Regulation of TGFβ receptor trafficking and signaling by atypical protein kinase C

Transforming growth factor beta (TGFβ) signaling is linked to the membrane trafficking of TGFβ receptors. The Protein Kinase C (PKC) family of serine/threonine kinases have been implicated in modulating the endocytic processes of various receptors. The present study investigated whether PKC activity plays a role in the trafficking, and signaling of TGFβ receptors, and further explored which PKC isoforms may be responsible for altered TGFβ signaling patterns. Using immunofluorescence microscopy and ¹²⁵I-TGFβ internalization assays, we show that the pharmacological inhibition of PKC activity alters TGFβ receptor trafficking and delays TGFβ receptor degradation. Consistent with these findings, we demonstrate that PKC inhibition extends TGFβ-dependent Smad2 phosphorylation. Previous studies have shown that PKCζ associates with TGFβ receptors to modulate cell plasticity. We therefore used siRNA directed at the atypical PKC isoforms to investigate if reducing PKCι and PKCζ protein levels would delay TGFβ receptor degradation and extend TGFβ signaling. Our findings suggest that atypical PKC isoforms regulate TGFβ signaling by altering cell surface TGFβ receptor trafficking and degradation.     

Transforming growth factor-β receptor expression on human skin fibroblasts: Dimeric complex formation of type I and type II receptors and identification of glycosyl phosphatidylinositol-anchored transforming growth factor-β binding proteins

Fibroblasts play a critical role in wound repair and in the development of fibrotic diseases, and transforming growth factor-β (TGF-β) has been shown to profoundly modulate fibroblast function. However, there is limited information on the TGF-β receptor types, isoform specificity, and complex formation in skin fibroblasts. Here, we report that normal adult human skin fibroblasts display two isoform-specific, cell surface glycosyl phosphatidylinositol (GPI)-anchored, TGF-β binding proteins in addition to the type I, II, and III TGF-β receptors. The identities of these proteins are confirmed on the basis of their affinity for TGF-β isoforms, immunoprecipitation with specific antireceptor antibodies, and other biochemical analyses. Immunoprecipitation results also indicated oligomeric complex formation between type I and II and between type II and III TGF-β receptors. Furthermore, by using affinity labeling and two-dimensional electrophoresis, we demonstrate the occurrence of type I and II heterodimers and type I homodimers of TGF-β receptors on these cells. Because the type I receptor does not bind TGF-β in the absence of type II receptor, these results indicate that one molecule of TGF-β induces the formation of a heterooligomeric complex containing more than one molecule each of type I and II TGF-β receptors on these cells. These cells respond to TGF-β by markedly down-regulating all five binding proteins and by potently augmenting DNA synthesis. These results allow the expansion of the proposed heteromeric TGF-β receptor signaling paradigm using mutantcells that are unresponsive to TGF-β and cell lines that have been transfected to overexpress these receptors, to include normal TGF-β-responsive cells. In addition, the definition of TGF-β receptor profiles in human skin fibroblasts provides important information for studying their alterations in these cells in various skin diseases. J. Cell. Physiol. 176:553–564, 1998. © 1998 Wiley-Liss, Inc.     

CD109-mediated degradation of TGF-β receptors and inhibition of TGF-β responses involve regulation of SMAD7 and Smurf2 localization and function

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates a wide variety of cellular processes including proliferation, differentiation, and extracellular matrix deposition. Dysregulation of TGF-β signaling is associated with several diseases such as cancer and tissue fibrosis. TGF-β signals through two transmembrane proteins known as the type I (TGFBR1) and type II (TGFBR2) receptors. The levels of these receptors at the cell surface are tightly regulated by several mechanisms, including degradation following recruitment of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor (Smurf) 2 by SMAD7. In addition, TGF-β co-receptors can modulate TGF-β signaling receptor activity in a cell-specific manner. We have previously identified a novel TGF-β co-receptor, CD109, a glycosyl phosphatidylinositol (GPI)-anchored protein that negatively regulates TGF-β signaling. Despite CD109's potential relevance as a regulator of TGF-β action in vivo, the mechanisms by which CD109 regulates TGF-β signaling are still incompletely understood. Previously, we have shown that CD109 downregulates TGF-β signaling by promoting TGF-β receptor localization into the lipid raft/caveolae compartment and by enhancing TGF-β receptor degradation. Here, we demonstrate that CD109 enhances SMAD7/Smurf2-mediated degradation of TGFBR1 in a ligand-dependent manner. Moreover, we show that CD109 regulates the localization and the association of SMAD7/Smurf2 with TGFBR1. Finally, we demonstrate that CD109's inhibitory effect on TGF-β signaling and responses require SMAD7 expression and Smurf2 ubiquitin ligase activity. Taken together, these results suggest that CD109 is an important regulator of SMAD7/Smurf2-mediated degradation of TGFBR1.     

