Integrin alphaVbeta6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1

Transforming growth factor-betas (TGF-beta) are secreted as inactive complexes containing the TGF-beta, the TGF-beta propeptide, also called the latency-associated protein (LAP), and the latent TGF-beta binding protein (LTBP). Extracellular activation of this complex is a critical but incompletely understood step in TGF-beta regulation. We have investigated the role of LTBP in modulating TGF-beta generation by the integrin alphaVbeta6. We show that even though alphavbeta6 recognizes an RGD on LAP, LTBP-1 is required for alphaVbeta6-mediated latent TGF-beta activation. The domains of LTBP-1 necessary for activation include the TGF-beta propeptide-binding domain and a basic amino acid sequence (hinge domain) with ECM targeting properties. Our results demonstrate an LTBP-1 isoform-specific function in alphaVbeta6-mediated latent TGF-beta activation; LTBP-3 is unable to substitute for LTBP-1 in this assay. The results reveal a functional role for LTBP-1 in latent TGF-beta activation and suggest that activation of specific latent complexes is regulated by distinct mechanisms that may be determined by the LTBP isoform and its potential interaction with the matrix.     

Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction

Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF-beta signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-beta type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF-beta/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF-beta/ALK1 signalling, which indirectly inhibits TGF-beta/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF-beta/ALK1 signalling is reduced and TGF-beta/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF-beta-induced growth arrest by indirectly reducing TGF-beta/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.     

Klotho inhibits transforming growth factor-beta1 (TGF-beta1) signaling and suppresses renal fibrosis and cancer metastasis in mice

Fibrosis is a pathological process characterized by infiltration and proliferation of mesenchymal cells in interstitial space. A substantial portion of these cells is derived from residing non-epithelial and/or epithelial cells that have acquired the ability to migrate and proliferate. The mesenchymal transition is also observed in cancer cells to confer the ability to metastasize. Here, we show that renal fibrosis induced by unilateral ureteral obstruction and metastasis of human cancer xenografts are suppressed by administration of secreted Klotho protein to mice. Klotho is a single-pass transmembrane protein expressed in renal tubular epithelial cells. The extracellular domain of Klotho is secreted by ectodomain shedding. Secreted Klotho protein directly binds to the type-II TGF-β receptor and inhibits TGF-β1 binding to cell surface receptors, thereby inhibiting TGF-β1 signaling. Klotho suppresses TGF-β1-induced epithelial-to-mesenchymal transition (EMT) responses in cultured cells, including decreased epithelial marker expression, increased mesenchymal marker expression, and/or increased cell migration. In addition to TGF-β1 signaling, secreted Klotho has been shown to inhibit Wnt and IGF-1 signaling that can promote EMT. These results have raised the possibility that secreted Klotho may function as an endogenous anti-EMT factor by inhibiting multiple growth factor signaling pathways simultaneously.     

Autocrine signaling via release of ATP and activation of P2X7 receptor influences motile activity of human lung cancer cells

Extracellular nucleotides, such as ATP, are released from cells and play roles in various physiological and pathological processes through activation of P2 receptors. Here, we show that autocrine signaling through release of ATP and activation of P2X7 receptor influences migration of human lung cancer cells. Release of ATP was induced by stimulation with TGF-β1, which is a potent inducer of cell migration, in human lung cancer H292 cells, but not in noncancerous BEAS-2B cells. Treatment of H292 cells with a specific antagonist of P2X7 receptor resulted in suppression of TGF-β1-induced migration. PC-9 human lung cancer cells released a large amount of ATP under standard cell culture conditions, and P2X7 receptor-dependent dye uptake was observed even in the absence of exogenous ligand, suggesting constitutive activation of P2X7 receptor in this cell line. PC-9 cells showed high motile activity, which was inhibited by treatment with ecto-nucleotidase and P2X7 receptor antagonists, whereas a P2X7 receptor agonist enhanced migration. PC-9 cells also harbor a constitutively active mutation in epidermal growth factor receptor (EGFR). Treatment with EGFR tyrosine kinase inhibitor AG1478 suppressed both cell migration and P2X7 receptor expression in PC-9 cells. Compared to control PC-9 cells, cells treated with P2X7 antagonist exhibited broadened lamellipodia around the cell periphery, while AG1478-treated cells lacked lamellipodia. These results indicate that P2X7-mediated signaling and EGFR signaling may regulate migration of PC-9 cells through distinct mechanisms. We propose that autocrine ATP-P2X7 signaling is involved in migration of human lung cancer cells through regulation of actin cytoskeleton rearrangement.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9411-x) contains supplementary material, which is available to authorized users.     

