Actinobacillus actinomycetemcomitans leukotoxin and

The study of FOP, a disabling genetic disorder of progressive heterotopic ossification, is hampered by the lack of readily available connective tissue progenitor cells. We isolated such cells from discarded primary teeth of patients with FOP and controls and discovered dysregulation of BMP signaling and rapid osteoblast differentiation in FOP cells compared with control cells.Introduction: Fibrodysplasia ossificans progressiva (FOP), the most disabling condition of progressive heterotopic ossification in humans, is caused by a recurrent heterozygous missense mutation in activin receptor IA (ACVR1), a bone morphogenetic protein (BMP) type I receptor, in all classically affected individuals. A comprehensive understanding of FOP has been limited, in part, by a lack of readily available connective tissue progenitor cells in which to study the molecular pathology of this disorder. Materials and Methods:We derived connective tissue progenitor cells from discarded primary teeth (SHED cells) of patients with FOP and controls and examined BMP signaling and osteogenic differentiation in these cells. Results: SHED cells transmitted BMP signals through both the SMAD and p38 mitogen-activated protein kinase (MAPK) pathways and responded to BMP4 treatment by inducing BMP responsive genes. FOP cells showed ligand-independent BMP signaling and ligand-dependent hyper-responsiveness to BMP stimulation. Furthermore, FOP cells showed more rapid differentiation to an osteogenic phenotype than control cells. Conclusions: This is the first study of BMP signaling and osteogenic differentiation in connective tissue progenitor cells from patients with FOP. Our data strongly support both basal and ligand-stimulated dysregulation of BMP signaling consistent with in silico studies of the mutant ACVR1 receptor in this condition. This study substantially extends our understanding of dysregulated BMP signaling in a progenitor cell population relevant to the pathogenesis of this catastrophic disorder of progressive ectopic ossification.

Heparan sulfate (HS) is a component of cell surface and matrix-associated proteoglycans (HSPGs) that collectively, play crucial roles in many physiologic processes including cell differentiation, organ morphogenesis and cancer. A key function of HS is to bind and interact with signaling proteins, growth factors, plasma proteins, immune-modulators and other factors. In so doing, the HS chains and HSPGs are able to regulate protein distribution, bio-availability and action on target cells and can also serve as cell surface co-receptors, facilitating ligand-receptor interactions. These proteins contain an HS/heparin-binding domain (HBD) that mediates their association and contacts with HS. HBDs are highly diverse in sequence and predicted structure, contain clusters of basic amino acids (Lys, Arg) and possess an overall net positive charge, most often within a consensus Cardin-Weintraub (CW) motif. Interestingly, other domains and residues are now known to influence protein-HS interactions, as well as interactions with other glycosaminoglycans, such as chondroitin sulfate. In this review we provide a description and analysis of HBDs in proteins including amphiregulin, fibroblast growth factor family members, heparanase, sclerostin and hedgehog protein family members. We discuss HBD structural and functional features and important roles carried out by other protein domains, and also provide novel conformational insights into the diversity of CW motifs present in Sonic, Indian and Desert hedgehogs. Finally, we review progress in understanding the pathogenesis of a rare pediatric skeletal disorder, Hereditary Multiple Exostoses (HME), characterized by HS deficiency and cartilage tumor formation. Advances in understanding protein-HS interactions will have broad implications for basic biology and translational medicine as well as for the development of HS-based therapeutics.

Cell surface heparan sulfate proteoglycans (HSPGs) have been implicated in bone morphogenetic protein (BMP)-mediated morphogenesis by regulating BMP activity and gradient formation. However, the direct role of HSPGs in BMP signaling is poorly understood. Here we show that HSPGs directly regulate BMP2-mediated transdifferentiation of C2C12 myoblasts into osteoblasts. HSPGs sequester BMP2 at the cell surface and mediate BMP2 internalization. Depletion of cell surface HSPGs by heparinase III treatment or decreased glycosaminoglycan chain sulfation with sodium chlorate enhances BMP2 morphogenetic bioactivity. The addition of exogenous heparin, a widely used anticoagulant, reduced BMP2 signaling. Our results suggest that cell surface HSPGs mediate BMP2 internalization and modulate BMP2 osteogenic activity.

Cells with osteogenic potential can be found in a variety of tissues. Here we show that circulating osteogenic precursor (COP) cells, a bone marrow-derived type I collagen+/CD45+ subpopulation of mononuclear adherent cells, are present in early pre-osseous fibroproliferative lesions in patients with fibrodysplasia ossificans progressiva (FOP) and nucleate heterotopic ossification (HO) in a murine in vivo implantation assay. Blood samples from FOP patients with active episodes of HO contain significantly higher numbers of clonally-derived COP cell colonies than patients with stable disease or unaffected individuals. The highest level of COP cells was found in a patient just prior to the clinical onset of an HO exacerbation. Our studies show that even COP cells derived from an unaffected individual can contribute to HO in genetically susceptible host tissue. The possibility that circulating, hematopoietic-derived cells with osteogenic potential can seed inflammatory sites has tremendous implications and, to our knowledge, represents the first example of their involvement in clinical HO. Thus, bone formation is not limited to cells of the mesenchymal lineage, and circulating cells of hematopoietic origin can also serve as osteogenic precursors at remote sites of tissue inflammation.

