Canonical TGF-β Signaling Pathway Research Articles

SMAD4 feedback regulates the canonical TGF-\u03b2 signaling pathway to control granulosa cell apoptosis

Canonical TGF-β signals are transduced from the cell surface to the cytoplasm, and then translocated into the nucleus, a process that involves ligands (TGF-β1), receptors (TGFBR2/1), receptor-activated SMADs (SMAD2/3), and the common SMAD (SMAD4). Here we provide evidence that SMAD4, a core component of the canonical TGF-β signaling pathway, regulates the canonical TGF-β signaling pathway in porcine granulosa cells (GCs) through a feedback mechanism. Genome-wide analysis and qRT-PCR revealed that SMAD4 affected miRNA biogenesis in GCs. Interestingly, TGFBR2, the type II receptor of the canonical TGF-β signaling pathway, was downregulated in SMAD4-silenced GCs and found to be a common target of SMAD4-inhibited miRNAs. miR-425, the most significantly elevated miRNA in SMAD4-silenced GCs, mediated the SMAD4 feedback regulation of the TGF-β signaling pathway. This was accomplished through a direct interaction between the transcription factor SMAD4 and the miR-425 promoter, and a direct interaction between miR-425 and the TGFBR2 3′-UTR. Furthermore, miR-425 enhanced GC apoptosis by targeting TGFBR2 and the canonical TGF-β signaling pathway, which was rescued by SMAD4 and TGF-β1. Overall, our findings demonstrate that a positive feedback mechanism exists within the canonical TGF-β signaling pathway. This study also provides new insights into mechanism underlying the canonical TGF-β signaling pathway, which regulates GC function and follicular development.

Reloading Promotes Recovery of Disuse Muscle Loss by Inhibiting TGFβ Pathway Activation in Rats After Hind Limb Suspension.

The purpose of this paper was to study the effect of transforming growth factor beta (TGFβ) signaling pathway on reloading-mediated restoration of disuse muscle loss induced by hind limb suspension in rats. Rats were divided into 4 groups: control group (CON), HLS group (hind limb suspension for 2 weeks), HLS + R group (hind limb suspension for 2 weeks followed by 2 weeks of natural reloading), and HRS + E group (hind limb suspension for 2 weeks followed by 2 weeks of treadmill exercise). Body weight, and weight and protein concentration of gastrocnemius were determined. The expression of members of canonical and noncanonical TGFβ signaling pathways, including TGFβ1, myostatin (MSTN), phospho-smad2/3, phospho-mitogen-activated protein kinases (p38, JNK1/2, and extracellular signal-regulated kinase 1 [ERK1]/ERK2), as well as the corresponding downstream effectors of muscle mass-p21, Pax7, MyoD, and MyoG-was determined at protein or messenger RNA (mRNA) levels. Reloading increased MyoD mRNA and restored the decreased gastrocnemius weight/body weight ratio, protein concentration of gastrocnemius, phospho-ERK2, Pax7 and the increased TGFβ1, MSTN, phospho-smad2/3, phospho-p38, phospho-JNK1/2, and p21 induced by hind limb suspension. Moreover, the effects of exercise reloading on the restoration of gastrocnemius weight/body weight ratio, TGFβ1, MSTN, phospho-smad2, phospho-p38, phospho-JNK2, Pax7, as well as the induction of MyoD mRNA were stronger than those of natural reloading. Disuse muscle loss can be recovered by reloading in an intensity-dependent manner through canonical and noncanonical TGFβ signaling pathways. Pax7 and MyoD might be the effectors of TGFβ pathway in mediating the recovery effect of reloading.

Sjögren's syndrome-associated microRNAs in CD14(+) monocytes unveils targeted TGFβ signaling.

