Endoglin differentially modulates antagonistic transforming growth factor-beta1 and BMP-7 signaling

Transforming growth factor-beta1 (TGF-beta1) and BMP-7 (bone morphogenetic protein-7; OP-1) play central, antagonistic roles in kidney fibrosis, a setting in which the expression of endoglin (CD105), an accessory TGF-beta type III receptor, is increased. So far, endoglin is known as a negative regulator of TGF-beta/ALK-5 signaling. Here we analyzed the effect of BMP-7 on TGF-beta1 signaling and the role of endoglin for both pathways in endoglin-deficient L(6)E(9) cells. In this myoblastic cell line, TGF-beta1 and BMPs are opposing cytokines, interfering with myogenic differentiation. Both induce specific target genes of which Id1 (for BMPs) and collagen I (for TGF-beta1) are two examples. TGF-beta1 activated two distinct type I receptors, ALK-5 and ALK-1, in these cells. Although the ALK-5/Smad3 signaling pathway mediated collagen I expression, ALK-1/Smad1/Smad5 signaling mediated a transient Id1 up-regulation. In contrast, BMP-7 exclusively activated Smad1/Smad5 resulting in a more prolonged Id1 expression. Although BMP-7 had no impact on collagen I abundance, it antagonized TGF-beta1-induced collagen I expression and (CAGA)(12)-MLP-Luc activity, effects that are mediated by the ALK-5/Smad3 pathway. Finally, we found that the transient overexpression of endoglin, previously shown to inhibit TGF-beta1-induced ALK-5/Smad3 signaling, enhanced the BMP-7/Smad1/Smad5 pathway.     

Angiopoietin 1, PDGF-B, and TGF-beta gene regulation in endothelial cell and smooth muscle cell interaction

The vascular wall is mainly composed of endothelial cells (ECs) and smooth muscle cells (SMCs). The crosstalking between these two cell types is critical in the vascular maturation process. Genetic studies suggest that the Tie2/angiopoietin 1 (Ang1) pathway regulates vascular remodeling. However, the molecular mechanism is unclear. PDGF is a potent chemoattractant for SMCs, and TGF-beta regulates SMC differentiation. Here, we examined gene regulation. PDGF-B stimulation upregulated Ang1 expression in SMCs through the PI3K and PKC pathways. PDGF-B stimulation also produced an acute induction of TGF-beta expression in SMCs through the MAPK/ERK pathway. Interestingly, TGF-beta negatively regulated Ang1 expression induced by the PDGF-B stimulation in SMCs. Reciprocally, we observed that stimulation of ECs with either Ang1 or TGF-beta slightly downregulated PDGF expression. A combination of both TGF-beta with Ang1 produced much stronger downregulation of PDGF. Our data showed complex gene regulations that include both positive and negative regulations between ECs and SMCs to maintain vascular homeostasis.     

Effects of transforming growth factor-beta 1 and ascorbic acid on differentiation of human bone-marrow-derived mesenchymal stem cells into smooth muscle cell lineage

Bone-marrow-derived mesenchymal stem cells (MSCs) can differentiate into a variety of cell types including smooth muscle cells (SMCs). We have attempted to demonstrate that, following treatment with transforming growth factor-beta 1 (TGF-beta1) and ascorbic acid (AA), human bone-marrow-derived MSCs differentiate into the SMC lineage for use in tissue engineering. Quantitative polymerase chain reaction for SMC-specific gene (alpha smooth muscle actin, h1-calponin, and SM22alpha) expression was performed on MSCs, which were cultured with various concentrations of TGF-beta1 or AA. TGF-beta1 had a tendency to up-regulate the expression of SMC-specific genes in a dose-dependent manner. The expression of SM22alpha was significantly up-regulated by 30 muM AA. We also investigated the additive effect of TGF-beta1 and AA for differentiation into SMCs and compared this effect with that of other factors including platelet-derived growth factor BB (PDGF-BB). In addition to SMC-specific gene expression, SMC-specific proteins increased by two to four times when TGF-beta1 and AA were used together compared with their administration alone. PDGF did not increase the expression of SMC-specific markers. MSCs cultured with TGF-beta1 and AA did not differentiate into osteoblasts and adipocytes. These results suggest that a combination of TGF-beta1 and AA is useful for the differentiation of MSCs into SMCs for use in tissue engineering.     

