ALK1 regulates the internalization of endoglin and the type III TGF-ß receptor.

Complex formation and endocytosis of transforming growth factor-ß (TGF-ß) receptors play important roles in signaling. However, their interdependence remained unexplored. Here, we demonstrate that ALK1, a TGF-ß type I receptor prevalent in endothelial cells, forms stable complexes at the cell surface with endoglin and with type III TGF-ß receptors (TßRIII). We show that ALK1 undergoes clathrin-mediated endocytosis (CME) faster than ALK5, type II TGF-ß receptor (TßRII), endoglin, or TßRIII. These complexes regulate the endocytosis of the TGF-ß receptors, with a major effect mediated by ALK1. Thus, ALK1 enhances the endocytosis of TßRIII and endoglin, while ALK5 and TßRII mildly enhance endoglin, but not TßRIII, internalization. Conversely, the slowly endocytosed endoglin has no effect on the endocytosis of either ALK1, ALK5, or TßRII, while TßRIII has a differential effect, slowing the internalization of ALK5 and TßRII, but not ALK1. Such effects may be relevant to signaling, as BMP9-mediated Smad1/5/8 phosphorylation is inhibited by CME blockade in endothelial cells. We propose a model that links TGF-ß receptor oligomerization and endocytosis, based on which endocytosis signals are exposed/functional in specific receptor complexes. This has broad implications for signaling, implying that complex formation among various receptors regulates their surface levels and signaling intensities.

Fluid shear stress generates a unique signaling response by activating multiple TGFß family type I receptors in osteocytes.

Bone is a dynamic tissue that constantly adapts to changing mechanical demands. The transforming growth factor beta (TGFß) signaling pathway plays several important roles in maintaining skeletal homeostasis by both coupling the bone-forming and bone-resorbing activities of osteoblasts and osteoclasts and by playing a causal role in the anabolic response of bone to applied loads. However, the extent to which the TGFß signaling pathway in osteocytes is directly regulated by fluid shear stress (FSS) is unknown, despite work suggesting that fluid flow along canaliculi is a dominant physical cue sensed by osteocytes following bone compression. To investigate the effects of FSS on TGFß signaling in osteocytes, we stimulated osteocytic OCY454 cells cultured within a microfluidic platform with FSS. We find that FSS rapidly upregulates Smad2/3 phosphorylation and TGFß target gene expression, even in the absence of added TGFß. Indeed, relative to treatment with TGFß, FSS induced a larger increase in levels of pSmad2/3 and Serpine1 that persisted even in the presence of a TGFß receptor type I inhibitor. Our results show that FSS stimulation rapidly induces phosphorylation of multiple TGFß family R-Smads by stimulating multimerization and concurrently activating several TGFß and BMP type I receptors, in a manner that requires the activity of the corresponding ligand. While the individual roles of the TGFß and BMP signaling pathways in bone mechanotransduction remain unclear, these results implicate that FSS activates both pathways to generate a downstream response that differs from that achieved by either ligand alone.

CtBP represses Dpp-dependent Mad activation during Drosophila eye development.

Complex networks of signaling pathways maintain the correct balance between positive and negative growth signals, ensuring that tissues achieve proper sizes and differentiation pattern during development. In Drosophila, Dpp, a member of the TGFß family, plays two main roles during larval eye development. In the early eye primordium, Dpp promotes growth and cell survival, but later on, it switches its function to induce a developmentally-regulated cell cycle arrest in the G1 phase and neuronal photoreceptor differentiation. To advance in the identification and characterization of regulators and targets of Dpp signaling required for retinal development, we carried out an in vivo eye-targeted double-RNAi screen to identify punt (Type II TGFß receptor) interactors. Using a set of 251 genes associated with eye development, we identified CtBP, Dad, Ago and Brk as punt genetic interactors. Here, we show that downregulation of Ago, or conditions causing increased tissue growth including overexpression of Myc or CyclinD-Cdk4 are sufficient to partially rescue punt-dependent growth and photoreceptor differentiation. Interestingly, we show a novel role for the transcriptional co-repressor CtBP in inhibiting Dpp-dependent Mad activation by phosphorylation, downstream or in parallel to Dad, the inhibitory Smad. Furthermore, CtBP downregulation activates JNK signaling pathway, implying a complex regulation of signaling pathways by CtBP during eye development.

