression is associated with stem cell-like properties and contributes to the malignant phenotype in vitro and in xenograft models. However, as a regulator of TGF-beta signaling, there is a striking lack of evidence for the role of tumor-expressed CD105 in the anti-tumor immune response and the tumor microenvironment. In this study, we report that tumor cell-expressed CD105 potentiates both the in vitro and in vivo tumorigenic potential of RCC in a syngeneic murine RCC tumor model. Importantly, we find that tumor cell-expressed CD105 sculpts the tumor microenvironment by enhancing the recruitment of immunosuppressive cell types and inhibiting the polyfunctionality of tumor-infiltrating CD4+ and CD8+ T cells. Finally, while CD105 expression by endothelial cells is a well-established contributor to tumor angiogenesis, we also find that tumor cell-expressed CD105 significantly contributes to tumor angiogenesis in RCC.

SAT-177 KLF11 DEFICIENCY EXACERBATES RENAL INJURY IN EXPERIMENTAL UNILATERAL URETERIC OBSTRUCTION

Kruppel-like factors (KLFs) are a family of zinc-finger transcription factors that play a critical role in regulation of growth and development, proliferation, and regeneration. Members of this family have been linked to podocyte dysfunction and renal fibrosis. In other systems, KLF11 has been shown to regulate TGF-beta signaling and fibrosis. Although KLF deficiency has been linked to endometriosis-related fibrosis, the role of KLF11 in mediating renal injury has not been previously established. We sought to test the hypothesis that KLF11 deficiency exacerbates renal damage in the murine unilateral ureteral obstruction (UUO) of renal fibrosis. We generated KLF11 knockout (KO), Smad 3 wild type (WT) mice, KLF11 WT Smad 3 KO, KLF11 KO Smad 3 KO, and KLF11/Smad3 WT mice and performed unilateral ureteral obstruction. Kidneys were harvested after 9 days (n=4 animals per group). Semiquantitative histopathologic analysis of renal atrophy, fibrosis, and inflammation was performed in a blinded fashion. Gene expression analysis was performed on renal cortex using Pathway Detect RNA Arrays. Renal atrophy (Figure 1) and inflammation (data not shown) were increased in KLF11 KO Smad3 WT mice compared to WT/WT mice subjected to UUO. As expected, Smad3 deficiency was associated with significantly less renal injury (Figure 1). Compared to WT/WT mice, KLF11 KO Smad3 WT mice showed marked upregulation of pro-inflammatory chemokines including CCL7 (250-fold compared to sham), Cxcl 11 (170-fold), Ccl12 (143-fold), Csf1 (117-fold), Ccl5 (96-fold), and Ccl2 (76-fold), inductions that were 2-5 fold higher than those observed in WT/WT mice. KLF11 deficiency was associated with modest upregulation of pro-fibrotic genes including Col1a2, Col1a1, and TGF-beta 1-3. Smad3 deficiency was associated with significant reduction of both pro-inflammatory and pro-fibrotic genes, both in the presence and absence of Klf11 deficiency. KLF11 deficiency exacerbates renal injury in the UUO model of renal fibrosis through upregulation of pro-inflammatory chemokine genes. To our knowledge, this is the first report linking KLF11 signaling to renal injury. Future studies will focus on potential mechanisms whereby KLF11 mediated chemokine signaling intersects with TGF-beta/Smad signaling to produce renal injury.

MiR-4668 as a Novel Potential Biomarker for Eosinophilic Esophagitis.

