Hem1 is essential for ruffled border formation in osteoclasts and efficient bone resorption.

Bone resorption is highly dependent on the dynamic rearrangement of the osteoclast actin cytoskeleton to allow formation of actin rings and a functional ruffled border. Hem1 is a hematopoietic-specific subunit of the WAVE-complex which regulates actin polymerization and is crucial for lamellipodia formation in hematopoietic cell types. However, its role in osteoclast differentiation and function is still unknown. Here, we show that although the absence of Hem1 promotes osteoclastogenesis, the ability of Hem1-/- osteoclasts to degrade bone was severely impaired. Global as well as osteoclast-specific deletion of Hem1 in vivo revealed increased femoral trabecular bone mass despite elevated numbers of osteoclasts in vivo. We found that the resorption defect derived from the morphological distortion of the actin-rich sealing zone and ruffled border deformation in Hem1-deficient osteoclasts leading to impaired vesicle transport and increased intracellular acidification. Collectively, our data identify Hem1 as a yet unknown key player in bone remodeling by regulating ruffled border formation and consequently the resorptive capacity of osteoclasts.

Lasp1 regulates adherens junction dynamics and fibroblast transformation in destructive arthritis.

The LIM and SH3 domain protein 1 (Lasp1) was originally cloned from metastatic breast cancer and characterised as an adaptor molecule associated with tumourigenesis and cancer cell invasion. However, the regulation of Lasp1 and its function in the aggressive transformation of cells is unclear. Here we use integrative epigenomic profiling of invasive fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and from mouse models of the disease, to identify Lasp1 as an epigenomically co-modified region in chronic inflammatory arthritis and a functionally important binding partner of the Cadherin-11/ß-Catenin complex in zipper-like cell-to-cell contacts. In vitro, loss or blocking of Lasp1 alters pathological tissue formation, migratory behaviour and platelet-derived growth factor response of arthritic FLS. In arthritic human TNF transgenic mice, deletion of Lasp1 reduces arthritic joint destruction. Therefore, we show a function of Lasp1 in cellular junction formation and inflammatory tissue remodelling and identify Lasp1 as a potential target for treating inflammatory joint disorders associated with aggressive cellular transformation.

Loss of Hem1 disrupts macrophage function and impacts migration, phagocytosis, and integrin-mediated adhesion.

Hematopoietic-specific protein 1 (Hem1) is an essential subunit of the WAVE regulatory complex (WRC) in immune cells. WRC is crucial for Arp2/3 complex activation and the protrusion of branched actin filament networks. Moreover, Hem1 loss of function in immune cells causes autoimmune diseases in humans. Here, we show that genetic removal of Hem1 in macrophages diminishes frequency and efficacy of phagocytosis as well as phagocytic cup formation in addition to defects in lamellipodial protrusion and migration. Moreover, Hem1-null macrophages displayed strong defects in cell adhesion despite unaltered podosome formation and concomitant extracellular matrix degradation. Specifically, dynamics of both adhesion and de-adhesion as well as concomitant phosphorylation of paxillin and focal adhesion kinase (FAK) were significantly compromised. Accordingly, disruption of WRC function in non-hematopoietic cells coincided with both defects in adhesion turnover and altered FAK and paxillin phosphorylation. Consistently, platelets exhibited reduced adhesion and diminished integrin αIIbß3 activation upon WRC removal. Interestingly, adhesion phenotypes, but not lamellipodia formation, were partially rescued by small molecule activation of FAK. A full rescue of the phenotype, including lamellipodia formation, required not only the presence of WRCs but also their binding to and activation by Rac. Collectively, our results uncover that WRC impacts on integrin-dependent processes in a FAK-dependent manner, controlling formation and dismantling of adhesions, relevant for properly grabbing onto extracellular surfaces and particles during cell edge expansion, like in migration or phagocytosis.

Importance of osteocyte-mediated regulation of bone remodelling in inflammatory bone disease.