Endoglin promotes transforming growth factor beta-mediated Smad 1/5/8 signaling and inhibits endothelial cell migration through its association with GIPC

Transforming growth factor beta (TGF-beta) signals through two distinct pathways to regulate endothelial cell proliferation, migration, and angiogenesis, the ALK-1/Smad 1/5/8 and ALK-5/Smad2/3 pathways. Endoglin is a co-receptor predominantly expressed in endothelial cells that participates in TGFbeta-mediated signaling with ALK-1 and ALK-5 and regulates critical aspects of cellular and biological responses. The embryonic lethal phenotype of knock-out mice because of defects in angiogenesis and disease-causing mutations resulting in human vascular diseases both support essential roles for endoglin, ALK-1, and ALK-5 in the vasculature. However, the mechanism by which endoglin mediates TGF-beta signaling through ALK-1 and ALK-5 has remained elusive. Here we describe a novel interaction between endoglin and GIPC, a scaffolding protein known to regulate cell surface receptor expression and trafficking. Co-immunoprecipitation and immunofluorescence confocal studies both demonstrate a specific interaction between endoglin and GIPC in endothelial cells, mediated by a class I PDZ binding motif in the cytoplasmic domain of endoglin. Subcellular distribution studies demonstrate that endoglin recruits GIPC to the plasma membrane and co-localizes with GIPC in a TGFbeta-independent manner, with GIPC-promoting cell surface retention of endoglin. Endoglin specifically enhanced TGF-beta1-induced phosphorylation of Smad 1/5/8, increased a Smad 1/5/8 responsive promoter, and inhibited endothelial cell migration in a manner dependent on the ability of endoglin to interact with GIPC. These studies define a novel mechanism for the regulation of endoglin signaling and function in endothelial cells and demonstrate a new role for GIPC in TGF-beta signaling.     

Interaction and functional interplay between endoglin and ALK-1, two components of the endothelial transforming growth factor-beta receptor complex

Transforming growth factor-beta (TGF-beta) signaling in endothelial cells is able to modulate angiogenesis and vascular remodeling, although the underlying molecular mechanisms remain poorly understood. Endoglin and ALK-1 are components of the TGF-beta receptor complex, predominantly expressed in endothelial cells, and mutations in either endoglin or ALK-1 genes are responsible for the vascular dysplasia known as hereditary hemorrhagic telangiectasia. Here we find that the extracellular and cytoplasmic domains of the auxiliary TGF-beta receptor endoglin interact with ALK-1 (a type I TGF-beta receptor). In addition, endoglin potentiates TGF-beta/ALK1 signaling, with the extracellular domain of endoglin contributing to this functional cooperation between endoglin and ALK-1. By contrast, endoglin appears to interfere with TGF-beta/ALK-5 signaling. These results suggest that the functional association of endoglin with ALK-1 is critical for the endothelial responses to TGF-beta.     

Platelet-derived growth factor BB modulates PCNA protein synthesis partially through the transforming growth factor beta signalling pathway in vascular smooth muscle cells.

PDGF-BB (Platelet-derived growth factor BB) and TGF-beta1(transforming growth factor beta1) are important growth factors in the modulation of vascular smooth muscle cell (VSMC) proliferation and PCNA (proliferating cell nuclear antigen) expression in VSMCs. PCNA expresses at a high level in proliferating cells. The present study aims to assess the effects of PDGF-BB-induced overexpression of TGF-beta1 on PCNA in VSMCs. The downstream proteins of the TGF-beta signalling system in VSMCs, including TGF-beta type I receptor (ALK-5 in VSMCs), Smurf2, Smad2, pSmad2/3, Smad4, and Smad7, were also investigated. Our results revealed that PDGF-BB significantly increased the expressions of TGF-beta1 and PCNA, and the increase in PCNA can be partially inhibited by neutralizing anti-TGF-beta1 antibody. Furthermore, PDGF-BB increased the expression of ALK-5, Smurf2, pSmad2/3, and Smad4, but lowered the levels of Smad2 and Smad7; these alterations were partially restored by neutralizing anti-TGF-beta1 antibody. These findings suggest that PDGF-BB promotes PCNA expression in VSMCs partially through TGF-beta1 overexpression, and that the TGF-beta signalling system involves the molecular mechanism of PDGF-BB in VSMCs.     