TGF-ß Signaling in Control of Cardiovascular Function.

Genetic studies in animals and humans indicate that gene mutations that functionally perturb transforming growth factor ß (TGF-ß) signaling are linked to specific hereditary vascular syndromes, including Osler-Rendu-Weber disease or hereditary hemorrhagic telangiectasia and Marfan syndrome. Disturbed TGF-ß signaling can also cause nonhereditary disorders like atherosclerosis and cardiac fibrosis. Accordingly, cell culture studies using endothelial cells or smooth muscle cells (SMCs), cultured alone or together in two- or three-dimensional cell culture assays, on plastic or embedded in matrix, have shown that TGF-ß has a pivotal effect on endothelial and SMC proliferation, differentiation, migration, tube formation, and sprouting. Moreover, TGF-ß can stimulate endothelial-to-mesenchymal transition, a process shown to be of key importance in heart valve cushion formation and in various pathological vascular processes. Here, we discuss the roles of TGF-ß in vasculogenesis, angiogenesis, and lymphangiogenesis and the deregulation of TGF-ß signaling in cardiovascular diseases.

ACVR2B/Fc counteracts chemotherapy-induced loss of muscle and bone mass.

Chemotherapy promotes the development of cachexia, a debilitating condition characterized by muscle and fat loss. ACVR2B/Fc, an inhibitor of the Activin Receptor 2B signaling, has been shown to preserve muscle mass and prolong survival in tumor hosts, and to increase bone mass in models of osteogenesis imperfecta and muscular dystrophy. We compared the effects of ACVR2B/Fc on muscle and bone mass in mice exposed to Folfiri. In addition to impairing muscle mass and function, Folfiri had severe negative effects on bone, as shown by reduced trabecular bone volume fraction (BV/TV), thickness (Tb.Th), number (Tb.N), connectivity density (Conn.Dn), and by increased separation (Tb.Sp) in trabecular bone of the femur and vertebra. ACVR2B/Fc prevented the loss of muscle mass and strength, and the loss of trabecular bone in femurs and vertebrae following Folfiri administration. Neither Folfiri nor ACVR2B/Fc had effects on femoral cortical bone, as shown by unchanged cortical bone volume fraction (Ct.BV/TV), thickness (Ct.Th) and porosity. Our results suggest that Folfiri is responsible for concomitant muscle and bone degeneration, and that ACVR2B/Fc prevents these derangements. Future studies are required to determine if the same protective effects are observed in combination with other anticancer regimens or in the presence of cancer.

microRNA-21 mediates stretch-induced osteogenic differentiation in human periodontal ligament stem cells.

microRNAs (miRNAs) are short 20- to 22-nucleotide noncoding RNAs that negatively regulate the expression of target genes at the post-transcriptional level. The expression of specific miRNAs and their roles in the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) exposed to mechanical stretch remain unclear. Here, we found that stretch induced both osteogenic differentiation and the differential expression of miR-21 in PDLSCs. Furthermore, we identified activin receptor type IIB (ACVR2B) as a target gene of miR-21. Luciferase reporter assays showed that miR-21 interacts directly with the 3'-untranslated repeat sequence of ACVR2B mRNA. Mechanical stretch suppressed ACVR2B protein levels in PDLSCs, and this suppressive effect was modulated when endogenous miR-21 levels were either enhanced or inhibited. Both stretch and the expression of miR-21 altered endogenous ACVR2B protein levels and thus the osteogenic differentiation of PDLSCs. In addition, gain- and loss of function of ACVR2B mediated the osteogenic differentiation of PDLSCs. This study demonstrates that miR-21 is a mechanosensitive gene that plays an important role in the osteogenic differentiation of PDLSCs exposed to stretch.

BMP9 regulates endoglin-dependent chemokine responses in endothelial cells.

