We have identified a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family, ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white-spotting ...mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was identified using Adamts9(+/-);bt/bt mice, which have a fully penetrant cleft palate. Palate closure was delayed, although eventually completed, in both Adamts9(+/-);bt/+ and bt/bt mice, demonstrating cooperation of these genes. Adamts20 is expressed in palatal mesenchyme, whereas Adamts9 is expressed exclusively in palate microvascular endothelium. Palatal shelves isolated from Adamts9(+/-);bt/bt mice fused in culture, suggesting an intact epithelial TGFβ3 signaling pathway. Cleft palate resulted from a temporally specific delay in palatal shelf elevation and growth towards the midline. Mesenchyme of Adamts9(+/-);bt/bt palatal shelves had reduced cell proliferation, a lower cell density and decreased processing of versican (VCAN), an extracellular matrix (ECM) proteoglycan and ADAMTS9/20 substrate, from E13.5 to E14.5. Vcan haploinsufficiency led to greater penetrance of cleft palate in bt mice, with a similar defect in palatal shelf extension as Adamts9(+/-);bt/bt mice. Cell density was normal in bt/bt;Vcan(hdf)(/+) mice, consistent with reduced total intact versican in ECM, but impaired proliferation persisted in palate mesenchyme, suggesting that ADAMTS-cleaved versican is required for cell proliferation. These findings support a model in which cooperative versican proteolysis by ADAMTS9 in vascular endothelium and by ADAMTS20 in palate mesenchyme drives palatal shelf sculpting and extension.
Acute trauma to the central nervous system (CNS) can result in permanent damage and loss of function related to the poor regeneration of injured axons. Injured axons encounter several barriers to ...regeneration, such as the glial scar at the injury site. The glial scar contains extracellular matrix (ECM) macromolecules deposited by reactive astrocytes in response to injury. The scar ECM is rich in chondroitin sulfate proteoglycans (CSPGs), macromolecules that inhibit axonal growth. CSPGs consist of a core protein with attachment sites for glycosaminoglycan (GAG) chains. An extensive literature demonstrates that enzymatic removal of the GAG chains by chondroitinase ABC permits some axonal regrowth; however, the remaining intact core proteins also possess inhibitory domains. Because metalloproteinases can degrade core proteins of CSPGs, we have evaluated five matrix metalloproteinases (MMPs) and a related protease—a disintegrin and metalloproteinase with thrombospondin motifs‐4 (ADAMTS‐4)—for their capacity to overcome CSPG inhibition of neuritic growth in culture. The metalloproteinases were selected for their known expression after CNS injuries. Of the MMPs, MMP‐3, ‐7 and ‐8 reduced or abolished inhibition of neurite outgrowth on a purified CSPG substrate and on an astrocyte‐derived ECM. ADAMTS‐4 also attenuated CSPG inhibition of neurites and had the additional benefits of neither degrading laminin nor causing neurotoxicity. The efficacy of ADAMTS‐4 matched that of blocking the EGFR signaling previously reported to mediate CSPG inhibition. These findings highlight ADAMTS‐4 as a superior protease for overcoming CSPG inhibition of axonal regeneration in the CNS.
Versican is a widely distributed chondroitin sulfate proteoglycan that forms large complexes with the glycosaminoglycan hyaluronan (HA). As a consequence of HA binding to its receptor CD44 and ...interactions of the versican C-terminal globular (G3) domain with a variety of extracellular matrix proteins, versican is a key component of well-defined networks in pericellular matrix and extracellular matrix. Versican is crucial for several developmental processes in the embryo ranging from cardiac development to digit separation, and there is an increasing interest in its roles in cancer and inflammation. Versican proteolysis by ADAMTS proteases is highly regulated, occurs at specific peptide bonds, and is relevant to several physiological and disease mechanisms. In this chapter, methods are described for the isolation and detection of intact and cleaved versican in tissues using morphologic and biochemical techniques. These, together with the methodologies for purification and analysis of recombinant versican and an N-terminal versican fragment named versikine that are provided here, are likely to facilitate further progress on the biology of versican and its proteolysis.
Focal adhesions anchor cells to extracellular matrix (ECM) and direct assembly of a pre-stressed actin cytoskeleton. They act as a cellular sensor and regulator, linking ECM to the nucleus. Here, we ...identify proteolytic turnover of the anti-adhesive proteoglycan versican as a requirement for maintenance of smooth muscle cell (SMC) focal adhesions. Using conditional deletion in mice, we show that ADAMTS9, a secreted metalloprotease, is required for myometrial activation during late gestation and for parturition. Through knockdown of ADAMTS9 in uterine SMC, and manipulation of pericellular versican via knockdown or proteolysis, we demonstrate that regulated pericellular matrix dynamics is essential for focal adhesion maintenance. By influencing focal adhesion formation, pericellular versican acts upstream of cytoskeletal assembly and SMC differentiation. Thus, pericellular versican proteolysis by ADAMTS9 balances pro- and anti-adhesive forces to maintain an SMC phenotype, providing a concrete example of the dynamic reciprocity of cells and their ECM.