BackgroundSjögren’s syndrome (SjS) monocytes have a pro-inflammatory phenotype, which may influence SjS pathogenesis. MicroRNAs (miRNAs) are small endogenously expressed molecules that can inhibit protein expression of their targeted genes and have important functions in regulating cell signaling responses. We profiled miRNAs in SjS monocytes to identify a SjS-specific miRNA profile and determine the potential roles of miRNAs in SjS pathogenesis.MethodsTotal RNA was extracted from healthy control (HC, n = 10), SjS (n = 18), systemic lupus erythematosus (SLE, n = 10), and rheumatoid arthritis (RA, n = 10) peripheral blood CD14+ monocytes for miRNA microarray analysis. To validate select miRNAs from the microarray analysis, the original cohort and a new cohort of monocyte RNA samples from HC (n = 9), SjS (n = 12), SLE (n = 8), and RA (n = 9) patients were evaluated by quantitative reverse transcription (RT)-PCR. Functional predictions of differentially expressed miRNAs were determined through miRNA target prediction database analyses. Statistical analyses performed included one-way analysis of variance with Bonferroni post tests, linear regression, and receiver operating characteristic curve analyses.ResultsMiRNAs were predominantly upregulated in SjS monocytes in comparison with controls. Quantitative RT-PCR confirmations supported co-regulation of miR-34b-3p, miR-4701-5p, miR-609, miR-300, miR-3162-3p, and miR-877-3p in SjS monocytes (13/30, 43.3 %) in comparison with SLE (1/17, 5.8 %) and RA (1/18, 5.6 %). MiRNA-target pathway predictions identified SjS-associated miRNAs appear to preferentially target the canonical TGFβ signaling pathway as opposed to pro-inflammatory interleukin-12 and Toll-like receptor/NFkB pathways.ConclusionsOur results underscore a novel underlying molecular mechanism where SjS-associated miRNAs may collectively suppress TGFβ signaling as opposed to pro-inflammatory interleukin-12 and Toll-like receptor/NFκB pathways in SjS pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-0987-0) contains supplementary material, which is available to authorized users.

Smad4 Deficiency in Smooth Muscle Cells Initiates the Formation of Aortic Aneurysm.

Aortic aneurysm is a life-threatening cardiovascular disorder caused by the predisposition for dissection and rupture. Genetic studies have proved the involvement of the transforming growth factor-β (TGF-β) pathway in aortic aneurysm. Smad4 is the central mediator of the canonical TGF-β signaling pathway. However, the exact role of Smad4 in smooth muscle cells (SMCs) leading to the pathogenesis of aortic aneurysms is largely unknown. To determine the role of smooth muscle Smad4 in the pathogenesis of aortic aneurysms. Conditional gene knockout strategy combined with histology and expression analysis showed that Smad4 or TGF-β receptor type II deficiency in SMCs led to the occurrence of aortic aneurysms along with an upregulation of cathepsin S and matrix metallopeptidase-12, which are proteases essential for elastin degradation. We further demonstrated a previously unknown downregulation of matrix metallopeptidase-12 by TGF-β in the aortic SMCs, which is largely abrogated in the absence of Smad4. Chemotactic assay and pharmacologic treatment demonstrated that Smad4-deficient SMCs directly triggered aortic wall inflammation via the excessive production of chemokines to recruit macrophages. Monocyte/macrophage depletion or blocking selective chemokine axis largely abrogated the progression of aortic aneurysm caused by Smad4 deficiency in SMCs. The findings reveal that Smad4-dependent TGF-β signaling in SMCs protects against aortic aneurysm formation and dissection. The data also suggest important implications for novel therapeutic strategies to limit the progression of the aneurysm resulting from TGF-β signaling loss-of-function mutations.

Role of Smad3 in platelet-derived growth factor-C-induced liver fibrosis.