BMP9 signaling has been implicated in hereditary hemorrhagic telangiectasia (HHT) and vascular remodeling, acting via the HHT target genes, endoglin and ALK1. This study sought to identify endothelial BMP9-regulated proteins that could affect the HHT phenotype. Gene ontology analysis of cDNA microarray data obtained after BMP9 treatment of primary human endothelial cells indicated regulation of chemokine, adhesion, and inflammation pathways. These responses included the up-regulation of the chemokine CXCL12/SDF1 and down-regulation of its receptor CXCR4. Quantitative mass spectrometry identified additional secreted proteins, including the chemokine CXCL10/IP10. RNA knockdown of endoglin and ALK1 impaired SDF1/CXCR4 regulation by BMP9. Because of the association of SDF1 with ischemia, we analyzed its expression under hypoxia in response to BMP9 in vitro, and during the response to hindlimb ischemia, in endoglin-deficient mice. BMP9 and hypoxia were additive inducers of SDF1 expression. Moreover, the data suggest that endoglin deficiency impaired SDF1 expression in endothelial cells in vivo. Our data implicate BMP9 in regulation of the SDF1/CXCR4 chemokine axis in endothelial cells and point to a role for BMP9 signaling via endoglin in a switch from an SDF1-responsive autocrine phenotype to an SDF1 nonresponsive paracrine state that represses endothelial cell migration and may promote vessel maturation.

A pair of transmembrane receptors essential for the retention and pigmentation of hair.

Hair follicles are simple, accessible models for many developmental processes. Here, using mutant mice, we show that Bmpr2, a known receptor for bone morphogenetic proteins (Bmps), and Acvr2a, a known receptor for Bmps and activins, are individually redundant but together essential for multiple follicular traits. When Bmpr2/Acvr2a function is reduced in cutaneous epithelium, hair follicles undergo rapid cycles of hair generation and loss. Alopecia results from a failure to terminate hair development properly, as hair clubs never form, and follicular retraction is slowed. Hair regeneration is rapid due to premature activation of new hair-production programs. Hair shafts differentiate aberrantly due to impaired arrest of medullary-cell proliferation. When Bmpr2/Acvr2a function is reduced in melanocytes, gray hair develops, as melanosomes differentiate but fail to grow, resulting in organelle miniaturization. We conclude that Bmpr2 and Acvr2a normally play cell-type-specific, necessary roles in organelle biogenesis and the shutdown of developmental programs and cell division.

New insights into the mechanisms of activin action and inhibition.

Like other members of the transforming growth factor-ß (TGF-ß) superfamily, activins are synthesised as precursor molecules comprising an N-terminal prodomain and C-terminal mature region. During synthesis, the prodomain interacts non-covalently with mature activin, maintaining the molecule in a conformation competent for dimerisation. Dimeric precursors are cleaved by proprotein convertases and activin is secreted from the cell non-covalently associated with its propeptide. Extracellularly, the propeptide interacts with heparan sulfate proteoglycans to regulate activin localization within tissues. The mature activin dimer exhibits the classic 'open-hand' structure of TGF-ß ligands with 'finger-like' domains projecting outward from the cysteine knot core of the molecule. These finger domains form the binding epitopes for type I and II serine/threonine kinase receptors. Activins ability to access its signalling receptors is regulated by the extracellular binding proteins, follistatin, follistatin-like-3, and by inhibins, which, in the presence of betaglycan, sequester type II receptors.

The expression of activins, their type II receptors and follistatin in human Fallopian tube during the menstrual cycle and in pseudo-pregnancy.