IntroductionEosinophilic esophagitis (EoE) is a clinico-pathological diagnosis characterized by esophageal dysfunction and eosinophilic infiltration of the esophagus. Demonstration of esophageal eosinophilia (more than 15 eosinophils/hpf) in biopsy specimen obtained by esophagogastroduodenoscopy (EGD) continues to be the gold standard for diagnosis and monitoring of response to therapy. There is a growing necessity for non-invasive biomarkers that can accurately diagnose this condition and assess response to therapy. While microRNAs (miRNA) are being investigated in allergic diseases, including EoE, not many studies have explored the role of salivary miRNAs in EoE. MiR-4668-5p is a particularly interesting candidate, as it is predicted to regulate TGF-beta signaling and has not previously been identified as a target in any allergy disease. We sought to further investigate the role of miR-4668 as a biomarker to characterize and monitor response to treatment with swallowed topical glucocorticoids.MethodsAfter IRB approval, twenty-two adult patients with EoE were randomly enrolled to provide a saliva sample before and after 2 months of swallowed fluticasone therapy. Differences of miRNA expression before and after treatment were analyzed by paired T-test. A significance cutoff of <0.05 was used for all analyses.ResultsExpression of miR-4668 was higher in EoE vs. non-EoE subjects. The level of miR-4668 decreased in all subjects except one, with a mean fold change 0.49 ± 0.25. There was an association between miRNA expression and number of positive aeroallergens. The miR-4668 high group had a higher number of positive aeroallergen tests, while the miR-4668 low group had a greater number of subjects with drug allergies.ConclusionsIn this study, we identified that salivary miRNAs may serve as biomarkers to characterize EoE and response to topical corticosteroids. We specifically identified miR-4668 as a novel potential biomarker, which was not previously discovered as a target in EoE or any other allergic disease.

Influence of microRNA on pulmonary fibrosis by regulating TGF-beta signaling pathways

Influence of microRNA on pulmonary fibrosis by regulating TGF-beta signaling pathways

Abstract 1865: Areca nut-induced JNK/ATF2/Jun axis regulates TGF-beta signaling contributing to pre-cancerous oral submucous fibrosis

Abstract Oral Submucous Fibrosis (OSF) is an inflammatory, pre-cancerous condition of the oral submucosa. It affects individuals with prolonged areca nut chewing habit and therefore areca nut is regarded as the etiological agent for OSF manifestation. Transforming Growth Factor beta is an established promoter of cancer and fibrosis. Up-regulation of TGF-β and its target genes has also been reported in OSF. However, the underlying mechanism for the regulation of TGF-β by areca nut has not been elucidated. The present study demonstrates that TGF-β pathway is activated in epithelial cells after two hour exposure to areca nut extract. Areca nut increases TGF-β transcript as well as protein and thereby the downstream canonical Smad2/3 signaling. Pre-treatment with various pathway inhibitors showed JNK pathway activation as a pre-requisite to TGF-β pathway activation by areca nut. Areca nut activated JNK pathway within half an hour of treatment and this was reversed by atropine treatment suggesting involvement of muscarinic acid receptor signaling. Second messengers upstream to JNK activation and regulated by areca nut were calcium/CAMKII and reactive oxygen species. Further, areca nut treatment resulted in JNK dependent phosphorylation of ATF2 and c-Jun transcription factors. These were found to bind TGF-β promoter, as revealed by chromatin immunoprecipitation experiments. Expectedly, knockdown of these transcription factors abrogated areca nut induced TGF-β pathway activation. Based on the data obtained a model is proposed to elucidate activation of TGF-β pathway by areca nut extract involving activation of JNK and ATF2, c-Jun resulting in the over expression of TGF-β in epithelial cells which may contribute to the manifestation of OSF. Thus, this study highlights ATF2/c-Jun as the critical nodes for therapeutic intervention to prevent TGF-β over production in OSF. Funding by DST and CSIR is acknowledged. Citation Format: Ila Pant, Paturu Kondaiah. Areca nut-induced JNK/ATF2/Jun axis regulates TGF-beta signaling contributing to pre-cancerous oral submucous fibrosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1865.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Disruption of Smad4 impairs TGF-β/Smad3 and Smad7 transcriptional regulation during renal inflammation and fibrosis in vivo and in vitro