Summary. Integrins are a ubiquitous family of non-covalently associated a/b transmembrane heterodimers linking extracellular ligands to intracellular signaling pathways [1] [Cell, 2002; 110: 673]. Platelets contain five integrins, three b1 integrins that mediate platelet adhesion to the matrix proteins collagen, fibronectin and laminin, and the b3 integrins avb3 and aIIbb3[2] [J Clin Invest, 2005; 115: 3363]. While there are only several hundred avb3 molecules per platelet, avb3 mediates platelet adhesion to osteopontin and vitronectin in vitro [3] [J Biol Chem, 1997; 272: 8137]; whether this occurs in vivo remains unknown. By contrast, the 80 000 aIIbb3 molecules on agoniststimulated platelets bind fibrinogen, von Willebrand factor, and fibronectin, mediating platelet aggregation when the bound proteins crosslink adjacent platelets [2] [J Clin Invest, 2005; 115: 3363]. Although platelet integrins are poised to shift from resting to active conformations, tight regulation of their activity is essential to prevent the formation of intravascular thrombi. This review focuses on the structure and function of the intensively studied b3 integrins, in particular aIIbb3, but reference will be made to other integrins where relevant.Keywords: glycoprotein IIb/IIIa, integrins, protein structure. The extracellular domain of b3 integrinsElectron microscopy (EM) of aIIbb3 isolated from platelets revealed an 8 · 12 nm nodular headpiece containing its ligand binding site and two 18 nm flexible legs containing transmembrane (TM) and cytoplasmic domains extending from one side [4]. Further, crystal structures for the extracellular portions of avb3 and aIIbb3 have revealed a complex domain structure that rearranges as the integrins switch from inactive resting to active ligand-binding conformations (Fig. 1) [5][6][7][8].The extracellular portions of aIIb and av are similar, consisting of an amino-terminal b-propeller domain followed by a ÔthighÕ and two ÔcalfÕ domains, whereas b3 is substantially more complicated [5,7]. Its amino-terminal portion consists of two tandem nested domains: a bA domain whose fold resembles that of integrin a subunit I-domains, which is inserted into a ÔhybridÕ domain whose fold is similar to an I-set Ig domain; the hybrid domain in turn is inserted into a PSI (plexin, semaphorin, integrin) domain that contains the N-terminus of b3 [7,9]. The C-terminus of the PSI domain is continuous with four tandem EGF-like repeats that make up the ÔlegÕ of b3, as well as a unique carboxyl-terminal bTD domain [8]. av and aIIb interact non-covalently with b3 via an interface between the a subunit b-propeller and the b3 bA domains that resembles the interface between the Ga and Gb subunits of G proteins and forms the surface for ligand binding [5,7].A surprising finding of the crystal structure of the extracellular portion of avb3 was a severe bend at ÔkneesÕ or ÔgenusÕ located between the first and second EGF-like repeats of b3 and the thigh and first calf domains of av [5,10]. Recent high resolution crystal structu...

Proper growth and development require the orderly synthesis and deposition of individual components of the extracellular matrix (ECM) into well ordered networks. Once formed, the ECM maintains tissue structure and houses resident cells. One ECM component, ␤ig-h3, is a highly conserved transforming growth factor-␤-inducible protein that has been hypothesized to function as a bifunctional linker between individual matrix components and resident cells. To gain insights into its physiological function, full-length ␤ig-h3 protein was produced using a baculovirus expression system and purified under native conditions. Human fibroblasts attached and spread on ␤ig-h3-coated plates and developed actin stress fibers. Purified ␤ig-h3 binds fibronectin (FN) and type I collagen (Col I) but does not bind gelatin. Using defined fragments of FN, we localized the ␤ig-h3 recognition region to the gelatin/collagen binding domain present in the N-terminal region of the FN molecule. Our results identify FN and Col I as two ligands of ␤ig-h3 in the ECM.

During limb skeletogenesis the cartilaginous long bone anlagen and their growth plates become delimited by perichondrium with which they interact functionally. Yet, little is known about how, despite being so intimately associated with cartilage, perichondrium acquires and maintains its distinct phenotype and exerts its border function. Because perichondrium becomes deranged and interrupted by cartilaginous outgrowths in Hereditary Multiple Exostoses (HME), a pediatric disorder caused by EXT mutations and consequent heparan sulfate (HS) deficiency, we asked whether EXT genes and HS normally have roles in establishing its phenotype and function. Indeed, conditional Ext1 ablation in perichondrium and lateral chondrocytes flanking the epiphyseal region of mouse embryo long bone anlagen – a region encompassing the groove of Ranvier – caused ectopic cartilage formation. A similar response was observed when HS function was disrupted in long bone anlagen explants by genetic, pharmacological or enzymatic means, a response preceded by ectopic BMP signaling within perichondrium. These treatments also triggered excess chondrogenesis and cartilage nodule formation and overexpression of chondrogenic and matrix genes in limb bud mesenchymal cells in micromass culture. Interestingly, the treatments disrupted the peripheral definition and border of the cartilage nodules in such a way that many nodules overgrew and fused with each other into large amorphous cartilaginous masses. Interference with HS function reduced the physical association and interactions of BMP2 with HS and increased the cell responsiveness to endogenous and exogenous BMP proteins. In sum, Ext genes and HS are needed to establish and maintain perichondrium’s phenotype and border function, restrain pro-chondrogenic signaling proteins including BMPs, and restrict chondrogenesis. Alterations in these mechanisms may contribute to exostosis formation in HME, particularly at the expense of regions rich in progenitor cells including the groove of Ranvier.