BACKGROUND: The Fallopian tube (FT) is the site of fertilization and early embryonic development. We have previously reported the expression of activins, their receptors and follistatin by the FT. Here, our aim was to study the expression of tubal activins, their type II receptors and follistatin during the menstrual cycle and following exposure to hCG in vivo. METHOD: A set of 30 FTs were collected from cycling women (n = 12) at different stages of the cycle (n = 4 in each stage) and pseudo-pregnant women (n = 3) at the time of hysterectomy for benign disease. The pseudo-pregnant women were injected with hCG in the days leading up to hysterectomy, and pseudo-pregnancy was confirmed by the persistence of amenorrhea, the presence of corpus luteum and decidualization of the endometrium. FT specimens were examined using immunohistochemistry and quantitative RT-PCR. RESULTS: The expression of activin ßA- and ßB-subunits, activin type IIA and IIB receptors, and follistatin varied throughout the menstrual cycle, being lowest in the follicular phase and highest in the luteal phase. These results were demonstrated at the mRNA and protein level by quantitative RT-PCR and immunohistochemistry (P< 0.05). HCG injection rescued the expression of the candidate molecules from falling to the follicular stage levels but the expression remained lower than in the luteal phase. CONCLUSIONS: We suggest that activins play a role in tubal physiology and early embryonic development. Additionally, exposure of the tubal epithelium to hCG modulates the expression of tubal activins.

Soluble activin receptor type IIB increases forward pulling tension in the mdx mouse.

INTRODUCTION: In this study we investigated the action of RAP-031, a soluble activin receptor type IIB (ActRIIB) comprised of a form of the ActRIIB extracellular domain linked to a murine Fc, and the NF-κB inhibitor, ursodeoxycholic acid (UDCA), on the whole body strength of mdx mice. METHODS: The whole body tension (WBT) method of assessing the forward pulling tension (FPT) exerted by dystrophic (mdx) mice was used. RESULTS: RAP-031 produced a 41% increase in body mass and a 42.5% increase in FPT without altering the FPT normalized for body mass (WBT). Coadministration of RAP-031 with UDCA produced increases in FPT that were associated with an increase in WBT. CONCLUSIONS: Myostatin inhibition increases muscle mass without altering the fundamental weakness characteristic of dystrophic muscle. Cotreatment with an NF-κB inhibitor potentiates the effects of myostatin inhibition in improving FPT in mdx mice.

Identification and analysis of type II TGF-ß receptors in BMP-9-induced osteogenic differentiation of C3H10T1/2 mesenchymal stem cells.

Our previous studies have demonstrated that bone morphogenetic protein 9 (BMP-9) is one of the most efficacious BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). However, the molecular mechanism underlying the BMP-9-induced osteogenic differentiation of MSCs remains to be fully elucidated. In this study, dominant negative (DN) type II TGF-ß receptors were constructed and introduced into C3H10T1/2 stem cells, then in vitro and in vivo assays were carried out to analyze and identify the type II TGF-ß receptors required for BMP-9-induced osteogenesis. We found that three DN type II TGF-ß receptors, DN-BMPRII, DN-ActRII, and DN-ActRIIB, diminished BMP-9-induced alkaline phosphatase (ALP) activity, led to a decrease in BMP-9-induced Smad binding element (SBE)-controled reporter activity, reduced BMP-9-induced expressions of Smad6 and Smad7, and decreased BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, finally resulted in decreased bone masses and immature osteogenesis. These findings strongly suggested that three wild-type II TGF-ß receptors, BMPRII, ActRII and ActRIIB, may play a functional role in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells. However, C3H10T1/2 stem cells can express BMPRII and ActRII, but not ActRIIB. Using RNA interference (RNAi), we found that luciferase reporter activity and ALP activity induced by BMP-9 were accordingly inhibited along with the knockdown of BMPRII and ActRII. Taken together, our results demonstrated that BMPRII and ActRII are the functional type II TGF-ß receptors in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells.

Overexpression of BMP3 in the developing skeleton alters endochondral bone formation resulting in spontaneous rib fractures.