The mechanism by which TGF-β regulates renal inflammation and fibrosis is largely unclear; however, it is well accepted that its biological effects are mediated through Smad2 and Smad3 phosphorylation. Following activation, these Smads form heteromeric complex with Smad4 and translocate into the nucleus to bind and regulate the expression of target genes. Here we studied the roles of Smad4 to regulate TGF-β signaling in a mouse model of unilateral ureteral obstruction using conditional Smad4 knockout mice and in isolated Smad4 mutant macrophages and fibroblasts. Disruption of Smad4 significantly enhanced renal inflammation as evidenced by a greater CD45(+) leukocyte and F4/80(+) macrophage infiltration and upregulation of IL-1β, TNF-α, MCP-1, and ICAM-1 in the obstructed kidney and in IL-1β-stimulated macrophages. In contrast, deletion of Smad4 inhibited renal fibrosis and TGF-β1-induced collagen I expression by fibroblasts. Further studies showed that the loss of Smad4 repressed Smad7 transcription, leading to a loss of functional protein. This, in turn, inhibited IκBα expression but enhanced NF-κB activation, thereby promoting renal inflammation. Interestingly, deletion of Smad4 influenced Smad3-mediated promoter activities and the binding of Smad3 to the COL1A2 promoter, but not Smad3 phosphorylation and nuclear translocation, thereby inhibiting the fibrotic response. Thus, Smad4 may be a key regulator for the diverse roles of TGF-β1 in inflammation and fibrogenesis by interacting with Smad7 and Smad3 to influence their transcriptional activities in renal inflammation and fibrosis.

Cancer degradomics: ADAM17 regulates TGF-beta signaling through the cleavage of vasorin

Cancer degradomics: ADAM17 regulates TGF-beta signaling through the cleavage of vasorin

Krüppel-like Factor 4 Promotes Differentiation by Transforming Growth Factor-β Receptor-mediated Smad and p38 MAPK Signaling in Vascular Smooth Muscle Cells

KLF4 (Krüppel-like factor 4) has been implicated in vascular smooth muscle cell (VSMC) differentiation induced by transforming growth factor beta (TGF-beta). However, the role of KLF4 and mechanism of KLF4 actions in regulating TGF-beta signaling in VSMCs remain unclear. In this study, we showed that TGF-beta1 inhibited cell cycle progression and induced differentiation in cultured rat VSMCs. This activity of TGF-beta1 was accompanied by up-regulation of KLF4, with concomitant increase in TbetaRI (TGF-beta type I receptor) expression. KLF4 was found to transduce TGF-beta1 signals via phosphorylation-mediated activation of Smad2, Smad3, and p38 MAPK. The activation of both pathways, in turn, increased the phosphorylation of KLF4, which enabled the formation of KLF4-Smad2 complex in response to TGF-beta1. Chromatin immunoprecipitation studies and oligonucleotide pull-down assays showed the direct binding of KLF4 to the KLF4-binding sites 2 and 3 of the TbetaRI promoter and the recruitment of Smad2 to the Smad-responsive region. Formation of a stable KLF4-Smad2 complex in the promoter's Smad-responsive region mediated cooperative TbetaRI promoter transcription in response to TGF-beta1. These results suggest that KLF4-dependent regulation of Smad and p38 MAPK signaling via TbetaRI requires prior phosphorylation of KLF4 through Smad and p38 MAPK pathways. This study demonstrates a novel mechanism by which TGF-beta1 regulates VSMC differentiation.

Annexin A1 regulates TGF-beta signaling and promotes metastasis formation of basal-like breast cancer cells.

Annexin A1 (AnxA1) is a candidate regulator of the epithelial- to mesenchymal (EMT)-like phenotypic switch, a pivotal event in breast cancer progression. We show here that AnxA1 expression is associated with a highly invasive basal-like breast cancer subtype both in a panel of human breast cancer cell lines as in breast cancer patients and that AnxA1 is functionally related to breast cancer progression. AnxA1 knockdown in invasive basal-like breast cancer cells reduced the number of spontaneous lung metastasis, whereas additional expression of AnxA1 enhanced metastatic spread. AnxA1 promotes metastasis formation by enhancing TGFbeta/Smad signaling and actin reorganization, which facilitates an EMT-like switch, thereby allowing efficient cell migration and invasion of metastatic breast cancer cells.