The interaction of endoglin with beta-arrestin2 regulates transforming growth factor-beta-mediated ERK activation and migration in endothelial cells

In endothelial cells, transforming growth factor beta (TGF-beta) signals through two distinct pathways to regulate endothelial cell proliferation and migration, the ALK-1/Smads 1/5/8 pathway and the ALK-5/Smads 2/3 pathway. TGF-beta signaling through these pathways is further regulated in endothelial cells by the endothelial specific TGF-beta superfamily co-receptor, endoglin. The importance of endoglin, ALK-1, and ALK-5 in endothelial biology is underscored by the embryonic lethal phenotypes of knock-outs in mice due to defects in angiogenesis, and by the presence of disease-causing mutations in these genes in human vascular diseases. However, the mechanism of action of endoglin is not well defined. Here we define a novel interaction between endoglin and the scaffolding protein beta-arrestin2. Both co-immunoprecipitation and fluorescence confocal studies demonstrate the specific interaction between endoglin and beta-arrestin2 in endothelial cells, enhanced by ALK-1 and to a lesser extent by the type II TGF-beta receptor. The endoglin/beta-arrestin2 interaction results in endoglin internalization and co-accumulation of endoglin and beta-arrestin2 in endocytic vesicles. Whereas endoglin did not have a direct impact on either Smad 2/3 or Smad 1/5/8 activation, endoglin antagonized TGF-beta-mediated ERK signaling, altered the subcellular distribution of activated ERK, and inhibited endothelial cell migration in a manner dependent on the ability of endoglin to interact with beta-arrestin2. Reciprocally, small interfering RNA-mediated silencing of endogenous beta-arrestin2 expression restored TGF-beta-mediated ERK activation and increased endothelial cell migration in an endoglin-dependent manner. These studies define a novel function for endoglin, and further expand the roles mediated by the ubiquitous scaffolding protein beta-arrestin2.     

Characterization of the activation of latent TGF-beta by co-cultures of endothelial cells and pericytes or smooth muscle cells: a self- regulating system

The conversion of latent transforming growth factor beta (LTGF-beta) to the active species, transforming growth factor beta (TGF-beta), has been characterized in heterotypic cultures of bovine aortic endothelial (BAE) cells and bovine smooth muscle cells (SMCs). The formation of TGF-beta in co-cultures of BAE cells and SMCs was documented by a specific radioreceptor competition assay, while medium from homotypic cultures of BAE cells or SMCs contained no active TGF-beta as determined by this assay. The concentration of TGF-beta in the conditioned medium of heterotypic co-cultures was estimated to be 400-1,200 pg/ml using the inhibition of BAE cell migration as an assay. Northern blotting of poly A+ RNA extracted from both homotypic and heterotypic cultures of BAE cells and SMCs revealed that BAE cells produced both TGF-beta 1 and TGF-beta 2, while SMCs produced primarily TGF-beta 1. No change in the expression of these two forms of TGF-beta was apparent after 24 h in heterotypic cultures. Time course studies on the appearance of TGF-beta indicated that most of the active TGF-beta was generated within the first 12 h after the establishment of co-cultures. The generation of TGF-beta in co-cultures stimulated the production of the protease inhibitor plasminogen activator inhibitor-1 (PAI-1). The inclusion of neutralizing antibodies to TGF-beta in the co-culture medium blocked the observed increase in PAI-1 levels. The increased expression of PAI-1 subsequent to TGF-beta formation blocked the activation of the protease required for conversion of LTGF-beta to TGF-beta as the inclusion of neutralizing antibodies to PAI-1 in the co-culture medium resulted in prolonged production of TGF-beta. This effect was lost upon removal of the PAI-1 antibodies. Thus, the activation of LTGF-beta appears to be a self-regulating system.     