Bone morphogenetic protein-3 (BMP) has been identified as a negative regulator in the skeleton as mice lacking BMP3 have increased bone mass. To further understand how BMP3 mediates bone formation, we created transgenic mice overexpressing BMP3 using the type I collagen promoter. BMP3 transgenic mice displayed spontaneous rib fractures that were first detected at E17.0. The fractures were due to defects in differentiation of the periosteum and late hypertrophic chondrocytes resulting in thinner cortical bone with decreased mineralization. As BMP3 modulates BMP and activin signaling through ActRIIB, we examined the ribs of ActRIIB receptor knockout mice and found they had defects in late chondrogenesis and mineralization similar to BMP3 transgenic mice. These data suggest that BMP3 exerts its effects in the skeleton by altering signaling through ActRIIB in chondrocytes and the periosteum, and this results in defects in bone collar formation and late hypertrophic chondrocyte maturation leading to decreased mineralization and less bone.

Functional analysis of saxophone, the Drosophila gene encoding the BMP type I receptor ortholog of human ALK1/ACVRL1 and ACVR1/ALK2.

In metazoans, bone morphogenetic proteins (BMPs) direct a myriad of developmental and adult homeostatic events through their heterotetrameric type I and type II receptor complexes. We examined 3 existing and 12 newly generated mutations in the Drosophila type I receptor gene, saxophone (sax), the ortholog of the human Activin Receptor-Like Kinase1 and -2 (ALK1/ACVRL1 and ALK2/ACVR1) genes. Our genetic analyses identified two distinct classes of sax alleles. The first class consists of homozygous viable gain-of-function (GOF) alleles that exhibit (1) synthetic lethality in combination with mutations in BMP pathway components, and (2) significant maternal effect lethality that can be rescued by an increased dosage of the BMP encoding gene, dpp+. In contrast, the second class consists of alleles that are recessive lethal and do not exhibit lethality in combination with mutations in other BMP pathway components. The alleles in this second class are clearly loss-of-function (LOF) with both complete and partial loss-of-function mutations represented. We find that one allele in the second class of recessive lethals exhibits dominant-negative behavior, albeit distinct from the GOF activity of the first class of viable alleles. On the basis of the fact that the first class of viable alleles can be reverted to lethality and on our ability to independently generate recessive lethal sax mutations, our analysis demonstrates that sax is an essential gene. Consistent with this conclusion, we find that a normal sax transcript is produced by saxP, a viable allele previously reported to be null, and that this allele can be reverted to lethality. Interestingly, we determine that two mutations in the first class of sax alleles show the same amino acid substitutions as mutations in the human receptors ALK1/ACVRl-1 and ACVR1/ALK2, responsible for cases of hereditary hemorrhagic telangiectasia type 2 (HHT2) and fibrodysplasia ossificans progressiva (FOP), respectively. Finally, the data presented here identify different functional requirements for the Sax receptor, support the proposal that Sax participates in a heteromeric receptor complex, and provide a mechanistic framework for future investigations into disease states that arise from defects in BMP/TGF-beta signaling.

Casein kinase 2beta as a novel enhancer of activin-like receptor-1 signaling.

ALK-1 is a transforming growth factor beta (TGF-beta) superfamily receptor that is predominantly expressed in endothelial cells and is essential for angiogenesis, as demonstrated by the embryonic lethal phentoype when targeted for deletion in mice and its mutation in the human disease hereditary hemorrhagic telangiectasia. Although ALK-1 and the endothelial-specific TGF-beta superfamily coreceptor, endoglin, form a heteromeric complex and bind similar TGF-beta superfamily ligands, their signaling mechanisms remain poorly characterized. Here we report the identification of CK2beta, the regulatory subunit of protein kinase CK2, as a novel enhancer of ALK-1 signaling. The cytoplasmic domain of ALK-1 specifically binds to CK2beta in vitro and in vivo. NAAIRS mutagenesis studies define amino acid sequences 181-199 of CK2beta and 207-212 of ALK-1 as the interaction domains, respectively. The ALK-1/CK2beta interaction specifically enhanced Smad1/5/8 phosphorylation and ALK-1-mediated reporter activation in response to TGF-beta1 and BMP-9 treatment. In a reciprocal manner, siRNA-mediated silencing of endogenous CK2beta inhibited TGF-beta1 and BMP-9-stimulated Smad1/5/8 phosphorylation and ALK-1-mediated reporter activation. Functionally, CK2beta enhanced the ability of activated or ligand-stimulated ALK-1 to inhibit endothelial cell migration. Similarly, ALK-1 and CK2beta antagonized endothelial tubule formation in Matrigel. These studies support CK2beta as an important regulator of ALK-1 signaling and ALK-1-mediated functions in endothelial cells.

