Collagenase-3 (MMP13), a member of the matrix metalloproteinase (MMP) family of neutral endopeptidases, is expressed in the skeleton during embryonic development and is highly overexpressed in human carcinomas and in chondrocytes and synovial cells in rheumatoid arthritis and osteoarthritis. To determine the functional roles of Mmp13, we generated Mmp13-null mice that showed profound defects in growth plate cartilage with markedly increased hypertrophic domains as well as delay in endochondral ossification and formation and vascularization of primary ossification centers. Absence of Mmp13 resulted in significant interstitial collagen accumulation due, in part, to the lack of appropriate collagenase-mediated cleavage that normally occurs in growth plates and primary ossification centers. Cartilaginous growth plate abnormalities persisted in adult mice and phenocopied defects observed in human hereditary chondrodysplasias. Our findings demonstrate a unique role of Mmp13 in skeletal development.collagen ͉ extracellular matrix ͉ vascularization C ollagenases, a group of matrix metalloproteinases (MMPs) that act at neutral pH (1-4), have been postulated to have a role in skeletal development and bone remodeling (5-8). The MMPs are members of a large family of proteinases that have several structural features in common including the presence of a conserved zinc-binding catalytic domain (1-4). Only the products of specific MMP genes, MMP1, -2, -8, -13, and -14, however, have the capacity to cleave native, undenatured, interstitial collagens at a specific helical locus (9-13). Of the collagenases, MMP13 (collagenase-3) has been considered to have an important role in skeletal biology in view of its exclusive presence in the skeleton during embryonic development in cartilaginous growth plates and primary centers of ossification (5-8). MMP13 is also a downstream target of parathyroid hormone (PTH)-related protein (PTHrP) (14) and the transcription factor Osf2͞Cbfa1͞Runx2 in growth plate chondrocytes (15,16). In contrast to humans, where MMP1 may be strongly expressed, e.g., in inflammation, the orthologue of MMP1, McolA (12), is expressed in mice only at low levels.To examine possible functional roles of collagenases during skeletal development in vivo, we targeted a null mutation to the Mmp13 gene in mice. Our targeting strategy resulted in splicing out exon 5 that encodes the zinc-binding residues in the catalytic domain. As described here, deletion of functional Mmp13 had profound effects on skeletal development. In Mmp13 Ϫ/Ϫ embryos compared with WT embryos, the growth plates were strikingly lengthened, a defect ascribable predominantly to a delay in terminal events in the growth plates, with failure to resorb collagens, as well as a delay in ossification at the primary centers. Materials and MethodsGeneration of Mmp13 ؊/؊ Mice. We isolated two Mmp13 genomic clones from a 129͞J1 library to construct the knockout vector.The first was a BamHI͞SalI fragment that spanned from Ϸ3.4 kb of promoter sequence through the first...
Type III collagen is a fibrillar forming collagen comprising three ␣1(III) chains and is expressed in early embryos and throughout embryogenesis. In the adult, type III collagen is a major component of the extracellular matrix in a variety of internal organs and skin. Mutations in the COL3A1 gene have been implicated as a cause of type IV Ehlers-Danlos syndrome, a disease leading to aortic rupture in early adult life. To directly study the role of Col3a1 in development and disease, we have inactivated the Col3a1 gene in embryonic stem cells by homologous recombination. The mutated allele was transmitted through the mouse germ line and homozygous mutant animals were derived from heterozygous intercrosses. About 10% of the homozygous mutant animals survived to adulthood but have a much shorter life span compared with wild-type mice. The major cause of death of mutant mice was rupture of the major blood vessels, similar to patients with type IV Ehlers-Danlos syndrome. Ultrastructural analysis of tissues from mutant mice revealed that type III collagen is essential for normal collagen I fibrillogenesis in the cardiovascular system and other organs.
SUMMARY A large and diverse array of chemoattractants control leukocyte trafficking, but how these apparently redundant signals collaborate in vivo is still largely unknown. We previously demonstrated an absolute requirement for the lipid chemoattractant leukotriene B4 (LTB4) and its receptor BLT1 for neutrophil recruitment into the joint in autoantibody-induced arthritis. We now demonstrate that BLT1 is required for neutrophils to deliver IL-1 into the joint to initiate arthritis. IL-1-expressing neutrophils amplify arthritis through the production of neutrophil-active chemokines from synovial tissue cells. CCR1 and CXCR2, two neutrophil chemokine receptors, operate non-redundantly to sequentially control the later phase of neutrophil recruitment into the joint and mediate all neutrophil chemokine activity in the model. Thus, we have uncovered a complex sequential relationship involving unique contributions from the lipid mediator LTB4, the cytokine IL-1, and CCR1 and CXCR2 chemokine ligands that are all absolutely required for effective neutrophil recruitment into the joint.