The role of microRNA-128a in regulating TGFbeta signaling in letrozole-resistant breast cancer cells

Resistance to endocrine therapy agents has presented a clinical obstacle in the treatment of hormone-dependent breast cancer. Our laboratory has initiated a study of microRNA regulation of signaling pathways that may result in breast cancer progression on aromatase inhibitors (AI). Microarray analysis of hormone refractory cell lines identified 115 differentially regulated microRNAs, of which 49 microRNAs were believed to be hormone-responsive. A group of microRNAs were inversely expressed in the AI-resistant lines versus LTEDaro and tamoxifen-resistant. We focused our work on hsa-miR-128a which was hormone-responsive and selectively up-regulated in the letrozole-resistant cell lines. Human miR-128a was predicted to target the TGFβ signaling pathway and indeed sensitivity to TGFβ was compromised in the letrozole-resistant cells, as compared to parental MCF-7aro. Human miR-128a was shown to negatively target TGFβRI protein expression by binding to the 3'UTR region of the gene. Inhibition of endogenous miR-128a resulted in resensitization of the letrozole-resistant lines to TGFβ growth inhibitory effects. These data suggest that the hormone-responsive miR-128a can modulate TGFβ signaling and survival of the letrozole-resistant cell lines. To our knowledge, this is the first study to address the role of microRNA regulation as well as TGFβ signaling in AI-resistant breast cancer cell lines. We believe that in addition to estrogen-modulation of gene expression, hormone-regulated microRNAs may provide an additional level of post-transcriptional regulation of signaling pathways critically involved in breast cancer progression and AI-resistance.

Prdm16 is required for normal palatogenesis in mice

Transcriptional cofactors are essential to the regulation of transforming growth factor beta (TGFbeta) superfamily signaling and play critical and widespread roles during embryonic development, including craniofacial development. We describe the cleft secondary palate 1 (csp1) N-ethyl-N-nitrosourea-induced mouse model of non-syndromic cleft palate (NSCP) that is caused by an intronic Prdm16 splicing mutation. Prdm16 encodes a transcriptional cofactor that regulates TGFbeta signaling, and its expression pattern is consistent with a role in palate and craniofacial development. The cleft palate (CP) appears to be the result of micrognathia and failed palate shelf elevation due to physical obstruction by the tongue, resembling human Pierre Robin sequence (PRS)-like cleft secondary palate. PRDM16 should be considered a candidate for mutation in human clefting disorders, especially NSCP and PRS-like CP.

Latent TGF-β–binding protein 4 modifies muscular dystrophy in mice

Most single-gene diseases, including muscular dystrophy, display a nonuniform phenotype. Phenotypic variability arises, in part, due to the presence of genetic modifiers that enhance or suppress the disease process. We employed an unbiased mapping approach to search for genes that modify muscular dystrophy in mice. In a genome-wide scan, we identified a single strong locus on chromosome 7 that influenced two pathological features of muscular dystrophy, muscle membrane permeability and muscle fibrosis. Within this genomic interval, an insertion/deletion polymorphism of 36 bp in the coding region of the latent TGF-beta-binding protein 4 gene (Ltbp4) was found. Ltbp4 encodes a latent TGF-beta-binding protein that sequesters TGF-beta and regulates its availability for binding to the TGF-beta receptor. Insertion of 12 amino acids into the proline-rich region of LTBP4 reduced proteolytic cleavage and was associated with reduced TGF-beta signaling, decreased fibrosis, and improved muscle pathology in a mouse model of muscular dystrophy. In contrast, a 12-amino-acid deletion in LTBP4 was associated with increased proteolysis, SMAD signaling, and fibrosis. These data identify Ltbp4 as a target gene to regulate TGF-beta signaling and modify outcomes in muscular dystrophy.