Chronic liver injury leads to fibrosis and cirrhosis. Cirrhosis, the end stage of chronic liver disease, is a leading cause of death worldwide and increases the risk of developing hepatocellular carcinoma. Currently, there is a lack of effective antifibrotic therapies to treat fibrosis and cirrhosis. Development of antifibrotic therapies requires an in-depth understanding of the cellular and molecular mechanisms involved in inflammation and fibrosis after hepatic injury. Two growth factor signaling pathways that regulate liver fibrosis are transforming growth factor-β (TGFβ) and platelet-derived growth factor (PDGF). However, their specific contributions to fibrogenesis are not well understood. Using a genetic model of liver fibrosis, we investigated whether the canonical TGFβ signaling pathway was necessary for fibrogenesis. PDGF-C transgenic (PDGF-C Tg) mice were intercrossed with mice that lack Smad3, and molecular and histological fibrosis was analyzed. PDGF-C Tg mice that also lacked Smad3 had less fibrosis and improved liver lobule architecture. Loss of Smad3 also reduced expression of collagen genes, which were induced by PDGF-C, but not the expression of genes frequently associated with hepatic stellate cell (HSC) activation. In vitro HSCs isolated from Smad3-null mice proliferated more slowly than cells from wild-type mice. Taken together, these findings indicate that PDGF-C activates TGFβ/Smad3 signaling pathways to regulate HSC proliferation, collagen production and ultimately fibrosis. In summary, these results suggest that inhibition of both PDGF and TGFβ signaling pathways may be required to effectively attenuate fibrogenesis in patients with chronic liver disease.

Abstract 4180: TGF-beta promotes angiogenesis in an RB-deficient, Kras-driven mouse model of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is projected to become the 2nd leading cause of cancer deaths by 2030. PDACs are associated with a high frequency of KRAS mutations (95%) and overexpression of many pro-angiogenic cytokines and growth factors. Using a genetically engineered mouse model (GEMM) that we established in which oncogenic Kras is combined with loss of RB (KRC), we have determined that PDACs arising in these mice (mPDACs) harbor many endothelial cells (ECs) and sinusoidal-like vessels that have blood flow, as determined by intravital confocal microscopy. Array analysis of pancreatic cancer cells (PCCs) derived from KRC tumors revealed gene expression profiles that correlate with active TGF-β signaling pathways, as determined by gene set enrichment analysis (GSEA), and include increased expression of multiple pro-angiogenic genes. In silico analysis indicated that many of these pro-angiogenic genes were TGF-β targets, and inhibition of the type I TGF-β receptor (TβRI) with SB505124 confirmed that TGF-βs drive pro-angiogenic gene expression in KRC PCCs. Moreover, TGF-β increased the levels of pro-angiogenic cytokines in conditioned media (CM) prepared from KRC PCCs, which when added to ECs, activated canonical TGF-β signaling pathways and stimulated EC proliferation and migration. Although SB505124 blocked TGF-β pathway activation in ECs, it failed to suppress CM-enhanced EC proliferation and migration. By contrast, SB505124 attenuated tumor angiogenesis, growth and metastasis in a syngeneic orthotopic mouse model using KRC PCCs. Taken together, these results suggest that TGF-β promotes angiogenesis in an indirect manner, by up-regulating pro-angiogenic factors in PCCs that act on ECs in a paracrine manner. Therefore, targeting TGF-β in PDAC could be a beneficial anti-angiogenic strategy. Citation Format: Jesse Gore, Kelly E. Craven, Julie L. Wilson, Murray Korc. TGF-beta promotes angiogenesis in an RB-deficient, Kras-driven mouse model of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4180. doi:10.1158/1538-7445.AM2015-4180

Role of dihydrotestosterone (DHT) on TGF-β1 signaling pathway in epithelial ovarian cancer cells.

PurposeOne of the hypotheses regarding the genesis of epithelial ovarian cancer involves the action of androgens on the proliferation of epithelial ovarian cells, as well as inclusion cysts. The purpose of the present study was to evaluate whether DHT causes changes in the TGF-β1 pathway that might modify the anti-proliferative effect of the latter.MethodsThe levels of TGF-β1 protein, of its receptors (TGFBR1 and TGFBR2), of Smad2/3 (canonical signaling pathway protein) and of p21 (cell cycle protein) were assessed in ovarian tissues, epithelial ovarian cancer cell lines (A2780) and control cell lines (HOSE) through the use of immunohistochemistry and immunocytochemistry. Additionally, cell lines were treated with 100 nmol/L DHT, 10 ng/mL of TGF-β1 and DHT + TGF-β1 during 72 h in the presence and absence of a siRNA against androgen receptor. After treatment, TGFBR1 and TGFBR2 levels were detected through Western blotting and p21 was assessed through immunocytochemistry.ResultsEpithelial ovarian cancer tissues showed a decrease in TGF-β1 I receptor (p < 0.05) and a change in Smad2/3 protein levels. Additionally, after treatment of cell lines with DHT, protein levels of TGF-β1 receptors (TGFBR1–TGFBR2) showed a decrease (p < 0.05) that might cause a potential disorder in TGF-β1 response, represented by the significant decrease in p21 protein levels in the presence of DHT (p < 0.001).ConclusionsOverall, our results indicate a defect in the canonical TGF-β signaling pathway in epithelial ovarian cancer caused by androgen action, thus suggesting eventual changes in such tissue proliferation rates.