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•ADAMTS9, a secreted metalloproteinase, is required for parturition in mice•ADAMTS9 siRNA impairs focal adhesion formation in uterine smooth muscle cells•ADAMTS9 ensures focal adhesion formation via pericellular versican turnover•Pericellular matrix proteolysis is a crucial regulator of focal adhesions
Mead et al. identify a proteolytic mechanism that actively maintains a pericellular microenvironment conducive to uterine smooth muscle activation prior to parturition. They show that pericellular matrix proteolysis by the secreted metalloprotease ADAMTS9 is crucial for maintenance of focal adhesions in uterine smooth muscle cells, and its absence impairs parturition.
In fetal valve maturation the mechanisms by which the relatively homogeneous proteoglycan-rich extracellular matrix (ECM) of endocardial cushions is replaced by a specialized and stratified ECM found ...in mature valves are not understood. Therefore, we reasoned that uncovering proteases critical for ‘remodeling’ the proteoglycan rich (extracellular matrix) ECM may elucidate novel mechanisms of valve development. We have determined that mice deficient in ADAMTS5, (A Disintegrin-like And Metalloprotease domain with ThromboSpondin-type 1 motifs) which we demonstrated is expressed predominantly by valvular endocardium during cardiac valve maturation, exhibited enlarged valves. ADAMTS5 deficient valves displayed a reduction in cleavage of its substrate versican, a critical cardiac proteoglycan. In vivo reduction of versican, in Adamts5−/− mice, achieved through Vcan heterozygosity, substantially rescued the valve anomalies. An increase in BMP2 immunolocalization, Sox9 expression and mesenchymal cell proliferation were observed in Adamts5−/− valve mesenchyme and correlated with expansion of the spongiosa (proteoglycan-rich) region in Adamts5−/− valve cusps. Furthermore, these data suggest that ECM remodeling via ADAMTS5 is required for endocardial to mesenchymal signaling in late fetal valve development. Although adult Adamts5−/− mice are viable they do not recover from developmental valve anomalies and have myxomatous cardiac valves with 100% penetrance. Since the accumulation of proteoglycans is a hallmark of myxomatous valve disease, based on these data we hypothesize that a lack of versican cleavage during fetal valve development may be a potential etiology of adult myxomatous valve disease.
► ADAMTS5 deficient mice develop enlarged cardiac valves by late fetal stages. ► Valve mesenchyme in Adamts5-/- mice display increased proliferation and intercellular space. ► In vivo reduction of the ADAMTS5 substrate versican rescues the valve phenotype in Adamts5-/- mice. ► Endothelial expression of ADAMTS5 is required for differentiation of cardiac valve mesenchyme. ► BMP2 and Sox9 expression is not down-regulated in Adamts5-/- cardiac valve cusps.
Fibroblasts produce the majority of collagen in the heart and are thought to regulate extracellular matrix (ECM) turnover. Although fibrosis accompanies many cardiac pathologies and is generally ...deleterious, the role of fibroblasts in maintaining the basal ECM network and in fibrosis in vivo is poorly understood. We genetically ablated fibroblasts in mice to evaluate the impact on homeostasis of adult ECM and cardiac function after injury. Fibroblast-ablated mice demonstrated a substantive reduction in cardiac fibroblasts, but fibrillar collagen and the ECM proteome were not overtly altered when evaluated by quantitative mass spectrometry and N-terminomics. However, the distribution and quantity of collagen VI, microfibrillar collagen that forms an open network with the basement membrane, was reduced. In fibroblast-ablated mice, cardiac function was better preserved following angiotensin II/phenylephrine (AngII/PE)-induced fibrosis and myocardial infarction (MI). Analysis of cardiomyocyte function demonstrated altered sarcomere shortening and slowed calcium decline in both uninjured and AngII/PE-infused fibroblast-ablated mice. After MI, the residual resident fibroblasts responded to injury, albeit with reduced proliferation and numbers immediately after injury. These results indicate that the adult mouse heart tolerates a significant degree of fibroblast loss with a potentially beneficial impact on cardiac function after injury. The cardioprotective effect of controlled fibroblast reduction may have therapeutic value in heart disease.