13-P138 Regulation of TGF beta signalling by Fbxo11, the gene mutated in the Jeff Otitis Media mouse mutant

13-P138 Regulation of TGF beta signalling by Fbxo11, the gene mutated in the Jeff Otitis Media mouse mutant

S21-02. Regulation of TGF-beta signalling by Dapper2 in vertebrate development

S21-02. Regulation of TGF-beta signalling by Dapper2 in vertebrate development

Aryl hydrocarbon receptor inhibition downregulates the TGF-β/Smad pathway in human glioblastoma cells

The dioxin/aryl hydrocarbon receptor (AhR) is a transcription factor, which has been attributed a role in human cancerogenesis, cell cycle progression and transforming growth factor-beta (TGF-beta) signaling. As TGF-beta is an important mediator of the malignant phenotype of human gliomas, we studied AhR expression and function in glioma cells. AhR was not only expressed in glioma cells in vitro, but was also detected in human gliomas in vivo by immunohistochemistry, with a predominantly nuclear staining in glioblastomas. The AhR agonist, 3-methylcholanthrene, induced AhR nuclear translocation and upregulated mRNA levels of the AhR target gene, cytochrome P450 1A1 (CYP1A1). Conversely, pharmacological inhibition of AhR using the novel AhR antagonist, CH-223191, or AhR gene silencing using small interfering RNA showed that constitutive AhR activity positively controls TGF-beta1, TGF-beta2 and latent TGF-beta-binding protein-1 protein levels in malignant glioma cells. Moreover, antagonism of AhR reduced clonogenic survival and invasiveness of glioma cells. In contrast, AhR regulates TGF-beta signaling negatively in non-neoplastic astrocytes. Thus, the pathogenesis of glioma formation may involve altered AhR regulation of the TGF-beta/Smad pathway, and AhR may represent a promising target for the treatment of human malignant gliomas and other diseases associated with pathological TGF-beta activity.

Caveolin-1, transforming growth factor-β receptor internalization, and the pathogenesis of systemic sclerosis

To review the scientific literature supporting the participation of caveolin-1 in the pathogenesis of tissue fibrosis and the notion that modulation of the caveolin-1 pathway may represent a novel treatment for systemic sclerosis and other fibrotic diseases. Caveolin-1 plays an important role in the regulation of transforming growth factor-beta (TGF-beta) signaling owing to its participation in TGF-beta receptor internalization. TGF-beta receptor internalized through caveolin-1 lipid rafts undergoes rapid degradation, effectively decreasing TGF-beta signaling. Studies have shown that caveolin-1 knockdown in vitro markedly increased collagen gene expression in normal human lung fibroblasts. Caveolin-1 was reduced in affected systemic sclerosis lungs and skin and in idiopathic pulmonary fibrosis lung tissues and fibroblasts. Increasing caveolin-1 expression markedly improved bleomycin-induced pulmonary fibrosis. Restoration of caveolin bioavailability employing penetratin, a cell-permeable peptide carrier for a bioactive caveolin-1 fragment, abrogated TGF-beta activation of cultured human dermal fibroblasts. Systemic administration of penetratin-caveolin-1 peptide to mice with bleomycin-induced lung fibrosis reduced fibrosis. Caveolin-1 plays an important role in the regulation of TGF-beta signaling and participates in the pathogenesis of systemic sclerosis and idiopathic pulmonary fibrosis. Restoration of caveolin function employing active caveolin-1 fragments coupled to cell-permeable carrier peptides may represent a novel approach for their treatment.

Opposite Effects of Dihydrosphingosine 1-Phosphate and Sphingosine 1-Phosphate on Transforming Growth Factor-β/Smad Signaling Are Mediated through the PTEN/PPM1A-dependent Pathway

Transforming growth factor-beta (TGF-beta) is an important regulator of physiological connective tissue biosynthesis and plays a central role in pathological tissue fibrosis. Previous studies have established that a biologically active lipid mediator, sphingosine 1-phosphate (S1P), mimics some of the profibrotic functions of TGF-beta through cross-activation of Smad signaling. Here we report that another product of sphingosine kinase, dihydrosphingosine 1-phosphate (dhS1P), has an opposite role in the regulation of TGF-beta signaling. In contrast to S1P, dhS1P inhibits TGF-beta-induced Smad2/3 phosphorylation and up-regulation of collagen synthesis. The effects of dhS1P require a lipid phosphatase, PTEN, a key modulator of cell growth and survival. dhS1P stimulates phosphorylation of the C-terminal domain of PTEN and its subsequent translocation into the nucleus. We demonstrate a novel function of nuclear PTEN as a co-factor of the Smad2/3 phosphatase, PPM1A. Complex formation of PTEN with PPM1A does not require the lipid phosphatase activity but depends on phosphorylation of the serine/threonine residues located in the C-terminal domain of PTEN. Upon complex formation with PTEN, PPM1A is protected from degradation induced by the TGF-beta signaling. Consequently, overexpression of PTEN abrogates TGF-beta-induced Smad2/3 phosphorylation. This study establishes a novel role for nuclear PTEN in the stabilization of PPM1A. PTEN-mediated cross-talk between the sphingolipid and TGF-beta signaling pathways may play an important role in physiological and pathological TGF-beta signaling.