TGF-beta/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage

Endochondral ossification begins from the condensation and differentiation of mesenchymal cells into cartilage. The cartilage then goes through a program of cell proliferation, hypertrophic differentiation, calcification, apoptosis, and eventually is replaced by bone. Unlike most cartilage, articular cartilage is arrested before terminal hypertrophic differentiation. In this study, we showed that TGF-beta/Smad3 signals inhibit terminal hypertrophic differentiation of chondrocyte and are essential for maintaining articular cartilage. Mutant mice homozygous for a targeted disruption of Smad3 exon 8 (Smad3(ex8/ex8)) developed degenerative joint disease resembling human osteoarthritis, as characterized by progressive loss of articular cartilage, formation of large osteophytes, decreased production of proteoglycans, and abnormally increased number of type X collagen-expressing chondrocytes in synovial joints. Enhanced terminal differentiation of epiphyseal growth plate chondrocytes was also observed in mutant mice shortly after weaning. In an in vitro embryonic metatarsal rudiment culture system, we found that TGF-beta1 significantly inhibits chondrocyte differentiation of wild-type metatarsal rudiments. However, this inhibition is diminished in metatarsal bones isolated from Smad3(ex8/ex8) mice. These data suggest that TGF-beta/Smad3 signals are essential for repressing articular chondrocyte differentiation. Without these inhibition signals, chondrocytes break quiescent state and undergo abnormal terminal differentiation, ultimately leading to osteoarthritis.     

5-azacytidine alters TGF-beta and BMP signaling and induces maturation in articular chondrocytes

Maintenance of the articular surface depends on the function of articular chondrocytes (ACs) which produce matrix and are constrained from undergoing the maturation program seen in growth plate chondrocytes. Only during pathologic conditions, such as in osteoarthritis, are maturational constraints lost causing recapitulation of the process that occurs during endochondral ossification. With the aim of establishing a model to identify regulatory mechanisms that suppress AC hypertrophy, we examined the capability of 5-azacytidine (Aza) to have an impact on the maturational program of these cells. Primary ACs do not spontaneously express markers of maturation and are refractory to treatment by factors that normally regulate chondrocyte maturation. However, following exposure to Aza, ACs (i) were induced to express type X collagen (colX), Indian hedgehog, and alkaline phosphatase and (ii) showed altered colX and AP expression in response to bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), and parathyroid hormone-related protein (PTHrP). Since Aza unmasked responsiveness of ACs to BMP-2 and TGF-beta, we examined the effect of Aza treatment on signaling via these pathways by assessing the expression of the TGF-beta Smads (2 and 3), the BMP-2 Smads (1 and 5), and the Smad2 and 3-degrading ubiquitin E3 ligase Smurf2. Aza-treated ACs displayed less Smad2 and 3 and increased Smad1, 5, and Smurf2 protein and showed a loss of TGF-beta signaling on the P3TP-luciferase reporter. Suggesting that Aza-induction of Smurf2 may be responsible for the loss of Smad2 and 3 protein via this pathway, immunoprecipitation and metabolic labeling experiments confirmed that Aza accelerated the ubiquitination and degradation of these targets. Overall, Aza-treated ACs represent a novel model for the study of mechanisms that regulate maturational potential of articular cartilage, with the data suggesting that maturation of these cells may be due to up-regulation of Smad1 and 5 coupled with a Smurf2-dependent degradation of Smad2 and 3 and loss of TGF-beta signaling.     

Endoglin in liver fibrosis

Liver fibrosis occurs in most types of chronic liver diseases and is characterized by excessive accumulation of extracellular matrix proteins, leading to disruption of tissue function and eventually organ failure. Transforming growth factor (TGF)-β represents an important pro-fibrogenic factor and aberrant TGF-β action has been implicated in many disease processes of the liver. Endoglin is a TGF-β co-receptor expressed mainly in endothelial cells that has been shown to differentially regulates TGF-β signal transduction by inhibiting ALK5-Smad2/3 signalling and augmenting ALK1-Smad1/5 signalling. Recent reports demonstrating upregulation of endoglin expression in pro-fibrogenic cell types such as scleroderma fibroblasts and hepatic stellate cells have led to studies exploring the potential involvement of this TGF-β co-receptor in organ fibrosis. A recent article by Meurer and colleagues now shows that endoglin expression is increased in transdifferentiating hepatic stellate cells in vitro and in two different models (carbon tetrachloride intoxication and bile duct ligation) of liver fibrosis in vivo. Moreover, they show that endoglin overexpression in hepatic stellate cells is associated with enhanced TGF-β-driven Smad1/5 phosphorylation and α-smooth muscle actin production without altering Smad2/3 signaling. These findings suggest that endoglin may play an important role in hepatic fibrosis by altering the balance of TGF-β signaling via the ALK1-Smad1/5 and ALK-Smad2/3 pathways and raise the possibility that targeting endoglin expression in transdifferentiating hepatic stellate cells may represent a novel therapeutic strategy for the treatment of liver fibrosis.     