ALK5 is essential for tooth germ differentiation during tooth development

ABSTRACT

The TGFβ superfamily of proteins participates in tooth development. TGFβ1 and TGFβ3 regulate odontoblast differentiation and dentin extracellular matrix synthesis. Although the expression of TGFβ family member ligands is well-characterized during mammalian tooth development, less is known about the TGFβ receptor, which is a heteromeric complex consisting of a type I and type II receptors. The molecular mechanism of ALK5 (TGFβR1) in the dental mesenchyme is not clear. We investigated the role of ALK5 in tooth germ mesenchymal cells (TGMCs) from the lower first molar tooth germs of day 15.5 embryonic mice. Human recombinant TGFβ3 protein or an ALK5 inhibitor (SD208) was added to the cells. Cell proliferation was inhibited by SD208 and promoted by TGFβ3. We found that SD208 inhibited TGMCs osteogenesis and dentinogenesis. Both canonical and noncanonical TGFβ signaling pathways participated in the process. TAK1, P-TAK1, p38 and P-p38 showed greater expression and SMAD4 showed less expression when ALK5 was inhibited. Our findings contribute to understanding the role of TGFβ signaling for the differentiation of mesenchymal stem cells derived from dental germ and suggest possible targets for optimizing the use of stem cells of dental origin for tissue regeneration.     

High resolution structures of the bone morphogenetic protein type II receptor in two crystal forms: implications for ligand binding

BMPRII is a type II TGF-beta serine threonine kinase receptor which is integral to the bone morphogenetic protein (BMP) signalling pathway. It is known to bind BMP and growth differentiation factor (GDF) ligands, and has overlapping ligand specificity with the activin type II receptor, ActRII. In contrast to activin and TGF-beta type ligands, BMPs bind to type II receptors with lower affinity than type I receptors. Crystals of the BMPRII ectodomain were grown in two different forms, both of which diffracted to high resolution. The tetragonal form exhibited some disorder, whereas the entire polypeptide was seen in the orthorhombic form. The two structures retain the basic three-finger toxin fold of other TGF-beta receptor ectodomains, and share the main hydrophobic patch used by ActRII to bind various ligands. However, they present different conformations of the A-loop at the periphery of the proposed ligand-binding interface, in conjunction with rearrangement of a disulfide bridge within the loop. This particular disulfide (Cys94-Cys117) is only present in BMPRII and activin receptors, suggesting that it is important for their likely shared mode of binding. Evidence is presented that the two crystal forms represent ligand-bound and free conformations of BMPRII. Comparison with the solved structure of ActRII bound to BMP2 suggests that His87, unique amongst TGF-beta receptors, may play a key role in ligand recognition.     

The regenerative capacity of the zebrafish heart is dependent on TGFβ signaling

Mammals respond to a myocardial infarction by irreversible scar formation. By contrast, zebrafish are able to resolve the scar and to regenerate functional cardiac muscle. It is not known how opposing cellular responses of fibrosis and new myocardium formation are spatially and temporally coordinated during heart regeneration in zebrafish. Here, we report that the balance between the reparative and regenerative processes is achieved through Smad3-dependent TGFβ signaling. The type I receptor alk5b (tgfbr1b) is expressed in both fibrotic and cardiac cells of the injured heart. TGFβ ligands are locally induced following cryoinjury and activate the signaling pathway both in the infarct area and in cardiomyocytes in the vicinity of the trauma zone. Inhibition of the relevant type I receptors with the specific chemical inhibitor SB431542 qualitatively altered the infarct tissue and completely abolished heart regeneration. We show that transient scar formation is an essential step to maintain robustness of the damaged ventricular wall prior to cardiomyocyte replacement. Taking advantage of the reversible action of the inhibitor, we dissected the multifunctional role of TGFβ signaling into three crucial processes: collagen-rich scar deposition, Tenascin C-associated tissue remodeling at the infarct-myocardium interface, and cardiomyocyte proliferation. Thus, TGFβ signaling orchestrates the beneficial interplay between scar-based repair and cardiomyocyte-based regeneration to achieve complete heart regeneration.     