S1700 Smurf2 Activity in T Lymphocytes Protects Mice from Experimental Colon Cancer

Introduction: TGF-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. Recent work from our group has shown that TGF-beta signalling in T cells is protective in a mouse model of colitis associated cancer. Smad ubiquitin regulating factors (Smurf) are ubiquitin ligases that are involved in the regulation of TGF-beta signalling. The aim of this study was to determine the function of Smurf2 expression in T-cells on the pathogenesis of experimental colitis associated colon cancer. Methodology: We could isolate a known splice variant of Smurf2 lacking an Exon in the C2-domain. To analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses Smurf2 in T-cells. Smurf2 expression were analysed by qPCR. Wild type (WT) and Transgenic (TG) mice were treated once with the mutagenic agent Azoxymethan (AOM) followed by three cycles of Dextran Sodiumsulfate (DSS). After each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. Results: Smurf2 expression was upregulated by TGF-beta stimulation in T-cells and Smurf2 was markedly upregulated in tumor infiltrating CD4+ lymphocytes in AOM/DSS treated mice. Whereas WT mice suffered from severe colitis resulting in colon tumors beginning at day 35, Smurf2 transgenic mice had less colitis and were significantly protected from tumor development. Interestingly, Tlymphocytes overexpressing Smurf2 showed an upregulation of Smurf2 target genes including the TGFbRII and an activation of Smad3 as compared towild-type T-lymphocytes, whichwere previously described as typical Smurf2 targets for degradation. In addition the transfection of Smurf2 and a CAGA-Luc Plasmid into Cos-cells for Smad3-Promotor studies yielded the same effect as shown by an upregulation of the Smad3 activity. Conclusion: Although, WTSmurf has been described as a negative regulator of the TGF-beta signalling pathway, our data show surprisingly that a Smurf2 splice variant upregulates the TGF-beta receptor expression and increases TGF-beta signalling effects. Due to immunosuppressive effects on T-cells Smurf2 has beneficial effects on mucosal inflammation and colon tumor development. These findings are probably due to autoubiquitination of WTSmurf2 via the splice variant of Smurf2. Smurf2 thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis.

Regulation of TGF‐beta signaling by RGS3

Regulator of G protein signaling RGS3 belongs to a family of RGS proteins that contain the homologous RGS domain which binds the alpha subunits of heterotrimeric G proteins and accelerates their GTPase activity. Through this mechanism RGS3 regulates the signaling mediated by a variety of Gq and Gi coupled receptors. In this study, we found that RGS3 interacts with the novel partners, Smad2, Smad3 and Smad4 ‐ the transcription factors that are activated through a transforming growth factor‐beta (TGF‐beta) receptor signaling. This interaction is mediated by Smad's Mad homology domain 2 (MH2) and by the region of RGS3 outside of the RGS domain. Overexpression of RGS3 results in inhibition of Smad‐mediated gene transcription. RGS3 does not affect TGF‐beta ‐ induced Smad phosphorylation, but it prevents heteromerization of Smad3 with Smad4, which is required for Smad's transcriptional activity. Functionally, this translates to inhibition of TGF‐beta – induced myofibroblast differentiation by adenovirus‐mediated overexpression of RGS3. In conclusion, this study identifies a novel, non‐canonical role of RGS3 in regulation of TGF‐beta signaling through its interaction with Smad transcription factors.