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Loss of MMP-2 in murine osteoblasts upregulates osteopontin and bone sialoprotein expression in a circuit regulating bone homeostasis.

ABSTRACT: Multicentric osteolysis with arthropathy (MOA; MIM 605156) is an inherited osteolyses and arthritis syndrome resulting from loss of matrix metalloproteinase 2 (MMP-2). We recently demonstrated that Mmp2(-/-) mice represent a unique model for the study of the human disease, sharing many features of the human syndrome including skeletal dysplasia and defects in osteoblast behavior. We therefore sought to explore the secondary molecular effects of MMP-2 loss, which coexist with the underlying skeletal and osteoblast phenotypes. We used quantitative real-time RT-PCR (qRT-PCR) to measure osteoblast-related gene expression through ex vivo osteoblast differentiation of bone marrow stromal cells (BMSC) from Mmp2(-/-) and Mmp2(+/+) mice. We used western blot to measure osteopontin (OPN) serum levels and immunohistochemical staining to examine bone expression. MMP-2 expression was inhibited in SaOS2 cells using siRNA, and decreased MMP-2 expression at both RNA and protein levels was confirmed by qRT-PCR and western blot, respectively. Mmp2(-/-) BMSC induced to differentiate into osteoblasts were shown to significantly upregulate OPN and bone sialoprotein (BSP) expression levels compared with controls. Transcriptional upregulation was maintained in vivo, as demonstrated by increased levels of OPN in serum and bone in Mmp2(-/-) mice. These effects are generalizable because siRNA-mediated inhibition in cultured cells also upregulated OPN and BSP. OPN and BSP are known to affect MMP-2 expression and activity but have not previously been shown to be regulated by MMP-2. Identification of this newly defined circuitry provides insight into the potential molecular landscape underlying the MOA phenotype and highlights a pathway that might play a role in normal bone homeostasis.

SUBMITTER: Mosig RA

PROVIDER: S-EPMC3597021 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Loss of MMP-2 in murine osteoblasts upregulates osteopontin and bone sialoprotein expression in a circuit regulating bone homeostasis.

Mosig Rebecca A RA Martignetti John A JA

Disease models & mechanisms 20120823 2

Multicentric osteolysis with arthropathy (MOA; MIM 605156) is an inherited osteolyses and arthritis syndrome resulting from loss of matrix metalloproteinase 2 (MMP-2). We recently demonstrated that Mmp2(-/-) mice represent a unique model for the study of the human disease, sharing many features of the human syndrome including skeletal dysplasia and defects in osteoblast behavior. We therefore sought to explore the secondary molecular effects of MMP-2 loss, which coexist with the underlying skele ...[more]

PMID: 22917927

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Project description:Bone and dentin are mineralized extracellular matrices produced by osteoblasts and odontoblasts, respectively, and their major organic portion is type I collagen. Dentinogenesis Imperfecta (DGI) is one of the most common clinically- and genetically-based disturbances of dentin formation, causing irreversible dentin defects. Among several types of DGI, patients with DGI type II exhibit opalescent dentin with partial or complete pulp obliteration. It has been previously reported that the non-sense mutation (c.133C>T) in Dentin Sialophosphoprotein (DSPP) was identified in DGI type II patients at glutamine residue 45, resulting in the premature stop codon (p.Q45X). DSPP is known to be synthesized as a single gene product and further processed at Gly462-Asp463, resulting in the production of Dentin Sialoprotein (DSP) and Dentin Phosphoprotein (DPP). We hypothesized that the shorter form (Q45X) of N-terminal Dentin Sialoprotein (N-DSP) may cause over-production of type I collagen protein as obliterated pulp is occupied by dentin. To test this hypothesis, we generated mouse recombinant Glutathione-S-Transferase (GST)-N-DSP fusion protein, and the effect of GST-N-DSP was investigated in calvarial bone explant culture and MC3T3-E1 osteoblastic culture systems. Here we show that a significant increase in calvarial bone formation is observed by GST-N-DSP. GST-N-DSP accelerates MC3T3-E1 osteoblast cell growth and proliferation and subsequent osteoblast differentiation by inducing the expression of certain osteogenic markers such as type I collagen, Runx2, Osterix and ATF4. Interestingly, GST-N-DSP significantly enhances dentinogenesis marker gene expression including Dspp and Dmp1 gene expression in non-odontogenic MC3T3-E1 cells. To rule out any artificial effect of GST-tag, we also used the synthetic peptide of N-DSP and confirmed the results of N-DSP peptide were essentially similar to those of GST-N-DSP. Taken together, our data suggest that N-DSP promotes bone formation by accelerating osteoblast cell proliferation and subsequent osteoblast differentiation accompanied by marked up-regulation of the dentin matrix markers, such as Dspp and Dmp1 genes.

Project description:Bone sialoprotein (BSP) is an extracellular matrix (ECM) protein associated with mineralized tissues, particularly bone and cementum. BSP includes functional domains implicated in collagen binding, hydroxyapatite nucleation, and cell signaling, although its function(s) in osteoblast and osteoclast differentiation and function remain incompletely understood. Genetic ablation of BSP in Ibsp knockout (Ibsp-/-) mice results in developmental bone mineralization and remodeling defects, with alveolar bone more severely affected than the femurs and tibias of the postcranial skeleton. The role of BSP in alveolar bone healing has not been studied. We hypothesized that BSP ablation would cause defective alveolar bone healing. We employed a maxillary first molar extraction socket healing model in 42-d postnatalIbsp-/- and wild-type (WT) control mice. Tissues were collected at 0, 7, 14, 21, and 56 d postprocedure (dpp) for analysis by micro-computed tomography (microCT), histology, in situ hybridization (ISH), immunohistochemistry (IHC), and quantitative polymerase chain reaction (qPCR) array. As expected, alveolar bone healing progressed in WT mice with increasing bone volume fraction (BV/TV), bone mineral density (BMD), and tissue mineral density (TMD), transitioning from woven to mature bone from 7 to 56 dpp. Ibsp messenger RNA (mRNA) and BSP protein were strongly expressed during alveolar bone healing in parallel with other osteogenic markers. Compared to WT, Ibsp-/- mice exhibited 50% to 70% reduced BV/TV and BMD at all time points, 7% reduced TMD at 21 dpp, abnormally increased Col1a1 and Alpl mRNA expression, and persistent presence of woven bone and increased bone marrow in healing sockets. qPCR revealed substantially dysregulated gene expression in alveolar bone of Ibsp-/- versus WT mice, with significantly disrupted expression of 45% of tested genes in functional groups, including markers for osteoblasts, osteoclasts, mineralization, ECM, cell signaling, and inflammation. We conclude that BSP is a critical and nonredundant factor for alveolar bone healing, and its absence disrupts multiple major pathways involved in appropriate healing.

Dataset Information

Loss of MMP-2 in murine osteoblasts upregulates osteopontin and bone sialoprotein expression in a circuit regulating bone homeostasis.

Publications

Loss of MMP-2 in murine osteoblasts upregulates osteopontin and bone sialoprotein expression in a circuit regulating bone homeostasis.

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