Although the impact of osteoblast-osteoclast crosstalk in bone remodelling has been intensively studied, the importance of osteocytes, descendants of osteoblasts, in this process has for a long time been neglected. During their embedding phase, osteocytes undergo considerable phenotypic transformation, from a cuboidal, highly metabolically active osteoblast secreting extracellular matrix to a small, stellate, quiescent osteocyte with numerous long dendrites. Osteocytes are encysted in cavities (lacunae) and their dendritic extensions are located in tunnels (canaliculi) forming a remarkable, highly branched, lacunar-canalicular signalling network that spans the entire bone matrix. Osteocytes and their dendrites can communicate directly with each other and through the release of effector proteins such as sclerostin and nuclear factor κB ligand (RANKL), influence osteoblast and osteoclast formation. This allows osteocytes embedded within the bone matrix to communicate and coordinate activity of cells on the bone surface to adapt to mechanical needs and hormonal changes. Besides their importance in sustaining physiological bone homeostasis, accumulating evidence suggests that dysregulated osteocyte function and alterations in the osteocyte lacunar-canalicular network structure are characteristics of skeletal diseases. This review highlights some aspects of osteocyte communication with osteoclasts and mesenchymal stromal cells, the importance of blood vessel-osteocyte interaction and describes central functions of these cells in rheumatoid arthritis, osteoarthritis, osteomyelitis and osteoporosis. Within the last decade new technologies and tools have facilitated the study of osteocyte biology and the search for therapeutic targets to address bone fragility in the near future.

Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes.

Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families. Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion.

Ter94/VCP is a novel component involved in BMP signaling.

Bone morphogenetic proteins (BMPs), a subgroup of the transforming growth factor (TGF)-ß family, transduce their signal through multiple components downstream of their receptors. Even though the components involved in the BMP signaling pathway have been intensely studied, many molecules mediating BMP signaling remain to be addressed. To identify novel components that participate in BMP signaling, RNA interference (RNAi)-based screening was established by detecting phosphorylated Mad (pMad) in Drosophila S2 cells. Ter94, a member of the family of AAA ATPases, was identified as a novel mediator of BMP signaling, which is required for the phosphorylation of Mad in Drosophila S2 cells. Moreover, the mammalian orthlog of Ter94 valosin-containing protein (VCP) plays a critical role in the BMP-Smad1/5/8 signaling pathway in mammalian cells. Genetic evidence suggests that Ter94 is involved in the dorsal-ventral patterning of the Drosophila early embryo through regulating decapentaplegic (Dpp)/BMP signals. Taken together, our data suggest that Ter94/VCP appears to be an evolutionarily conserved component that regulates BMP-Smad1/5/8 signaling.

Antisense-oligonucleotide mediated exon skipping in activin-receptor-like kinase 2: inhibiting the receptor that is overactive in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare heritable disease characterized by progressive heterotopic ossification of connective tissues, for which there is presently no definite treatment. A recurrent activating mutation (c.617G→A; R206H) of activin receptor-like kinase 2 (ACVR1/ALK2), a BMP type I receptor, has been shown as the main cause of FOP. This mutation constitutively activates the BMP signaling pathway and initiates the formation of heterotopic bone. In this study, we have designed antisense oligonucleotides (AONs) to knockdown mouse ALK2 expression by means of exon skipping. The ALK2 AON could induce exon skipping in cells, which was accompanied by decreased ALK2 mRNA levels and impaired BMP signaling. In addition, the ALK2 AON potentiated muscle differentiation and repressed BMP6-induced osteoblast differentiation. Our results therefore provide a potential therapeutic approach for the treatment of FOP disease by reducing the excessive ALK2 activity in FOP patients.

Anti-Sclerostin antibody inhibits internalization of Sclerostin and Sclerostin-mediated antagonism of Wnt/LRP6 signaling.

Sclerosteosis is a rare high bone mass disease that is caused by inactivating mutations in the SOST gene. Its gene product, Sclerostin, is a key negative regulator of bone formation and might therefore serve as a target for the anabolic treatment of osteoporosis. The exact molecular mechanism by which Sclerostin exerts its antagonistic effects on Wnt signaling in bone forming osteoblasts remains unclear. Here we show that Wnt3a-induced transcriptional responses and induction of alkaline phosphatase activity, an early marker of osteoblast differentiation, require the Wnt co-receptors LRP5 and LRP6. Unlike Dickkopf1 (DKK1), Sclerostin does not inhibit Wnt-3a-induced phosphorylation of LRP5 at serine 1503 or LRP6 at serine 1490. Affinity labeling of cell surface proteins with [(125)I]Sclerostin identified LRP6 as the main specific Sclerostin receptor in multiple mesenchymal cell lines. When cells were challenged with Sclerostin fused to recombinant green fluorescent protein (GFP) this was internalized, likely via a Clathrin-dependent process, and subsequently degraded in a temperature and proteasome-dependent manner. Ectopic expression of LRP6 greatly enhanced binding and cellular uptake of Sclerostin-GFP, which was reduced by the addition of an excess of non-GFP-fused Sclerostin. Finally, an anti-Sclerostin antibody inhibited the internalization of Sclerostin-GFP and binding of Sclerostin to LRP6. Moreover, this antibody attenuated the antagonistic activity of Sclerostin on canonical Wnt-induced responses.