Assignment of transforming growth factor beta1 and beta3 and a third new ligand to the type I receptor ALK-1

Germ line mutations in one of two distinct genes, endoglin or ALK-1, cause hereditary hemorrhagic telangiectasia (HHT), an autosomal dominant disorder of localized angiodysplasia. Both genes encode endothelial cell receptors for the transforming growth factor beta (TGF-beta) ligand superfamily. Endoglin has homology to the type III receptor, betaglycan, although its exact role in TGF-beta signaling is unclear. Activin receptor-like kinase 1 (ALK-1) has homology to the type I receptor family, but its ligand and corresponding type II receptor are unknown. In order to identify the ligand and type II receptor for ALK-1 and to investigate the role of endoglin in ALK-1 signaling, we devised a chimeric receptor signaling assay by exchanging the kinase domain of ALK-1 with either the TGF-beta type I receptor or the activin type IB receptor, both of which can activate an inducible PAI-1 promoter. We show that TGF-beta1 and TGF-beta3, as well as a third unknown ligand present in serum, can activate chimeric ALK-1. HHT-associated missense mutations in the ALK-1 extracellular domain abrogate signaling. The ALK-1/ligand interaction is mediated by the type II TGF-beta receptor for TGF-beta and most likely through the activin type II or type IIB receptors for the serum ligand. Endoglin is a bifunctional receptor partner since it can bind to ALK-1 as well as to type I TGF-beta receptor. These data suggest that HHT pathogenesis involves disruption of a complex network of positive and negative angiogenic factors, involving TGF-beta, a new unknown ligand, and their corresponding receptors.     

A short loop on the ALK-2 and ALK-4 activin receptors regulates signaling specificity but cannot account for all their effects on early Xenopus development

Activin, a member of the transforming growth factor beta (TGF-beta) superfamily, signals through a heteromeric complex of type I and type II serine-threonine kinase receptors. The two activin type I receptors previously identified, ALK-2 (ActR-I) and ALK-4 (ActR-IB), have distinct effects on gene expression, differentiation and morphogenesis in the Xenopus animal cap assay. ALK-4 reproduces the effects of activin treatment including the dose-dependent induction of progressively more dorso-anterior mesodermal and endodermal markers, whereas ALK-2 induces only ventral mesodermal markers and counteracts the effects of ALK-4. To identify regions of the receptors that determine signaling specificity we have generated chimeras of the constitutively active ALK-2 and ALK-4 receptors (termed ALK-2* and ALK-4*). The effects of these chimeric receptors on gene expression and morphogenetic movements implicate the loop between kinase subdomains IV and V in mediating the strong dorsal gene-inducing properties of ALK-4*; when the seven amino acids comprising this loop are transferred from ALK-4* to ALK-2*, the resulting chimeric receptor is capable of inducing the expression of dorsal-specific genes. In contrast, when the equivalent region of ALK-2* is transferred to the ALK-4* backbone it cannot effectively counteract the dorsalizing effects of ALK-4*, suggesting that other regions of type I receptors are also involved in determining signal specificity.     

Role of PKC and TGF-beta receptor in glucose-induced proliferation of smooth muscle cells

The role of protein kinase C (PKC) and transforming growth factor (TGF)-beta in the proliferation of vascular smooth muscle cells (SMCs) under a high glucose condition was investigated. [3H]-thymidine incorporation under 20 mM glucose was significantly accelerated compared with that under 5.5 mM glucose, and this increase was inhibited by an anti-TGF-beta antibody or a PKC-beta specific inhibitor, LY333531. The amount of active and total TGF-beta1 in the conditioned media did not differ between 5.5 and 20 mM glucose. However, the expression of TGF-beta receptor type II under 20 mM glucose was significantly increased, but that of the TGF-beta receptor type I was not. This increased expression of the TGF-beta receptor type II was prevented by LY333531. These observations suggest that the increased expression of the TGF-beta receptor type II via PKC-beta plays an important role in the accelerated proliferation of SMCs under a high glucose condition, leading to the development of diabetic macroangiopathy.     