Elevated expression levels of lysyl oxidases protect against aortic aneurysm progression in Marfan syndrome

Patients with Marfan syndrome (MFS) are at high risk of life-threatening aortic dissections. The condition is caused by mutations in the gene encoding fibrillin-1, an essential component in the formation of elastic fibers. While experimental findings in animal models of the disease have shown the involvement of transforming growth factor-β (TGF-β)- and angiotensin II-dependent pathways, alterations in the vascular extracellular matrix (ECM) may also play a role in the onset and progression of the aortic disease. Lysyl oxidases (LOX) are extracellular enzymes, which initiates the formation of covalent cross-linking of collagens and elastin, thereby contributing to the maturation of the ECM. Here we have explored the role of LOX in the formation of aortic aneurysms in MFS. We show that aortic tissue from MFS patients and MFS mouse model (Fbn1C1039G/+) displayed enhanced expression of the members of the LOX family, LOX and LOX-like 1 (LOXL1), and this is associated with the formation of mature collagen fibers. Administration of a LOX inhibitor for 8weeks blocked collagen accumulation and aggravated elastic fiber impairment, and these effects correlated with the induction of a strong and rapidly progressing aortic dilatation, and with premature death in the more severe MFS mouse model, Fbn1mgR/mgR, without any significant effect on wild type animals. This detrimental effect occurred preferentially in the ascending portion of the aorta, with little or no involvement of the aortic root, and was associated to an overactivation of both canonical and non-canonical TGF-β signaling pathways. The blockade of angiotensin II type I receptor with losartan restored TGF-β signaling activation, normalized elastic fiber impairment and prevented the aortic dilatation induced by LOX inhibition in Fbn1C1039G/+ mice. Our data indicate that LOX enzymes and LOX-mediated collagen accumulation play a critical protective role in aneurysm formation in MFS.

Erratum to: Angiotensin II stimulates canonical TGF-β signaling pathway through angiotensin type 1 receptor to induce granulation tissue contraction

Erratum to: Angiotensin II stimulates canonical TGF-β signaling pathway through angiotensin type 1 receptor to induce granulation tissue contraction

Anti-tumor activity of the TGF-β receptor kinase inhibitor galunisertib (LY2157299 monohydrate) in patient-derived tumor xenografts.

PurposeThe transforming growth factor-beta (TGF-β) signaling pathway is known to play a critical role in promoting tumor growth. Consequently, blocking this pathway has been found to inhibit tumor growth. In order to achieve an optimal anti-tumor effect, however, it remains to be established whether blocking the TGF-β signaling pathway alone is sufficient, or whether the tumor microenvironment plays an additional, possibly synergistic, role.MethodsTo investigate the relevance of blocking TGF-β signaling in tumor cells within the context of their respective tissue microenvironments, we treated a panel of patient-derived xenografts (PDX) with the selective TGF-β receptor kinase inhibitor LY2157299 monohydrate (galunisertib) and assessed both the in vitro and in vivo effects.ResultsGalunisertib was found to inhibit the growth in an in vitro clonogenic assay in 6.3 % (5/79) of the examined PDX. Evaluation of the expression profiles of a number of genes, representing both canonical and non-canonical TGF-β signaling pathways, revealed that most PDX exhibited expression changes affecting TGF-β downstream signaling. Next, we subjected 13 of the PDX to an in vivo assessment and, by doing so, observed distinct response patterns. These results suggest that, next to intrinsic, also extrinsic or microenvironmental factors can affect galunisertib response. pSMAD2 protein expression and TGF-βRI mRNA expression levels were found to correlate with the in vivo galunisertib effects.ConclusionsFrom our data we conclude that intrinsic, tumor-dependent TGF-β signaling does not fully explain the anti-tumor effect of galunisertib. Hence, in vivo xenograft models may be more appropriate than in vitro clonogenic assays to assess the anti-tumor activity of TGF-β inhibitors such as galunisertib.Electronic supplementary materialThe online version of this article (doi:10.1007/s13402-014-0210-8) contains supplementary material, which is available to authorized users.

