TGFβ signaling in cartilage development and maintenance

Members of the transforming growth factor beta (TGFβ) superfamily of secreted factors play essential roles in nearly every aspect of cartilage formation and maintenance. However, the mechanisms by which TGFβs transduce their effects in cartilage in vivo remain poorly understood. Mutations in several TGFβ family members, their receptors, extracellular modulators, and intracellular transducers have been described, and these usually impact the development of the cartilaginous skeleton. Furthermore, genome‐wide association studies have linked components of the (TGFβ) superfamily to susceptibility to osteoarthritis. This review focuses on recent discoveries from genetic studies in the mouse regarding the regulation of TGFβ signaling in developing growth plate and articular cartilage, as well as the different modes of crosstalk between canonical and noncanonical TGFβ signaling. These new insights into TGFβ signaling in cartilage may open new prospects for therapies that maintain healthy articular cartilage. Birth Defects Research (Part C) 102:37–51, 2014. © 2014 Wiley Periodicals, Inc.     

β‐Catenin and Rho GTPases as downstream targets of TGF‐β1 during pulp repair

TGF‐β1 (transforming growth factor‐β1) plays a central role in regulating proliferation, migration and differentiation of dental pulp cells during the repair process after tooth injury. Our previous study showed that p38 mitogen‐activated protein kinase may act downstream of TGF‐β1 signalling to effect the differentiation of dental pulp cells. However, the molecular mechanisms that trigger and regulate the process remain to be elucidated. TGF‐β1 interacts with signalling pathways such as Wnt/β‐catenin and Rho to induce diverse biological effects. TGF‐β1 activates β‐catenin signalling, increases β‐catenin nuclear translocation and interacts with LEF/TCF to regulate gene expression. Morphologic changes in response to TGF‐β1 are associated with activation of Rho GTPases, but are abrogated by inhibitors of Rho‐associated kinase, a major downstream target of Rho. These results suggest that the Wnt/β‐catenin and Rho pathways may mediate the downstream events of TGF‐β1 signalling.     

Kips off to Myc: Implications for TGFβ signaling

Loss of sensitivity to the negative growth regulator transforming growth factor β (TGFβ) is a feature of many different tumor types and is likely involved in tumor progression. In some cases this loss of sensitivity to TGFβ has been shown to be manifest in the absence of membrane-associated TGFβ receptor complexes, thus preventing initiation of antiproliferative signals from the cell surface. In others, loss of sensitivity to TGFβ-induced inhibitory signals has been attributed to loss of function of intracellular effectors of TGFβ-induced inhibitory signals due to mutation or allelic loss of effector genes and their products. The intracellular effectors of TGFβ inhibitory signals have been shown to be involved in the normal regulation of progression through the cell cycle, specifically during G₁ phase. In this manner, elucidation of the mechanisms by which TGFβ inhibits cell growth not only helps us identify steps involved in tumor progression, but also allows us to better understand how cells regulate progression through the cell cycle. J. Cell. Biochem. 66:427–432, 1997. © 1997 Wiley-Liss, Inc.     

TGFβ1‐induced leucine limitation uncovered by differential ribosome codon reading

Cancer cells modulate their metabolic networks to support cell proliferation and a higher demand of building blocks. These changes may restrict the availability of certain amino acids for protein synthesis, which can be utilized for cancer therapy. However, little is known about the amino acid demand changes occurring during aggressive and invasive stages of cancer. Recently, we developed diricore, an approach based on ribosome profiling that can uncover amino acid limitations. Here, we applied diricore to a cellular model in which epithelial breast cells respond rapidly to TGFβ1, a cytokine essential for cancer progression and metastasis, and uncovered shortage of leucine. Further analyses indicated that TGFβ1 treatment of human breast epithelial cells reduces the expression of SLC3A2, a subunit of the leucine transporter, which diminishes leucine uptake and inhibits cell proliferation. Thus, we identified a specific amino acid limitation associated with the TGFβ1 response, a vulnerability that might be associated with aggressiveness in cancer.     