Structural and functional characterizations of an Activin type II receptor orthologue from the pacific oyster Crassostrea gigas.

Members of the Transforming Growth factor beta (TGF-beta) superfamily of cell signalling polypeptides are known to play important roles in cell proliferation and differentiation during development and in various physiological processes of most animal clades. Recent findings in the mollusc Crassostrea gigas demonstrate the occurrence of a diversity of TGF-beta signalling components including various ligands, three type I receptors but only a single type II receptor. This report describes the characterization of Cg-ActRII, a new type II receptor displaying homology with vertebrate and Drosophila Activin type II receptors. The use of zebrafish embryo as a reporter organism revealed that, in a way similar to its zebrafish counterpart, overexpression of Cg-ActRII or its dominant negative acting truncated form resulted in a dose dependent range of dorsoventral defects coupled with anterior disorders. Expression pattern of Cg-ActRII transcripts examined by real time PCR and in situ PCR in C. gigas showed high levels of Cg-ActRII transcripts in early embryonic stages and in the developing larval central nervous system. Except for a high expression in the visceral ganglia, most oyster adult tissues displayed rather low levels of transcripts. Altogether, the data suggest a high degree of conservation at both the structural and functional levels during evolution for this class of receptors.

Bone morphogenetic protein-9 is a circulating vascular quiescence factor.

Angiogenesis is a complex process, requiring a finely tuned balance between numerous stimulatory and inhibitory signals. ALK1 (activin receptor like-kinase 1) is an endothelial-specific type 1 receptor of the transforming growth factor-beta receptor family. Heterozygotes with mutations in the ALK1 gene develop hereditary hemorrhagic telangiectasia type 2 (HHT2). Recently, we reported that bone morphogenetic protein (BMP)9 and BMP10 are specific ligands for ALK1 that potently inhibit microvascular endothelial cell migration and growth. These data lead us to suggest that these factors may play a role in the control of vascular quiescence. To test this hypothesis, we checked their presence in human serum. We found that human serum induced Smad1/5 phosphorylation. To identify the active factor, we tested neutralizing antibodies against BMP members and found that only the anti-BMP9 inhibited serum-induced Smad1/5 phosphorylation. The concentration of circulating BMP9 was found to vary between 2 and 12 ng/mL in sera and plasma from healthy humans, a value well above its EC(50) (50 pg/mL). These data indicated that BMP9 is circulating at a biologically active concentration. We then tested the effects of BMP9 in 2 in vivo angiogenic assays. We found that BMP9 strongly inhibited sprouting angiogenesis in the mouse sponge angiogenesis assay and that BMP9 could inhibit blood circulation in the chicken chorioallantoic membrane assay. Taken together, our results demonstrate that BMP9, circulating under a biologically active form, is a potent antiangiogenic factor that is likely to play a physiological role in the control of adult blood vessel quiescence.

TGF-beta signaling is dynamically regulated during the alveolarization of rodent and human lungs.

Although transforming growth factor-beta (TGF-beta) signaling negatively regulates branching morphogenesis in early lung development, few studies to date have addressed the role of this family of growth factors during late lung development. We describe here that the expression, tissue localization, and activity of components of the TGF-beta signaling machinery are dynamically regulated during late lung development in the mouse and human. Pronounced changes in the expression and localization of the TGF-beta receptors Acvrl1, Tgfbr1, Tgfbr2, Tgfbr3, and endoglin, and the intracellular messengers Smad2, Smad3, Smad4, Smad6, and Smad7 were noted as mouse and human lungs progressed through the canalicular, saccular, and alveolar stages of development. TGF-beta signaling, assessed by phosphorylation of Smad2, was detected in the vascular and airway smooth muscle, as well as the alveolar and airway epithelium throughout late lung development. These data suggest that active TGF-beta signaling is required for normal late lung development.