Overactive bone morphogenetic protein signaling in heterotopic ossification and Duchenne muscular dystrophy.

Bone morphogenetic proteins (BMPs) are important extracellular cytokines that play critical roles in embryogenesis and tissue homeostasis. BMPs signal via transmembrane type I and type II serine/threonine kinase receptors and intracellular Smad effector proteins. BMP signaling is precisely regulated and perturbation of BMP signaling is connected to multiple diseases, including musculoskeletal diseases. In this review, we will summarize the recent progress in elucidation of BMP signal transduction, how overactive BMP signaling is involved in the pathogenesis of heterotopic ossification and Duchenne muscular dystrophy, and discuss possible therapeutic strategies for treatment of these diseases.

Deregulated bone morphogenetic protein receptor signaling underlies fibrodysplasia ossificans progressiva.

Transforming growth factor-ß family members, which include TGF-ßs, activins and bone morphogenetic proteins (BMPs), play important roles in development and maintaining tissue homeostasis. The extracellular TGF-ß family members signal across the plasmamembrane by activating type I and type II serine/threonine kinase receptors. Pertubation in TGF-ß family receptor signaling has been implicated in certain diseases, including musculo-skeletal disorders. Fibrodysplasia ossificans progressiva (FOP) is a rare disorder characterized by progressive formation of ectopic bone and congenital malformations of the great toes. At present no curative therapy is available, therefore prevention of heterotopic ossification is the hallmark of FOP management. FOP has been linked to an autosomal dominant mutation on chromosome 2, to the gene encoding activin receptor-like kinase 2 (ALK2), a BMP type I receptor. This mutation is found in almost all classically affected FOP patients and causes the FOP phenotype. This discovery has paved the way for further investigations into the molecular basis underlying FOP and has recently pointed towards potential strategies to treat this devastating disease.

Cell-type specific regulation of myostatin signaling.

The transforming growth factor (TGF)-ß family member myostatin is an important regulator of myoblast, adipocyte, and fibroblast growth and differentiation, but the signaling mechanisms remain to be established. We therefore determined the contribution of myostatin type I receptors activin receptor-like kinase-4 (ALK4) and -5 (ALK5) and different coreceptors in C2C12 myoblasts, C3H10T1/2 mesenchymal stem cells, and 3T3-L1 fibroblasts, as well as in primary myoblast and fibroblasts. We performed siRNA-mediated knockdown of each receptor and measured signaling activity using Smad3-dependent luciferase and Smad2 phosphorylation assays with nontargeting siRNA as control. We find that myostatin utilizes ALK4 in myoblasts, whereas it has a preference for ALK5 in nonmyogenic cells. Notably, our results show that coreceptor Cripto is expressed in myoblasts but not in the nonmyogenic cells and that it regulates myostatin activity. More specifically, myostatin requires Cripto in myoblasts, whereas Cripto represses activin activity and TGF-ß signaling is Cripto independent. Cripto-mediated myostatin signaling is dependent on both epidermal growth factor (EGF)-like and Cripto-FRL1-cryptic (CFC) domains, whereas activin signaling is solely conferred by the CFC domain. Furthermore, Cripto down-regulation enhances myoblast differentiation, showing its importance in myostatin signaling. Together, our results identify a molecular mechanism that explains the cell-type specific aspects of signaling by myostatin and other TGF-ß family members.

Use and efficacy of bone morphogenetic proteins in fracture healing.

PURPOSE: This review evaluates the application of bone morphogenetic proteins (BMPs) in delayed bone repair, aiming at a broad audience from clinicians to scientists. Next to an overview of the role of the different BMPs, their antagonists and their current applications, special attention is focused on new scientific developments improving the effects of BMP-based therapy for bone repair. METHODS: Publication searches in PubMed and Embase revealed 850 relevant articles on the criteria 'BMP' AND 'bone repair' (as of May 2011). The abstracts were carefully reviewed and papers were selected according to the content. RESULTS: The resulting publications showed that BMP-2 and BMP-7 are clearly the most extensively evaluated BMPs, in general with positive results on bone healing, comparable to the use of unspecific preparations such as autologous bone grafts or platelet-rich plasma. CONCLUSIONS: Although the efficacy of BMPs as stimulators of bone repair has been demonstrated in model systems and clinical studies, the use of BMPs to enhance fracture healing in the clinical setting is still controversial. Issues such as when, where and how much of which BMP is the most effective and profitable to use still have to be elucidated. But optimisation of the BMP products used in combination with cheaper production methods will inevitably stimulate the clinical use of BMPs for bone fracture healing in the near future.