Abstract. Degradation of type I collagen, the most abundant collagen, is initiated by collagenase cleavage at a highly conserved site between Gly775 and I1e776 of the od(I) chain. Mutations at or around this site render type I collagen resistant to collagenase digestion in vitro. We show here that mice carrying a collagenaseresistant mutant Colla-1 transgene die late in embryogenesis, ascribable to overexpression of the transgene, since the same mutation introduced into the endogenous Colla-1 gene by gene targeting permitted normal development of mutant mice to young adulthood. With increasing age, animals carrying the targeted mutation developed marked fibrosis of the dermis similar to that in human scleroderma. Postpartum involution of the uterus in the mutant mice was also impaired, with persistence of collagenous nodules in the uterine wall. AIthough type I collagen from the homozygous mutant mice was resistant to cleavage by human or rat fibroblast collagenases at the helical site, only the rat collagenase cleaved collagen trimers at an additional, novel site in the nonhelical N-telopeptide domain. Our results suggest that cleavage by murine collagenase at the N-telopeptide site could account for resorption of type I collagen during embryonic and early adult life. During intense collagen resorption, however, such as in the immediate postpartum uterus and in the dermis later in life, cleavage at the helical site is essential for normal collagen turnover. Thus, type I collagen is degraded by at least two differentially controlled mechanisms involving collagenases with distinct, but overlapping, substrate specificities.T YPE I collagen is among the most abundant components of the extracellular matrix of many tissues, particularly in skin, tendons, ligaments, uterus, large blood vessels and bone. The helical trimeric molecules of type I collagen comprise two al(I) chains and one ~2(I) chain, encoded by two separate genes, Colla-1 and Colla-2, respectively (50). In mice, type I collagen is first synthesized in the mesenchymal stroma of the head, heart and somites at day 8 of gestation (E8) and its production continues throughout development and postnatal life (27). It has been demonstrated that type I collagen is critical for bone development (9, 10), hematopoiesis (29, 37), integrity of the vascular system (29) and for mesenchymal-epithelial induction in organogenesis (6).The collagen content of different tissues during development and in adult animals is tightly regulated by coordinated processes of synthesis and degradation (2,8,57 teoporosis or inflammatory joint diseases) or excessive deposition (e.g. pulmonary fibrosis or scleroderma) (32, 39). The degradation of type I collagen requires the action of specific collagenases since native, triple-helical molecules are resistant to attack by proteolytic enzymes at 37°C and neutral pH. The collagenases are members of a family of proteinases, the metalloproteinases or matrixins, all of which contain a catalytic zinc-binding domain that includes the sequence moti...
Clearance of apoptotic cells is critical for control of tissue homeostasis however the full range of receptor(s) on phagocytes responsible for recognition of apoptotic cells remains to be identified. Here we show that dendritic cells (DCs), macrophages and endothelial cells use scavenger receptor type F family member 1 (SCARF1) to recognize and engulf apoptotic cells via C1q. Loss of SCARF1 impairs uptake of apoptotic cells. Consequently, in SCARF1-deficient mice, dying cells accumulate in tissues leading to a lupus-like disease with the spontaneous generation of autoantibodies to DNA-containing antigens, immune cell activation, dermatitis and nephritis. The discovery of SCARF1 interactions with C1q and apoptotic cells provides insights into molecular mechanisms involved in maintenance of tolerance and prevention of autoimmune disease.Clearance of apoptotic cells is one of the most important processes of the immune system and is necessary for the homeostatic maintenance of healthy tissues and removal of infected or damaged cells [1][2][3] . Several types of cells are capable of apoptotic cell uptake, including Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms * Correspondence and requests for materials should be addressed to: T.K. M. means.terry@mgh.harvard.edu, 617-726-6497). Note: Supplementary information is available on the Nature Immunology website. AUTHORS CONTRIBUTIONS T.K.M., Z.G.R., and W.F.P. III planned the research, analyzed and interpreted data and wrote the manuscript. Z.G.R. performed most of the experiments. C.J.B helped with mouse breeding and genotyping. W.F.P. III, A.P., and T.I. performed and analyzed ELISAs, PCR, and mouse pathology. N.H. and A.D.L. analyzed and interpreted data. T.K.M., J.E.K., and M.H.B. contributed to the generation of SCARF1-deficient mice. All authors participated in editing the manuscript into its final form. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript both professional scavengers (macrophages, DCs) and non-professional phagocytes (fibroblasts, endothelial, and epithelial cells). In vivo, phagocytes are responsible for the rapid removal of dying cells before necrosis, a post-apoptotic stage accompanied by loss of membrane integrity and leakage of noxious intracellular molecules into the surrounding tissues 4,5 . Phagocyte engulfment of apoptotic cells occurs via an immunologically silent process by activating immunosuppressive pathways and the production of anti-inflammatory cytokines to prevent an immune response against self-antigens 6 . Consequently, defects in recognition and/or engulfment of apoptotic cells can lead to chronic inflammatory diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis, glomerulonephritis and at...