Anti-human activin receptor-like kinase 1 (ALK1) antibody attenuates bone morphogenetic protein 9 (BMP9)-induced ALK1 signaling and interferes with endothelial cell sprouting

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor β (TGF-β) type I receptor, and endoglin, a TGF-β co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for antiangiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti-hALK1 antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 and TGF-β to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation, and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.     

Dual roles of immune cells and their factors in cancer development and progression

Traditional wisdom holds that intact immune responses, such as immune surveillance or immunoediting, are required for preventing and inhibiting tumor development; but recent evidence has also indicated that unresolved immune responses, such as chronic inflammation, can promote the growth and progression of cancer. Within the immune system, cytotoxic CD8(+) and CD4(+) Th1 T cells, along with their characteristically produced cytokine IFN-γ, function as the major anti-tumor immune effector cells, whereas tumor associated macrophages (TAM) or myeloid-derived suppressive cells (MDSC) and their derived cytokines IL-6, TNF, IL-1β and IL-23 are generally recognized as dominant tumor-promoting forces. However, the roles played by Th17 cells, CD4(+) CD25(+) Foxp3(+) regulatory T lymphocytes and immunoregulatory cytokines such as TGF-β in tumor development and survival remain elusive. These immune cells and the cellular factors produced from them, including both immunosuppressive and inflammatory cytokines, play dual roles in promoting or discouraging cancer development, and their ultimate role in cancer progression may rely heavily on the tumor microenvironment and the events leading to initial propagation of carcinogenesis.     

Characterization of latent recombinant TGF-beta 2 produced by Chinese hamster ovary cells.

Latent recombinant transforming growth factor-beta 2 (LrTGF-beta 2) complex has been purified from serum-free media conditioned by Chinese hamster ovary cells transfected with a plasmid encoding the TGF-beta 2 (414) precursor. Under neutral conditions, LrTGF-beta 2 had an apparent molecular weight of 130 kDa. The complex contained both mature and pro-region sequences. Acidification of LrTGF-beta 2 resulted in the release of mature 24 kDa TGF-beta 2 from the high molecular weight pro-region-containing complex, suggesting that TGF-beta 2 was non-covalently associated with this complex. These results were confirmed by crosslinking experiments performed on partially purified LrTGF-beta 2. Protein sequence analysis of the purified TGF-beta 2 pro-region indicated that signal peptide cleavage occurred between ser(20) and leu(21). The pro-region, which previously was found to contain mannose-6-phosphate, bound to the mannose-6-phosphate receptor. Proteolytic cleavage of mature TGF-beta 2 from pro-TGF-beta 2 was inhibited by monensin and chloroquine suggesting that binding to this receptor and subsequent transport to acidic vesicles may be involved in the processing of rTGF-beta 2 precursor.     

Roles of collagen and periostin expression by cranial neural crest cells during soft palate development

The tissue in the palatal region can be divided into the hard and the soft palates, each having a specialized function such as occlusion, speech, or swallowing. Therefore, an understanding of the mechanism of palatogenesis in relation to the function of each region is important. However, in comparison with the hard palate, there is still a lack of information about the mechanisms of soft palate development. In this study, the authors investigated the contribution of cranial neural crest (CNC) cells to development of both hard and soft palates. They also demonstrated a unique pattern of periostin expression during soft palate development, which was closely related to that of collagen type I (Col I) in palatine aponeurosis. Furthermore, organ culture analysis showed that exogenous transforming growth factor-β (TGF-β) induced the expression of both periostin and Col I. These novel patterns of expression in the extracellular matrix (ECM) induced by CNC cells suggest that these cells may help to determine the character of both the hard and soft palates through ECM induction. TGF-β signaling appears to be one of the mediators of Col I and periostin expression in the formation of functional structures during soft palate development.