Proteolysis of the Glu441-Ala442 bond in the glycosaminoglycan (GAG) β domain of the versican-V1 variant by a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif (ADAMTS) ...proteases is required for proper embryo morphogenesis. However, the processing mechanism and the possibility of additional ADAMTS-cleaved processing sites are unknown. We demonstrate here that if Glu441 is mutated, ADAMTS5 cleaves inefficiently at a proximate upstream site but normally does not cleave elsewhere within the GAGβ domain. Chondroitin sulfate (CS) modification of versican is a prerequisite for cleavage at the Glu441-Ala442 site, as demonstrated by reduced processing of CS-deficient or chondroitinase ABC-treated versican-V1. Site-directed mutagenesis identified the N-terminal CS attachment sites Ser507 and Ser525 as essential for processing of the Glu441-Ala442 bond by ADAMTS5. A construct including only these two GAG chains, but not downstream GAG attachment sites, was cleaved efficiently. Therefore, CS chain attachment to Ser507 and Ser525 is necessary and sufficient for versican proteolysis by ADAMTS5. Mutagenesis of Glu441 and an antibody to a peptide spanning Thr432-Gly445 (i.e. containing the scissile bond) reduced versican-V1 processing. ADAMTS5 lacking the C-terminal ancillary domain did not cleave versican, and an ADAMTS5 ancillary domain construct bound versican-V1 via the CS chains. We conclude that docking of ADAMTS5 with two N-terminal GAG chains of versican-V1 via its ancillary domain is required for versican processing at Glu441-Ala442. V1 proteolysis by ADAMTS1 demonstrated a similar requirement for the N-terminal GAG chains and Glu441. Therefore, versican cleavage can be inhibited substantially by mutation of Glu441, Ser507, and Ser525 or by an antibody to the region of the scissile bond.
Mutations in the secreted glycoprotein ADAMTSL2 cause recessive geleophysic dysplasia (GD) in humans and Musladin-Lueke syndrome (MLS) in dogs. GD is a severe, often lethal, condition presenting with ...short stature, brachydactyly, stiff skin, joint contractures, tracheal-bronchial stenosis and cardiac valve anomalies, whereas MLS is non-lethal and characterized by short stature and severe skin fibrosis. Although most mutations in fibrillin-1 (FBN1) cause Marfan syndrome (MFS), a microfibril disorder leading to transforming growth factor-β (TGFβ) dysregulation, domain-specific FBN1 mutations result in dominant GD. ADAMTSL2 has been previously shown to bind FBN1 and latent TGFβ-binding protein-1 (LTBP1). Here, we investigated mice with targeted Adamtsl2 inactivation as a new model for GD (Adamtsl2(-/-) mice). An intragenic lacZ reporter in these mice showed that ADAMTSL2 was produced exclusively by bronchial smooth muscle cells during embryonic lung development. Adamtsl2(-/-) mice, which died at birth, had severe bronchial epithelial dysplasia with abnormal glycogen-rich inclusions in bronchial epithelium resembling the cellular anomalies described previously in GD. An increase in microfibrils in the bronchial wall was associated with increased FBN2 and microfibril-associated glycoprotein-1 (MAGP1) staining, whereas LTBP1 staining was increased in bronchial epithelium. ADAMTSL2 was shown to bind directly to FBN2 with an affinity comparable to FBN1. The observed extracellular matrix (ECM) alterations were associated with increased bronchial epithelial TGFβ signaling at 17.5 days of gestation; however, treatment with TGFβ-neutralizing antibody did not correct the epithelial dysplasia. These investigations reveal a new function of ADAMTSL2 in modulating microfibril formation, and a previously unsuspected association with FBN2. Our studies suggest that the bronchial epithelial dysplasia accompanying microfibril dysregulation in Adamtsl2(-/-) mice cannot be reversed by TGFβ neutralization, and thus might be mediated by other mechanisms.
• Combined genetic-pharmacologic ADAMTS1 and ADAMTS5 inactivation in mice.• Degradomics identification of putative ADAMTS1 and ADAMTS5 substrates.• A requirement for versican proteolysis and ...glypican-6 in heart development.• Novel roles for ADAMTS1, ADAMTS5 and glypican-6 in hedgehog signaling in the heart.