Angiotensin II stimulates canonical TGF-β signaling pathway through angiotensin type 1 receptor to induce granulation tissue contraction.

Hypertrophic scar contraction (HSc) is caused by granulation tissue contraction propagated by myofibroblast and fibroblast migration and contractility. Identifying the stimulants that promote migration and contractility is key to mitigating HSc. Angiotensin II (AngII) promotes migration and contractility of heart, liver, and lung fibroblasts; thus, we investigated the mechanisms of AngII in HSc. Human scar and unwounded dermis were immunostained for AngII receptors angiotensin type 1 receptor (AT1 receptor) and angiotensin type 2 receptor (AT2 receptor) and analyzed for AT1 receptor expression using Western blot. In vitro assays of fibroblast contraction and migration under AngII stimulation were conducted with AT1 receptor, AT2 receptor, p38, Jun N-terminal kinase (JNK), MEK, and activin receptor-like kinase 5 (ALK5) antagonism. Excisional wounds were created on AT1 receptor KO and wild-type (WT) mice treated with AngII ± losartan and ALK5 and JNK inhibitors SB-431542 and SP-600125, respectively. Granulation tissue contraction was quantified, and wounds were analyzed by immunohistochemistry. AT1 receptor expression was increased in scar, but not unwounded tissue. AngII induced fibroblast contraction and migration through AT1 receptor. Cell migration was inhibited by ALK5 and JNK, but not p38 or MEK blockade. In vivo experiments determined that absence of AT1 receptor and chemical AT1 receptor antagonism diminished granulation tissue contraction while AngII stimulated wound contraction. AngII granulation tissue contraction was diminished by ALK5 inhibition, but not JNK. AngII promotes granulation tissue contraction through AT1 receptor and downstream canonical transforming growth factor (TGF)-β signaling pathway, ALK5. Further understanding the pathogenesis of HSc as an integrated signaling mechanism could improve our approach to establishing effective therapeutic interventions. AT1 receptor expression is increased in scar tissue compared to unwounded tissue. AngII stimulates expression of proteins that confer cell migration and contraction. AngII stimulates fibroblast migration and contraction through AT1 receptor, ALK5, and JNK. AngII-stimulated in vivo granulation tissue contraction is AT1 receptor and ALK5 dependent.

Chemosensation of Bacterial Secondary Metabolites Modulates Neuroendocrine Signaling and Behavior of C. elegans

Discrimination between pathogenic and beneficial microbes is essential for host organism immunity and homeostasis. Here, we show that chemosensory detection of two secondary metabolites produced by Pseudomonas aeruginosa modulates a neuroendocrine signaling pathway that promotes avoidance behavior in the simple animal host Caenorhabditis elegans. Secondary metabolites phenazine-1-carboxamide and pyochelin activate a G-protein-signaling pathway in the ASJ chemosensory neuron pair that induces expression of the neuromodulator DAF-7/TGF-β. DAF-7, in turn, activates a canonical TGF-β signaling pathway in adjacent interneurons to modulate aerotaxis behavior and promote avoidance of pathogenic P. aeruginosa. Our data provide a chemical, genetic, and neuronal basis for how the behavior and physiology of a simple animal host can be modified by the microbial environment and suggest that secondary metabolites produced by microbes may provide environmental cues that contribute to pathogen recognition and host survival.

Cutting the brakes and flooring the gas: how TMEPAI turns TGF-β into a tumor promoter.