Biphasic effects of transforming growth factor ß on bone morphogenetic protein-induced osteoblast differentiation.

Bone morphogenetic proteins (BMPs) exert an important role in skeletal development, adult bone homeostasis, and fracture healing and have demonstrated clinical utility for bone regeneration. However, BMPs fall short as regenerative agents because high doses need to be used to obtain therapeutic effects. Determining the molecular mechanisms controlling BMP-induced bone formation may lead to the development of more effective BMP-based therapies. To identify kinases mediating BMP-induced osteoblast differentiation, we performed an siRNA screen to find kinases modulating BMP-6-induced alkaline phosphatase (ALP) activity. Surprisingly, although transforming growth factor ß (TGF-ß) generally is considered to antagonize BMP-induced osteoblast differentiation, C2C12 cells transfected with siRNAs targeting TGF-ß receptors displayed reduced BMP-6-induced ALP activity. Furthermore, pharmacologic inhibitors blocking the TGF-ß type I receptor impaired BMP-induced ALP activity in KS483 and C2C12 cells and mineralization of KS483 cells. Consistently, costimulation with BMPs and TGF-ß further increased expression of osteoblast-specific genes, ALP activity, and mineralization of KS483 cells and primary mesenchymal stem cells compared with BMPs alone. The stimulatory and inhibitory effects of TGF-ß were found to depend on timing and duration of the costimulation. TGF-ß inhibited BMP-induced activation of a BMP-Smad-dependent luciferase reporter, suggesting that the stimulatory effect of TGF-ß is not due to increased BMP-Smad activity. TGF-ß also inhibited the BMP-induced expression of the BMP antagonist noggin and prolonged BMP activity. In conclusion, TGF-ß, besides acting as an inhibitor, also can, by dampening the noggin-mediated negative-feedback loop, enhance BMP-induced osteoblast differentiation, which might be beneficial in fracture healing.

Dual exon skipping in myostatin and dystrophin for Duchenne muscular dystrophy.

BACKGROUND: Myostatin is a potent muscle growth inhibitor that belongs to the Transforming Growth Factor-ß (TGF-ß) family. Mutations leading to non functional myostatin have been associated with hypermuscularity in several organisms. By contrast, Duchenne muscular dystrophy (DMD) is characterized by a loss of muscle fibers and impaired regeneration. In this study, we aim to knockdown myostatin by means of exon skipping, a technique which has been successfully applied to reframe the genetic defect of dystrophin gene in DMD patients. METHODS: We targeted myostatin exon 2 using antisense oligonucleotides (AON) in healthy and DMD-derived myotubes cultures. We assessed the exon skipping level, transcriptional expression of myostatin and its target genes, and combined myostatin and several dystrophin AONs. These AONs were also applied in the mdx mice models via intramuscular injections. RESULTS: Myostatin AON induced exon 2 skipping in cell cultures and to a lower extent in the mdx mice. It was accompanied by decrease in myostatin mRNA and enhanced MYOG and MYF5 expression. Furthermore, combination of myostatin and dystrophin AONs induced simultaneous skipping of both genes. CONCLUSIONS: We conclude that two AONs can be used to target two different genes, MSTN and DMD, in a straightforward manner. Targeting multiple ligands of TGF-beta family will be more promising as adjuvant therapies for DMD.

BMP antagonists enhance myogenic differentiation and ameliorate the dystrophic phenotype in a DMD mouse model.

Duchenne Muscular Dystrophy (DMD) is an X-linked lethal muscle wasting disease characterized by muscle fiber degeneration and necrosis. The progressive pathology of DMD can be explained by an insufficient regenerative response resulting in fibrosis and adipose tissue formation. BMPs are known to inhibit myogenic differentiation and in a previous study we found an increased expression of a BMP family member BMP4 in DMD myoblasts. The aim of the current study was therefore to investigate whether inhibition of BMP signaling could be beneficial for myoblast differentiation and muscle regeneration processes in a DMD context. All tested BMP inhibitors, Noggin, dorsomorphin and LDN-193189, were able to accelerate and enhance myogenic differentiation. However, dorsomorphin repressed both BMP and TGFß signaling and was found to be toxic to primary myoblast cell cultures. In contrast, Noggin was found to be a potent and selective BMP inhibitor and was therefore tested in vivo in a DMD mouse model. Local adenoviral-mediated overexpression of Noggin in muscle resulted in an increased expression of the myogenic regulatory genes Myog and Myod1 and improved muscle histology. In conclusion, our results suggest that repression of BMP signaling may constitute an attractive adjunctive therapy for DMD patients.

Measurement of constitutive activity of BMP type I receptors.