MicroRNA‐30c suppresses the pro‐fibrogenic effects of cardiac fibroblasts induced by TGF‐β1 and prevents atrial fibrosis by targeting TGFβRII

Atrial fibrosis serves as an important contributor to atrial fibrillation (AF). Recent data have suggested that microRNA‐30c (miR‐30c) is involved in fibrotic remodelling and cancer development, but the specific role of miR‐30c in atrial fibrosis remains unclear. The purpose of this study was to investigate the role of miR‐30c in atrial fibrosis and its underlying mechanisms through in vivo and in vitro experiments. Our results indicate that miR‐30c is significantly down‐regulated in the rat abdominal aortic constriction (AAC) model and in the cellular model of fibrosis induced by transforming growth factor‐β1 (TGF‐β1). Overexpression of miR‐30c in cardiac fibroblasts (CFs) markedly inhibits CF proliferation, differentiation, migration and collagen production, whereas decrease in miR‐30c leads to the opposite results. Moreover, we identified TGFβRII as a target of miR‐30c. Finally, transferring adeno‐associated virus 9 (AAV9)‐miR‐30c into the inferior vena cava of rats attenuated fibrosis in the left atrium following AAC. These data indicate that miR‐30c attenuates atrial fibrosis via inhibition of CF proliferation, differentiation, migration and collagen production by targeting TGFβRII, suggesting that miR‐30c might be a novel potential therapeutic target for preventing atrial fibrosis.     

Cardiac malformations and alteration of TGFβ signaling system in diabetic embryopathy

BACKGROUND : Cardiovascular defects are the most common anomalies in diabetic embryopathy. The mechanisms underlying the manifestation of the defects remain to be addressed. METHODS : Female mice were administered streptozotocin to induce diabetes. Embryos from euglycemic (control) and hyperglycemic groups were examined for morphological and histological evaluation of malformations. Cell proliferation and programmed cell death (apoptosis) were assessed using mitotic markers (BrdU and Ki67) and TUNEL assay, respectively. Expression of eight four genes in the TGFβ signaling system was analyzed using real‐time RT‐PCR. RESULTS : Structural abnormalities were observed in the heart and neural tube in diabetic groups, with significantly higher malformation rates than in control groups. Moreover, malformation rates in the heart were higher than those in the neural tube. Cardiac abnormalities including dilated heart tube, smaller ventricles, conotruncal stenosis, and abnormal heart looping were seen during early morphogenesis prior to cardiac septation [embryonic day (E) 9.5–11.5]. Histological examinations showed hypoplastic myocardium and endocardial cushions. After cardiac septation (E15.5), ventricular septal defects were observed, which were manifested in the non‐muscular portion of the septum. Significant decreases in cell proliferation with no differences in apoptosis were observed in the myocardium and endocardial cushions in diabetic compared to control groups. Factors in the TGFβ signaling that regulate heart development were downregulated by maternal diabetes. CONCLUSIONS : Maternal diabetes causes malformations in the heart of the embryo. The heart is more susceptible to maternal diabetic insults than the neural tube. Malformations in the heart prior to septation are associated with decreased cell proliferation, but not increased apoptosis. The TGFβ signaling is involved in cardiac malformations in diabetic embryopathy. Birth Defects Res (Part B) 89:97–105, 2010. © 2010 Wiley‐Liss, Inc.     

TGFβ‐stimulated Smad1/5 phosphorylation requires the ALK5 L45 loop and mediates the pro‐migratory TGFβ switch

During the course of breast cancer progression, normally dormant tumour‐promoting effects of transforming growth factor β (TGFβ), including migration, invasion, and metastasis are unmasked. In an effort to identify mechanisms that regulate the pro‐migratory TGFβ ‘switch’ in mammary epithelial cells in vitro, we found that TGFβ stimulates the phosphorylation of Smad1 and Smad5, which are typically associated with bone morphogenetic protein signalling. Mechanistically, this phosphorylation event requires the kinase activity and, unexpectedly, the L45 loop motif of the type I TGFβ receptor, ALK5, as evidenced by studies using short hairpin RNA‐resistant ALK5 mutants in ALK5‐depleted cells and in vitro kinase assays. Functionally, Smad1/5 co‐depletion studies demonstrate that this phosphorylation event is essential to the initiation and promotion of TGFβ‐stimulated migration. Moreover, this phosphorylation event is preferentially detected in permissive environments such as those created by tumorigenic cells or oncogene activation. Taken together, our data provide evidence that TGFβ‐stimulated Smad1/5 phosphorylation, which occurs through a non‐canonical mechanism that challenges the notion of selective Smad phosphorylation by ALK5, mediates the pro‐migratory TGFβ switch in mammary epithelial cells.     