Extracellular matrix remodeling mechanisms are understudied in cardiac development and congenital heart defects. We show that matrix-degrading metalloproteases ADAMTS1 and ADAMTS5, are extensively co-expressed during mouse cardiac development. The mouse mutants of each gene have mild cardiac anomalies, however, their combined genetic inactivation to elicit cooperative roles is precluded by tight gene linkage. Therefore, we coupled Adamts1 inactivation with pharmacologic ADAMTS5 blockade to uncover stage-specific cooperative roles and investigated their potential substrates in mouse cardiac development. ADAMTS5 blockade was achieved in Adamts1 null mouse embryos using an activity-blocking monoclonal antibody during distinct developmental windows spanning myocardial compaction or cardiac septation and outflow tract rotation. Synchrotron imaging, RNA in situ hybridization, immunofluorescence microscopy and electron microscopy were used to determine the impact on cardiac development and compared to Gpc6 and ADAMTS-cleavage resistant versican mutants. Mass spectrometry-based N-terminomics was used to seek relevant substrates. Combined inactivation of ADAMTS1 and ADAMTS5 prior to 12.5 days of gestation led to dramatic accumulation of versican-rich cardiac jelly and inhibited formation of compact and trabecular myocardium, which was also observed in mice with ADAMTS cleavage-resistant versican. Combined inactivation after 12.5 days impaired outflow tract development and ventricular septal closure, generating a tetralogy of Fallot-like defect. N-terminomics of combined ADAMTS knockout and control hearts identified a cleaved glypican-6 peptide only in the controls. ADAMTS1 and ADAMTS5 expression in cells was associated with specific glypican-6 cleavages. Paradoxically, combined ADAMTS1 and ADAMTS5 inactivation reduced cardiac glypican-6 and outflow tract Gpc6 transcription. Notably, Gpc6−/− hearts demonstrated similar rotational defects as combined ADAMTS inactivated hearts and both had reduced hedgehog signaling. Thus, versican proteolysis in cardiac jelly at the canonical Glu441-Ala442 site is cooperatively mediated by ADAMTS1 and ADAMTS5 and required for proper ventricular cardiomyogenesis, whereas, reduced glypican-6 after combined ADAMTS inactivation impairs hedgehog signaling, leading to outflow tract malrotation.
Geleophysic dysplasia is a rare, frequently lethal condition characterized by severe short stature with progressive joint contractures, cardiac, pulmonary, and skin anomalies. Geleophysic dysplasia ...results from dominant fibrillin-1 (FBN1) or recessive ADAMTSL2 mutations, suggesting a functional link between ADAMTSL2 and fibrillin microfibrils. Mice lacking ADAMTSL2 die at birth, which has precluded analysis of postnatal limb development and mechanisms underlying the skeletal anomalies of geleophysic dysplasia. Here, detailed expression analysis of Adamtsl2 using an intragenic lacZ reporter shows strong Adamtsl2 expression in limb tendons. Expression in developing and growing bones is present in regions that are destined to become articular cartilage but is absent in growth plate cartilage. Consistent with strong tendon expression, Adamtsl2 conditional deletion in limb mesenchyme using Prx1-Cre led to tendon anomalies, albeit with normal collagen fibrils, and distal limb shortening, providing a mouse model for geleophysic dysplasia. Unexpectedly, conditional Adamtsl2 deletion using Scx-Cre, a tendon-specific Cre-deleter strain, which does not delete in cartilage, also impaired skeletal growth. Recombinant ADAMTSL2 is shown here to colocalize with fibrillin microfibrils in vitro, and enhanced staining of fibrillin-1 microfibrils was observed in Prx1-Cre Adamtsl2 tendons. The findings show that ADAMTSL2 specifically regulates microfibril assembly in tendons and that proper microfibril composition in tendons is necessary for tendon growth. We speculate that reduced bone growth in geleophysic dysplasia may result from external tethering by short tendons rather than intrinsic growth plate anomalies. Taken together with previous work, we suggest that GD results from abnormal microfibril assembly in tissues, and that ADAMTSL2 may limit the assembly of fibrillin microfibrils.
•Adamtsl2 is expressed in several musculoskeletal tissues, such as tendon and muscle, but not in the growth plate.•Deletion of ADAMTSL2 in the limb resulted in a geleophysic dysplasia-like phenotype, e.g. shorter bones with a more severe shortening of the distal skeletal elements.•ADAMTSL2-deficient Achilles tendons are shorter than wild-type tendons.•Conditional deletion of ADAMTSL2 in tendon resulted in tendon shortening and short limb phenotype, suggesting an autonomous role for ADAMTSL2 in tendon growth and a non-autonomous role in regulating bone growth.•ADAMTSL2 deficiency in tendon results in a disarray of tenocytes and an accumulation of fibrillin-1 in the vicnity of tenocytes.•ADAMTSL2 colocalizes with fibrillin microfibrils assembled by cultured fibroblasts.
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