Influence of the photoperiod on TGF-β1 and p15 expression in hamster Leydig cells

Adult hamsters exposed to short photoperiods show a marked atrophy of their internal reproductive organs, including a reduction in size, though not number of Leydig cells. Transforming growth factor-β1 (TGF-β1) is involved in the regulation of growth and proliferation of different cell types. The aim of the present study was to examine the influence of photoperiod on the protein and gene expression of TGF-β1 and its receptors as well as gene expression of p15. The effect of TGF-β1 on the expression of p15 in purified Leydig cells from regressed and non-regressed hamster testes was also tested. Protein and gene expression of TGF-β1 was detected in both regressed and non-regressed testes. In contrast to the activin receptor-like kinase 1 (ALK-1), the TGF-β1, the activin receptor-like kinase 5 (ALK-5) and the co-receptor endoglin all showed a greater basal expression in regressed than non-regressed hamster testes. Melatonin induced the TGF-β1 mRNA expression in purified Leydig cells from non-regressed testes. The p15 mRNA level was greater in regressed than non-regressed testes. A high dose of TGF-β1 during a short incubation period increased the p15 mRNA level in Leydig cells from non-regressed testes. ALK-5 and mitogen-activated protein kinase (MAPK) p38 might have played a role in this process. In regressed hamster testes, the p15 mRNA level increased due to a low dose of TGF-β1 after short incubation periods and to a high dose after longer incubation periods; in both instances, ALK-5, ERK 1/2 and p38 were involved. Collectively, these results suggest that the alterations in p15 expression, mediated by MAPK, are involved in the shift between the active and inactive states in hamster Leydig cells.     

Specificity of the inhibitory effects of Dad on TGF-beta family type I receptors, Thickveins, Saxophone, and Baboon in Drosophila

In mammals, two inhibitory Smads (I-Smads), Smad6 and Smad7, play pivotal roles in negative regulation of TGF-beta family signaling. Smad7 ubiquitously inhibits TGF-beta family signaling, whereas Smad6 inhibits signaling from the ALK-3/6 subfamily in preference to that from the ALK-1/2 and ALK-4/5/7 subfamilies of TGF-beta family type I receptors. In Drosophila, only one I-Smad, Dad, has been identified. Here we examined inhibitory effects of Dad on type I receptors in Drosophila. Dad inhibited Saxophone (ALK-1/2 orthologue) and Thickveins (ALK-3/6 orthologue) but not Baboon (ALK-4/5/7 orthologue). The differential modes of action of I-Smads in mammals and Drosophila are discussed.     

Functional roles of the bone morphogenetic protein system in thyrotropin signaling in porcine thyroid cells

We uncovered a new regulation of thyrocyte function by bone morphogenetic protein (BMP) under the influence of thyrotropin (TSH) using primary culture of porcine thyrocytes. The BMP type I receptors, ALK-2 (ActRIA), -3 (BMPRIA), and -6 (BMPRIB), were expressed in porcine thyrocytes, while ALK-6 was not detected in human thyroid. Treatment with BMP-2, -4, -6, -7, and TGF-beta1 exhibited a dose-dependent suppression of DNA synthesis by porcine thyrocytes. BMP-2, -4, -6, -7, and TGF-beta1 suppressed TSH receptor mRNA expression on thyrocytes, which was consistent with their suppressive effect on TSH-induced cAMP synthesis and TSH-induced insulin-like growth factor-1 expression. Activin exhibited minimal suppression of thyrocyte DNA synthesis and did not exhibit suppressive effects on TSH receptor mRNA expression. Phosphorylated Smad1/5/8 was detected in the lysates of porcine thyrocytes treated with BMP-2, -4, -6, and -7. However, in the presence of TSH, BMP-6 and -7 failed to activate Smad1/5/8 phosphorylation and 3TP-reporter activity, whereas BMP-2 and -4 maintained clear activation of the BMP signaling regardless of the presence of TSH. This diverged regulation of thyroid BMP system by TSH is most likely due to the reduction of ALK-6 expression caused by TSH. Thus, the thyroid BMP system is functionally linked to TSH actions through modulating TSH receptor expression and TSH, in turn, selectively inhibits BMP signaling. Given that BMP system is present in human thyroid and the expression pattern of ALK-2 and BMPRII is different between follicular adenomas and normal thyroid tissues, the endogenous BMP system may be involved in regulating thyrocyte growth and TSH sensitivity of human thyroid adenomas.