In normal or nonmalignant cells, TGF-β inhibits cellular proliferation through activation of the SMAD-dependent canonical signaling pathway. Recent findings demonstrate that the protein TMEPAI1 can block the cytostatic effects of the canonical TGF-β signaling pathway, while activating cellular proliferation through the noncanonical, SMAD-independent TGF-β signaling pathway. As TMEPAI1 shows increased expression in the poor prognosis basal and HER2 intrinsic subtypes of breast cancer, these findings point to a new avenue of targeted therapy with considerable therapeutic potential.

Abstract 969: TGF-beta cross-talks with the EGF receptor family to promote proliferation of pancreatic cancer cells with dysfunctional RB

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy, associated with a high frequency of KRAS mutations (95%) and loss of negative growth constraints, due, in part, to frequent CDKN2A (85%), TP53 (75%) and SMAD4 (55%) mutations. PDACs also overexpress all three TGF-β isoforms, and high levels of TGF-βs correlates with decreased survival. Recently, we showed that in human PDAC and in murine PDAC (mPDAC) arising in biologically aggressive, oncogenic Kras-driven genetically engineered mouse models (GEMMs), proliferating pancreatic cancer cells (PCCs) often exhibit abundant phosphorylated Smad2 and RB indicating that in these PCCs, canonical TGF-β signaling pathways are active, whereas RB is inactive. Using PCCs derived from a GEMM that expresses oncogenic Kras, but lacks RB (KRC cells), we showed that TGF-β1 activates canonical and non-canonical (MEK, Src, PI3K) pathways, and enhances growth in 3-dimensional (3D) culture. This mitogenic effect is abrogated by type I TGF-β receptor (TβRI) kinase inhibition with SB505124, combined inhibition by MEK/Src or MEK/PI3K, or re-expression of wild-type, but not mutant RB. Thus, loss of RB function enables TGF-β to directly enhance PCC growth. Given that the EGF receptor (EGFR) family can activate these non-canonical pathways and that TGF-β cross-talks with EGFR, we investigated the role of EGFR family members in mediating the mitogenic effects of TGF-β on RB-deficient PCCs. In 3D culture, TGF-β1-enhanced proliferation was inhibited by targeting EGFR with erlotinib and completely suppressed by EGFR/HER2 inhibition with lapatinib. Moreover, lentiviral-mediated silencing of HER2 markedly attenuated TGF-β1-stimulated KRC growth, and this inhibitory effect was potentiated by erlotinib. Thus, TGF-β1's mitogenic effects are mediated, in part, through transactivation of EGFR/HER2, raising the possibility that concomitant use of TβRI inhibitors and lapatinib to inhibit EGFR/HER2 and may represent a novel therapeutic approach in PDAC. Citation Format: Jesse Gore, Samantha L. Deitz, Julie L. Wilson, Murray Korc. TGF-beta cross-talks with the EGF receptor family to promote proliferation of pancreatic cancer cells with dysfunctional RB. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 969. doi:10.1158/1538-7445.AM2014-969