Transforming growth factor β receptor type 1 is essential for female reproductive tract integrity and function

The transforming growth factor β (TGFβ) superfamily proteins are principle regulators of numerous biological functions. Although recent studies have gained tremendous insights into this growth factor family in female reproduction, the functions of the receptors in vivo remain poorly defined. TGFβ type 1 receptor (TGFBR1), also known as activin receptor-like kinase 5, is the major type 1 receptor for TGFβ ligands. Tgfbr1 null mice die embryonically, precluding functional characterization of TGFBR1 postnatally. To study TGFBR1-mediated signaling in female reproduction, we generated a mouse model with conditional knockout (cKO) of Tgfbr1 in the female reproductive tract using anti-Müllerian hormone receptor type 2 promoter-driven Cre recombinase. We found that Tgfbr1 cKO females are sterile. However, unlike its role in growth differentiation factor 9 (GDF9) signaling in vitro, TGFBR1 seems to be dispensable for GDF9 signaling in vivo. Strikingly, we discovered that the Tgfbr1 cKO females develop oviductal diverticula, which impair embryo development and transit of embryos to the uterus. Molecular analysis further demonstrated the dysregulation of several cell differentiation and migration genes (e.g., Krt12, Ace2, and MyoR) that are potentially associated with female reproductive tract development. Moreover, defective smooth muscle development was also revealed in the uteri of the Tgfbr1 cKO mice. Thus, TGFBR1 is required for female reproductive tract integrity and function, and disruption of TGFBR1-mediated signaling leads to catastrophic structural and functional consequences in the oviduct and uterus.     

Receptor binding competition: A paradigm for regulating TGF-β family action

The transforming growth factor (TGF)-β family is a group of structurally related, multifunctional growth factors, or ligands that are crucially involved in the development, regulation, and maintenance of animal tissues. In humans, the family counts over 33 members. These secreted ligands typically form multimeric complexes with two type I and two type II receptors to activate one of two distinct signal transduction branches. A striking feature of the family is its promiscuity, i.e., many ligands bind the same receptors and compete with each other for binding to these receptors. Although several explanations for this feature have been considered, its functional significance has remained puzzling. However, several recent reports have promoted the idea that ligand-receptor binding promiscuity and competition are critical features of the TGF-β family that provide an essential regulating function. Namely, they allow a cell to read and process multi-ligand inputs. This capability may be necessary for producing subtle, distinctive, or adaptive responses and, possibly, for facilitating developmental plasticity. Here, we review the molecular basis for ligand competition, with emphasis on molecular structures and binding affinities. We give an overview of methods that were used to establish experimentally ligand competition. Finally, we discuss how the concept of ligand competition may be fundamentally tied to human physiology, disease, and therapy.     

Coordinate regulation of neural tube patterning and proliferation by TGFbeta and WNT activity

Pattern formation and growth must be tightly coupled during embryonic development. In vertebrates, however, little is known of the molecules that serve to link these two processes. Here we show that bone morphogenetic proteins (BMP) coordinate the acquisition of pattern information and the stimulation of proliferation in the embryonic spinal neural tube. We have blocked BMP and transforming growth factor-β superfamily (TGFβ) function in the chick embryo using Noggin, a BMP antagonist, and siRNA against Smad4. We show that BMPs/TGFβs are necessary to regulate pattern formation and the specification of neural progenitor populations in the dorsal neural tube. BMPs also serve to establish discrete expression domains of Wnt ligands, receptors, and antagonists along the dorsal-ventral axis of the neural tube. Using the extracellular domain of Frizzled 8 to block Wnt signaling and Wnt3a ligand misexpression to activate WNT signaling, we demonstrate that the Wnt pathway acts mitogenically to expand the populations of neuronal progenitor cells specified by BMP. Thus, BMPs, acting through WNTs, couple patterning and growth to generate dorsal neuronal fates in the appropriate proportions within the neural tube.     

Specificity and Structure of a High Affinity Activin Receptor-like Kinase 1 (ALK1) Signaling Complex

Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-β superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-β family ligands BMP9 and BMP10 as well as the three type II TGF-β family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.     

Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation

Functions of bone morphogenetic proteins (BMPs) are initiated by signaling through specific type I and type II serine/threonine kinase receptors. In previous studies, we have demonstrated that the type IB BMP receptor (BMPR-IB) plays an essential and specific role in osteoblast commitment and differentiation. To determine the role of BMP receptor signaling in bone formation in vivo, we generated transgenic mice, which express a truncated dominant-negative BMPR-IB targeted to osteoblasts using the type I collagen promoter. The mice are viable and fertile. Tissue-specific expression of the truncated BMPR-IB was demonstrated. Characterization of the phenotype of these transgenic mice showed impairment of postnatal bone formation in 1-mo-old homozygous transgenic mice. Bone mineral density, bone volume, and bone formation rates were severely reduced, but osteoblast and osteoclast numbers were not significantly changed in the transgenic mice. To determine whether osteoblast differentiation is impaired, we used primary osteoblasts isolated from the transgenic mice and showed that BMP signaling is blocked and BMP2-induced mineralized bone matrix formation was inhibited. These studies show the effects of alterations in BMP receptor function targeted to the osteoblast lineage and demonstrate a necessary role of BMP receptor signaling in postnatal bone growth and bone formation in vivo.     

转化因子-β 受体III

转化生长因子-β(transforming growth factor-β,TGF-β)受体Ⅲ,又称为β蛋白聚糖(betaglycan),是一种膜锚定蛋白。TGF-β受体Ⅲ是表达最为丰富的TGF-β受体,曾被认为是TGF-β超家族(包括TGF-β、激活素和抑制素等)的辅助受体。后来研究表明,它在介导和调节TGF-β的信号转导中具有非常重要的、不可替代的作用。它通过与TGF-β形成复合体来介导对靶细胞的作用。在没有TGF配体的情况下,TGF-β受体Ⅲ可以激活p38信号,表明这一受体可能与不依赖TGF-β的信号通路相互作用。TGFβ受体Ⅲ还可以结合并调节抑制素的信号转导。TGFβ受体Ⅲ与抑制素A结合,形成一个稳定的高亲和复合物。体外研究表明,TGFβ受体III还结合抑制素B和强化抑制素与Ⅱ型激活素受体的关系。有关报道显示TGFβ受体Ⅲ在卵巢癌中具有肿瘤抑制的作用。研究表明,在上皮源性卵巢癌中,TGFβ受体Ⅲ mRNA和蛋白质表达降低或丢失,丢失的程度与肿瘤分级相关。有很多因素可以影响并调节该受体的表达,如雌激素、卵泡刺激素(FSH)、TGF-β1等,深入开展相关机制的研究,对于癌症的治疗和预防将会起到一定的推动作用。     

TGFβ signaling and congenital heart disease: Insights from mouse studies

Transforming growth factor β (TGFβ) regulates one of the major signaling pathways that control tissue morphogenesis. In vitro experiments using heart explants indicated the importance of this signaling pathway for the generation of cushion mesenchymal cells, which ultimately contribute to the valves and septa of the mature heart. Recent advances in mouse genetics have enabled in vivo investigation into the roles of individual ligands, receptors, and coreceptors of this pathway, including investigation of the tissue specificity of these roles in heart development. This work has revealed that (1) cushion mesenchyme can form in the absence of TGFβ signaling, although mesenchymal cell numbers may be misregulated; (2) TGFβ signaling is essential for correct remodeling of the cushions, particularly those of the outflow tract; (3) TGFβ signaling also has a role in ensuring accurate remodeling of the pharyngeal arch arteries to form the mature aortic arch; and (4) mesenchymal cells derived from the epicardium require TGFβ signaling to promote their differentiation to vascular smooth muscle cells to support the coronary arteries. In addition, a mouse genetics approach has also been used to investigate the disease pathogenesis of Loeys-Dietz syndrome, a familial autosomal dominant human disorder characterized by a dilated aortic root, and associated with mutations in the two TGFβ signaling receptor genes, TGFBR1 and TGFBR2. Further important insights are likely as this exciting work progresses.     

A new model for growth factor activation: type II receptors compete with the prodomain for BMP-7

Bone morphogenetic proteins (BMPs) are morphogens with long-range signaling activities. BMP-7 is secreted as a stable complex consisting of a growth factor noncovalently associated with two propeptides. In other transforming growth factor-β-like growth factor complexes, the prodomain (pd) confers latency to the complex. However, we detected no difference in signaling capabilities between the growth factor and the BMP-7 complex in multiple in vitro bioactivity assays. Biochemical and biophysical methods elucidated the interaction between the BMP-7 complex and the extracellular domains of its type I and type II receptors. Results showed that type II receptors, such as BMP receptor II, activin receptor IIA, and activin receptor IIB, competed with the pd for binding to the growth factor and displaced the pd from the complex. In contrast, type I receptors interacted with the complex without displacing the pd. These studies suggest a new model for growth factor activation in which proteases or other extracellular molecules are not required and provide a molecular mechanism consistent with a role for BMP receptors in the establishment of early morphogen gradients.     