Collagenase (matrix metalloproteinase 1) cleaves type I, II, and III collagen helices at a specific site between Gly-Ile or Gly-Leu bonds (residues 775 and 776, Pj-P1'). To understand the mechanism of collagen processing, mutations around the cleavage site have been introduced into the cloned murine proal(I) collagen (Collal) The collagenases cleave native type I, II, and III collagens by hydrolyzing the peptide bond between residues Gly-Ile (or Leu) located at residues 775 and 776 of the helical portion of the al(I) chain to yield a larger three-quarter length fragment (TCA) and a smaller one-quarter length fragment (TCB) (1,4,17). The region around the cleavage site is more hydrophobic than other parts of the collagen molecule and deficient in the hydroxyproline and proline residues that stabilize the triple helix. Our 5888The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Multiple EGF-like domains 10 (Megf10) is a class F scavenger receptor (SR-F3) expressed on astrocytes and myosatellite cells, and recessive mutations in humans result in early-onset myopathy, areflexia, respiratory distress, and dysphagia (EMARDD). Here we report that Megf10-deficient mice have increased apoptotic cells in the developing cerebellum and have impaired phagocytosis of apoptotic cells by astrocytes ex vivo. We also report that cells transfected with Megf10 gain the ability to phagocytose apoptotic neurons and that Megf10 binds with high affinity to C1q, an eat-me signal for apoptotic cells. In contrast, cells expressing Megf10 with EMARDD mutations have impaired apoptotic cell clearance and impaired binding to C1q. Our studies reveal that Megf10 is a receptor for C1q and identify a novel role for Megf10 in clearance of apoptotic cells in the mammalian developing brain with potential relevance to EMARDD patients and other CNS disorders.
Parathyroid hormone (PTH) induces hypercalcemia in part through increasing bone resorption mediated by osteoclasts. Receptors for PTH are present not on osteoclasts, however, but on mesenchymal cells of the osteoblast lineage and stromal cells in the bone marrow (1). PTH must therefore act directly on these mesenchymal cells, which then -through direct cell-cell contact mediated by cellbound ligands such as osteoclast differentiation factor (ODF) and/or production of soluble ligands -modulates the activity of existing osteoclasts and the differentiation of osteoclasts from precursor cells (2, 3).Shortly after the discovery of the animal collagenases by Gross and Lapière (4), Walker et al. (5) found that bones removed from mice injected with doses of PTH sufficient to elevate serum calcium levels by ∼4 mg/dl produce collagenase activity at levels much higher than in bones removed from uninjected mice. The time course of the effects on inducing hypercalcemia and collagenase production was different in that increased collagenase production was not detected until several hours after serum calcium levels reached their peak (6). It had also been shown that the sustained hypercalcemia induced by PTH was obviated by inhibitors of mRNA synthesis (actinomycin D) (7,8) or protein synthesis (puromycin) (9), suggesting that, at least in part, PTH-induced hypercalcemia is dependent upon the synthesis of a protein in bone; collagenase was a candidate. In subsequent studies from several laboratories it was shown that in organ cultures of bone fragments, collagenase was released into the ambient medium and collagenase production could be stimulated by PTH (10-14). Later, it was possible to clone a collagenase gene from a cDNA library prepared from a rat osteogenic sarcoma cell line stimulated with PTH (15). A mouse collagenase cDNA was subsequently cloned (16) using the rat cDNA probe (15), and it was determined that the rodent enzymes had only ∼50% amino acid sequence similarity to human collagenase-1 (matrix metalloproteinase-1 [MMP-1]) but had high similarity to what was later identified as collagenase-3 (MMP-13) in humans (17). Osteoclasts, in addition to producing cysteine proteinases, also produce MMPs such as the 92-kDa gelatinase (gelatinase B) (18) and one of the membrane-bound (MT) MMPs, . In all but a few reported studies (e.g., ref. 20), however, it has not been possible to identify the expression of specific collagenases in osteoclasts using cDNA or cRNA probes. In contrast, several investigators have been readily able to measure collagenase produced by osteoblasts or stromal fibroblasts from different species, as described above, either constitutively or induced by several ligands, including PTH. When osteoblasts are exposed to PTH, they start producing collagenase and stop synthesizing collagen (21).During normal embryonic development, collagenase is expressed in cells (osteoblasts, stromal cells) in the bone shaft and in hypertrophic chondrocytes of the distal growth plate (22-25). More proximal hypertrophic cho...
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