Abstract 1137: Bub1 is a key regulator of TGF-β signaling

Abstract The transforming growth factor-beta (TGF-β) family of cytokines regulates many processes such as immune suppression, angiogenesis, wound healing and epithelial to mesenchymal transition (EMT. Early in tumorigenesis, when epithelial cells retain exquisite growth sensitivity to this ligand, TGF-β signaling elicits a tumor suppressing activity. However, transformed cells become refractory to TGF-β-mediated growth inhibition and acquire a phenotype wherein the intracellular signaling circuitry of the cells is altered, leading to tumorigenic and metastatic effects in response to TGF-β exposure. Although TGF-β activating pathways have been studied, the molecular participants are poorly defined. Here we identify budding uninhibited by benzimidazoles-1 (Bub1) as an integral component of canonical and non-canonical TGF-β signaling pathways, where Bub1 is required for TGFBRI-TGFBRII complex formation and activation. Bub1-depleted cells exhibited reductions in TGF-β dependent Smad2/3 phosphorylation, recruitment of Smad2/3 to the TGFBRI-II complex, PI3K/Akt and p38MAPK activation, Smad binding element driven promoter activity (SBE4-Luc), and invasion and migration. Furthermore, a targeted small molecule inhibitor of Bub1 kinase activity (2OH-BNPP1), as well as an inactive kinase mutant of Bub1, abrogated ligand mediated TGF-β signaling and phenotypic response. These studies demonstrate a role for the Bub1 kinase in mediating TGF-β dependent signaling beyond its established function in cell-cycle regulation and chromosome cohesion and uncover the underlying basis for the pleiotropic cellular response commonly observed upon activation of the pathway. Citation Format: Shyam Nyati, Katrina Schinske-Sebolt, Sethuramasundaram Pitchiaya, Katerina Chekhovskiy, Areeb Chator, Nauman Chaudhry, Joseph Dosch, Marcian E. Van Dort, Varambally, Kumar-Sinha, Nyati, Ray, Walter, Sooryanarayana Varambally, Chandan Kumar-Sinha, Mukesh K. Nyati, Dipankar Ray, Nils G. Walter, Hongtao Yu, Brian D. Ross, Alnawaz Rehemtulla. Bub1 is a key regulator of TGF-β signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1137. doi:10.1158/1538-7445.AM2014-1137

Angiotensin II Stimulates Canonical TGFβ Signaling Pathway through Angiotensin Receptor 1 to Induce Granulation Tissue Contraction

Angiotensin II Stimulates Canonical TGFβ Signaling Pathway through Angiotensin Receptor 1 to Induce Granulation Tissue Contraction

Transcriptional Regulation of Seprase in Invasive Melanoma Cells by Transforming Growth Factor-β Signaling

The tumor invasive phenotype driven by seprase expression/activity has been widely examined in an array of malignant tumor cell types; however, very little is known about the transcriptional regulation of this critical protease. Seprase (also named fibroblast activation protein-α, antiplasmin-cleaving enzyme, and dipeptidyl prolyl peptidase 5) is expressed at high levels by stromal fibroblast, endothelial, and tumor cells in a variety of invasive tumors but is undetectable in the majority of normal adult tissues. To examine the transcriptional regulation of the gene, we cloned the human seprase promoter and demonstrated that endogenous seprase expression and exogenous seprase promoter activity are high in invasive melanoma cells but not in non-invasive melanoma cells/primary melanocytes. In addition, we identified a crucial TGF-β-responsive cis-regulatory element in the proximal seprase promoter region that enabled robust transcriptional activation of the gene. Treatment of metastatic but not normal/non-invasive cells with TGF-β1 caused a rapid and profound up-regulation of endogenous seprase mRNA, which coincided with an abolishment of the negative regulator c-Ski, and an increase in binding of Smad3/4 to the seprase promoter in vivo. Blocking TGF-β signaling in invasive melanoma cells through overexpression of c-Ski, chemically using SB-431542, or with a neutralizing antibody against TGF-β significantly reduced seprase mRNA levels. Strikingly, RNAi of seprase in invasive cells greatly diminished their invasive potential in vitro as did blocking TGF-β signaling using SB-431542. Altogether, we found that seprase is transcriptionally up-regulated in invasive melanoma cells via the canonical TGF-β signaling pathway, supporting the roles of both TGF-β and seprase in tumor invasion and metastasis.

Proteinase-activated Receptor-2 Transactivation of Epidermal Growth Factor Receptor and Transforming Growth Factor-β Receptor Signaling Pathways Contributes to Renal Fibrosis