PMEPA1 induces EMT via a non‐canonical TGF‐β signalling in colorectal cancer

Prostate transmembrane protein androgen induced 1 (PMEPA1) has been reported to promote cancer progression. Metastasis is the main factor leading to cancer progression and poor prognosis, and at the beginning of metastasis, epithelial‐to‐mesenchymal transition (EMT) is a crucial activation. However, the relationship between PMEPA1 and EMT in colorectal cancer metastasis is still poorly understood. In this study, we first testified that PMEPA1 expresses higher in tumour than normal tissue in Gene Expression Omnibus database, in the Cancer Genome Atlas (TCGA) as well as in the clinical data we collected. Moreover, the higher expression was associated with poor prognosis. We furthermore demonstrated PMEPA1 promotes colorectal cancer metastasis and EMT in vivo and in vitro. We found that PMEPA1 activates the bone morphogenetic proteins (BMP) signalling of TGF‐β signalling resulting in promoting EMT and accelerating the proliferation and metastasis of colorectal cancer.     

TGFβ regulation of membrane mucin Muc4 via proteosome degradation

Muc4 is a heterodimeric membrane mucin implicated in epithelial differentiation and tumor progression. It is expressed from a single gene as a 300 kDa precursor protein which is cleaved in the endoplasmic reticulum to its two subunits. Our previous work has shown that Muc4 is regulated by TGFβ, which represses the precursor cleavage. Working with Muc4‐transfected A375 tumor cells, we now show that Muc4 undergoes proteosomal degradation. Proteosome inhibitors prolong the life of the precursor, shunt the Muc4 into cytoplasmic aggresomes, increase the level of Muc4 associated with the endoplasmic reticulum chaperones calnexin and calreticulin and increase the levels of ubiquitinated Muc4. Most importantly, proteosome inhibitors repress the TGFβ inhibition of Muc4 expression. These results suggest a model in which TGFβ inhibits precursor cleavage, shunting the precursor into the proteosomal degradation pathway. Thus, the cells have evolved a mechanism to use the quality control pathway for glycoproteins to control the quantity of the protein produced. J. Cell. Biochem. 107: 797–802, 2009. © 2009 Wiley‐Liss, Inc.     

Hepsin regulates TGFβ signaling via fibronectin proteolysis

Transforming growth factor‐beta (TGFβ) is a multifunctional cytokine with a well‐established role in mammary gland development and both oncogenic and tumor‐suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFβ activity by acting as a storage compartment of latent‐TGFβ, but how TGFβ is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFβ signaling through the release of latent‐TGFβ from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFβ signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent‐TGFβ1, while overexpression of hepsin in mammary tumors increased TGFβ signaling. Cell‐free and cell‐based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent‐TGFβ and, importantly, that the ability of hepsin to activate TGFβ signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFβ pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.     

TGFβ2 is a prognostic‐related biomarker and correlated with immune infiltrates in gastric cancer

TGFβ2 is an essential regulator of immune cell functionality, but the mechanisms whereby it drives immune infiltration in gastric cancer remain uncertain. The Oncomine and Tumor Immunoassay Resource (TIMER) databases were used for assessing the expression of TGFβ2, after which TIMER was used to explore the relationship between TGFβ2 and tumour immune infiltration. Finally, we assessed how TGFβ2 expression correlated with the expression of a set of marker genes associated with immune infiltration using TIMER and GEPIA. We determined TGFβ2 expression to be significantly correlated with outcome in multiple types of cancer in the Cancer Genome Atlas (TCGA), with the effect being particularly pronounced in gastric cancer. Furthermore, elevated TGFβ2 expression was found to be significantly correlated with gastric cancer N staging, and with the expression of a variety of immune markers associated with particular immune cell subsets. These results indicate that TGFΒ2 is associated with patient outcome and tumour immune cell infiltration in multiple cancer types. This suggests that TGFβ2 is a key factor which governs immune cell recruitment to gastric cancer tumours, potentially playing a vital role in governing immune cell infiltration and thus representing a valuable prognostic biomarker in gastric cancer patients.