Bone morphogenetic proteins (BMPs) are pleiotropic cytokines controlling a multitude of processes, among which bone formation. BMPs function by binding and activating BMP type I and type II receptors, resulting in activation of Smad transcription factors and expression of BMP target genes. Mutations in the BMP type I receptor ALK2 were identified in patients suffering from Fibrodysplasia Ossificans Progressiva (FOP). The mutation found in all patients displaying classical FOP symptoms, the ALK2 R206H mutation, renders ALK2 constitutively active. Here, we provide a detailed description of how to determine whether other ALK2 mutations will confer constitutive activity to the receptor.

Distinct modes of inhibition by sclerostin on bone morphogenetic protein and Wnt signaling pathways.

Sclerostin is expressed by osteocytes and has catabolic effects on bone. It has been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity, although at present the underlying mechanisms are unclear. Consistent with previous findings, Sclerostin opposed direct Wnt3a-induced but not direct BMP7-induced responses when both ligand and antagonist were provided exogenously to cells. However, we found that when both proteins are expressed in the same cell, sclerostin can antagonize BMP signaling directly by inhibiting BMP7 secretion. Sclerostin interacts with both the BMP7 mature domain and pro-domain, leading to intracellular retention and proteasomal degradation of BMP7. Analysis of sclerostin knock-out mice revealed an inhibitory action of sclerostin on Wnt signaling in both osteoblasts and osteocytes in cortical and cancellous bones. BMP7 signaling was predominantly inhibited by sclerostin in osteocytes of the calcaneus and the cortical bone of the tibia. Our results suggest that sclerostin exerts its potent bone catabolic effects by antagonizing Wnt signaling in a paracrine and autocrine manner and antagonizing BMP signaling selectively in the osteocytes that synthesize simultaneously both sclerostin and BMP7 proteins.

ALK2 R206H mutation linked to fibrodysplasia ossificans progressiva confers constitutive activity to the BMP type I receptor and sensitizes mesenchymal cells to BMP-induced osteoblast differentiation and bone formation.

Fibrodysplasia ossificans progressiva (FOP) is a rare disabling disease characterized by heterotopic ossification for which there is currently no treatment available. FOP has been linked recently to a heterozygous R206H mutation in the bone morphogenetic protein (BMP) type I receptor activin receptor-like kinase 2 (ALK2). Expression of the mutant ALK2-R206H receptor (FOP-ALK2) results in increased phosphorylation of the downstream Smad1 effector proteins and elevated basal BMP-dependent transcriptional reporter activity, indicating that FOP-ALK2 is constitutively active. FOP-ALK2-induced transcriptional activity could be blocked by overexpressing either of the inhibitory Smads, Smad6 or -7, or by treatment with the pharmacological BMP type I receptor inhibitor dorsomorphin. However, in contrast to wild-type ALK2, FOP-ALK2 is not inhibited by the negative regulator FKBP12. Mesenchymal cells expressing the FOP-ALK2 receptor are more sensitive to undergoing BMP-induced osteoblast differentiation and mineralization. In vivo bone formation was assessed by loading human mesenchymal stem cells (hMSCs) expressing the ALK2-R206H receptor onto calcium phosphate scaffolds and implantation in nude mice. Compared with control cells FOP-ALK2-expressing cells induced increased bone formation. Taken together, the R206H mutation in ALK2 confers constitutive activity to the mutant receptor, sensitizes mesenchymal cells to BMP-induced osteoblast differentiation, and stimulates new bone formation. We have generated an animal model that can be used as a stepping stone for preclinical studies aimed at inhibiting the heterotopic ossification characteristic of FOP.

Osteocyte-derived sclerostin inhibits bone formation: its role in bone morphogenetic protein and Wnt signaling.

Sclerosteosis and Van Buchem disease are rare, high-bone-mass disorders that have been linked to deficiency in the SOST gene, encoding sclerostin. Sclerostin belongs to the DAN family of glycoproteins, of which multiple family members have been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity. Sclerostin is specifically expressed by osteocytes and inhibits BMP-induced osteoblast differentiation and ectopic bone formation. Sclerostin binds only weakly to BMPs and does not inhibit direct BMP-induced responses. Instead, sclerostin antagonizes canonical Wnt signaling by binding to Wnt coreceptors, low-density lipoprotein receptor-related protein 5 and 6. Several lipoprotein receptor-related protein-5 mutants that cause the high-bone-mass trait are defective in sclerostin binding. Thus, high bone mass in sclerosteosis and Van Buchem disease may result from increased Wnt signaling due to the absence of or insensitivity to sclerostin.