Chronic kidney diseases cause significant morbidity and mortality in the population. During renal injury, kidney-localized proteinases can signal by cleaving and activating proteinase-activated receptor-2 (PAR2), a G-protein-coupled receptor involved in inflammation and fibrosis that is highly expressed in renal tubular cells. Following unilateral ureteric obstruction, PAR2-deficient mice displayed reduced renal tubular injury, fibrosis, collagen synthesis, connective tissue growth factor (CTGF), and α-smooth muscle actin gene expression at 7 days, compared with wild-type controls. In human proximal tubular epithelial cells in vitro, PAR2 stimulation with PAR2-activating peptide (PAR2-AP) alone significantly up-regulated the expression of CTGF, a potent profibrotic cytokine. The induction of CTGF by PAR2-AP was synergistically increased when combined with transforming growth factor-β (TGF-β). Consistent with these findings, treating human proximal tubular epithelial cells with PAR2-AP induced Smad2/3 phosphorylation in the canonical TGF-β signaling pathway. The Smad2 phosphorylation and CTGF induction required signaling via both the TGFβ-receptor and EGF receptor suggesting that PAR2 utilizes transactivation mechanisms to initiate fibrogenic signaling. Taken together, our data support the hypothesis that PAR2 synergizes with the TGFβ signaling pathway to contribute to renal injury and fibrosis.

Chondrogenesis of Mesenchymal Stem Cells in an Osteochondral Environment Is Mediated by the Subchondral Bone

In articular cartilage repair, cells that will be responsible for the formation of repair tissue are often exposed to an osteochondral environment. To study cartilage repair mechanisms in vitro, we have recently developed a bovine osteochondral biopsy culture model in which cartilage defects can be simulated reproducibly. Using this model, we now aimed at studying the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (hBMSCs) in an osteochondral environment. In contrast to standard in vitro chondrogenesis, it was found that supplementing transforming growth factor beta (TGFβ) to culture medium was not required to induce chondrogenesis of hBMSCs in an osteochondral environment. hBMSC culture in defects created in osteochondral biopsies or in bone-only biopsies resulted in comparable levels of cartilage-related gene expression, whereas culture in cartilage-only biopsies did not induce chondrogenesis. Subcutaneous implantation in nude mice of osteochondral biopsies containing hBMSCs in osteochondral defects resulted in the formation of more cartilaginous tissue than hBMSCs in chondral defects. The subchondral bone secreted TGFβ; however, the observed results could not be attributed to TGFβ, as either capturing TGFβ with an antibody or blocking the canonical TGFβ signaling pathway did not result in significant changes in cartilage-related gene expression of hBMSCs in the osteochondral culture model. Inhibition of BMP signaling did not prevent chondrogenesis. In conclusion, we demonstrate that chondrogenesis of hBMSCs is induced by factors secreted from the bone. We have strong indications that this is not solely mediated by members of the TGFβ family but other, yet unknown, factors originating from the subchondral bone appeared to play a key role.

Tuftsin signals through its receptor neuropilin‐1 via the transforming growth factor beta pathway

Tuftsin (Thr-Lys-Pro-Arg) is a natural immunomodulating peptide found to stimulate phagocytosis in macrophages/microglia. Tuftsin binds to the receptor neuropilin-1 (Nrp1) on the surface of cells. Nrp1 is a single-pass transmembrane protein, but its intracellular C-terminal domain is too small to signal independently. Instead, it associates with a variety of coreceptors. Despite its long history, the pathway through which tuftsin signals has not been described. To investigate this question, we employed various inhibitors to Nrp1's coreceptors to determine which route is responsible for tuftsin signaling. We use the inhibitor EG00229, which prevents tuftsin binding to Nrp1 on the surface of microglia and reverses the anti-inflammatory M2 shift induced by tuftsin. Furthermore, we demonstrate that blockade of transforming growth factor beta (TGFβ) signaling via TβR1 disrupts the M2 shift similar to EG00229. We report that tuftsin promotes Smad3 phosphorylation and reduces Akt phosphorylation. Taken together, our data show that tuftsin signals through Nrp1 and the canonical TGFβ signaling pathway. Despite the 40-year history of the tetrapeptide tuftsin (TKPR), a macrophage and microglial activator, its mechanism of action has not been defined. Here, we report that the tuftsin-mediated anti-inflammatory M2 shift in microglia is caused specifically by tuftsin binding to the receptor neuropilin-1 (Nrp1) and signaling through TGFβ receptor-1, a coreceptor of Nrp1. We further show that tuftsin signals via the canonical TGFβ pathway and promotes TGFβ release from target cells.