PDGF mediates TGFβ-induced migration during development of the spinous process

Mechanisms mediating closure of the dorsal vertebrae are not clear. Previously, we showed that deletion of TGFβ type II receptor (Tgfbr2) in sclerotome in mice results in failure in the formation of the spinous process, mimicking spina bifida occulta, a common malformation in humans. In this study, we aimed to determine whether missing dorsal structures in Tgfbr2 mutant mice were due to defects in mesenchymal migration and to clarify mechanism of TGFβ-mediated migration. First, we showed that gross alterations in dorsal vertebrae were apparent by E16.5days in Tgfbr2 mutants. In addition, histological staining showed that the mesenchyme adjacent to the developing cartilage was thin compared to controls likely due to reduced proliferation and migration of these cells. Next, we used a chemotaxis migration assay to show that TGFβ promotes migration in mixed cultures of embryonic sclerotome and associated mesenchyme. TGFβ stimulated expression of PDGF ligands and receptors in the cultures and intact PDGF signaling was required for TGFβ-mediated migration. Since PDGF ligands are expressed in the sclerotome-derived cartilage where Tgfbr2 is deleted and the receptors are predominantly expressed in the adjacent mesenchyme, we propose that TGFβ acts on the sclerotome to regulate expression of PDGF ligands, which then act on the associated mesenchyme in a paracrine fashion to mediate proliferation, migration and subsequent differentiation of the adjacent sclerotome.     

Type II transforming growth factor-beta receptor recycling is dependent upon the clathrin adaptor protein Dab2

Transforming growth factor (TGF)-β family proteins form heteromeric complexes with transmembrane serine/threonine kinases referred to as type I and type II receptors. Ligand binding initiates a signaling cascade that generates a variety of cell type-specific phenotypes. Whereas numerous studies have investigated the regulatory activities controlling TGF-β signaling, there is relatively little information addressing the endocytic and trafficking itinerary of TGF-β receptor subunits. In the current study we have investigated the role of the clathrin-associated sorting protein Disabled-2 (Dab2) in TGF-β receptor endocytosis. Although small interfering RNA-mediated Dab2 knockdown had no affect on the internalization of various clathrin-dependent (i.e., TGF-β, low-density lipoprotein, or transferrin) or -independent (i.e., LacCer) cargo, TGF-β receptor recycling was abrogated. Loss of Dab2 resulted in enlarged early endosomal antigen 1-positive endosomes, reflecting the inability of cargo to traffic from the early endosome to the endosomal recycling compartment and, as documented previously, diminished Smad2 phosphorylation. The results support a model whereby Dab2 acts as a multifunctional adaptor in mesenchymal cells required for TGF-β receptor recycling as well as Smad2 phosphorylation.     

Blood–brain barrier dysfunction promotes astrocyte senescence through albumin-induced TGFβ signaling activation

Blood–brain barrier dysfunction (BBBD) and accumulation of senescent astrocytes occur during brain aging and contribute to neuroinflammation and disease. Here, we explored the relationship between these two age-related events, hypothesizing that chronic hippocampal exposure to the blood-borne protein serum albumin could induce stress-induced premature senescence (SIPS) in astrocytes via transforming growth factor beta 1 (TGFβ) signaling. We found that 1 week of albumin exposure significantly increased TGFβ signaling and senescence marker expression in cultured rat hippocampal astrocytes. These changes were preventable by pharmacological inhibition of the type I TGFβ receptor (TGFβR) ALK5. To study these effects in vivo, we utilized an animal model of BBBD in which albumin was continuously infused into the lateral ventricles of adult mice. Consistent with our in vitro results, 1 week of albumin infusion significantly increased TGFβ signaling activation and the burden of senescent astrocytes in hippocampal tissue. Pharmacological inhibition of ALK5 TGFβR or conditional genetic knockdown of astrocytic TGFβR prior to albumin infusion was sufficient to prevent albumin-induced astrocyte senescence. Together, these results establish a link between TGFβ signaling activation and astrocyte senescence and suggest that prolonged exposure to serum albumin due to BBBD can trigger these phenotypic changes.