MMP-13 stimulates osteoclast differentiation and activation in tumour breast bone metastases

INTRODUCTION: The increased bone degradation in osteolytic metastases depends on stimulation of mature osteoclasts and on continuous differentiation of new pre-osteoclasts. Metalloproteinases (MMP)-13 is expressed in a broad range of primary malignant tumours and it is emerging as a novel biomarker. Recent data suggest a direct role of MMP-13 in dissolving bone matrix complementing the activity of MMP-9 and other enzymes. Tumour-microenvironment interactions alter gene expression in malignant breast tumour cells promoting osteolytic bone metastasis. Gene expression profiles revealed that MMP-13 was among the up-regulated genes in tumour-bone interface and its abrogation reduced bone erosion. The precise mechanism remained not fully understood. Our purpose was to further investigate the mechanistic role of MMP-13 in bone osteolytic lesions. METHODS: MDA-MB-231 breast cancer cells that express MMP-13 were used as a model for in vitro and in vivo experiments. Conditioned media from MDA-MB-231 cells were added to peripheral blood mononuclear cultures to monitor pre-osteoclast differentiation and activation. Bone erosion was evaluated after injection of MMP-13-silenced MDA-MB-231 cells into nude mice femurs. RESULTS: MMP-13 was co-expressed by human breast tumour bone metastases with its activator MT1-MMP. MMP-13 was up-regulated in breast cancer cells after in vitro stimulation with IL-8 and was responsible for increased bone resorption and osteoclastogenesis, both of which were reduced by MMP inhibitors. We hypothesized that MMP-13 might be directly involved in the loop promoting pre-osteoclast differentiation and activity. We obtained further evidence for a direct role of MMP-13 in bone metastasis by a silencing approach: conditioned media from MDA-MB-231 after MMP-13 abrogation or co-cultivation of silenced cells with pre-osteoclast were unable to increase pre-osteoclast differentiation and resorption activity. MMP-13 activated pre-MMP-9 and promoted the cleavage of galectin-3, a suppressor of osteoclastogenesis, thus contributing to pre-osteoclast differentiation. Accordingly, MMP-13 abrogation in tumour cells injected into the femurs of nude mice reduced the differentiation of TRAP positive cells in bone marrow and within the tumour mass as well as bone erosion. CONCLUSIONS: These results indicate that within the inflammatory bone microenvironment MMP-13 production was up-regulated in breast tumour cells leading to increased pre-osteoclast differentiation and their subsequent activation

Endostatin inhibits VEGF-A induced osteoclastic bone resorption in vitro

BACKGROUND: Endostatin is a C-terminal fragment of collagen XVIII which is a component of basement membranes with the structural properties of both collagens and proteoglycans. Endostatin has a major role in angiogenesis which is intimately associated with bone development and remodeling. Signaling between the endothelial cells and the bone cells, for example, may have a role in recruitment of osteoclastic precursor cells. Our study aims at exploring a possibility that endostatin, either as a part of basement membrane or as a soluble molecule, may control osteoclastogenesis and osteoclastic bone resorption in vitro. METHODS: Rat pit formation assay was employed in order to examine the effect of endostatin alone or in combination with vascular endothelial growth factor-A (VEGF-A) on bone resorption in vitro. Effect of these agents on osteoclast differentiation in vitro was also tested. Osteoclastogenesis and the number of osteoclasts were followed by tartrate resistant acid phosphatase (TRACP) staining and resorption was evaluated by measuring the area of excavated pits. RESULTS: Endostatin inhibited the VEGF-A stimulated osteoclastic bone resorption, whereas endostatin alone had no effect on the basal resorption level in the absence of VEGF-A. In addition, endostatin could inhibit osteoclast differentiation in vitro independent of VEGF-A. CONCLUSION: Our in vitro data indicate that collagen XVIII/endostatin can suppress VEGF-A induced osteoclastic bone resorption to the basal level. Osteoclastogenesis is also inhibited by endostatin. The regulatory effect of endostatin, however, is not critical since endostatin alone does not modify the basal bone resorption

Disease modifying and antiangiogenic activity of 2-Methoxyestradiol in a murine model of rheumatoid arthritis

<p>Abstract</p> <p>Background</p> <p>A critical component of disease progression in rheumatoid arthritis (RA) involves neovascularization associated with pannus formation. 2-methoxyestradiol (2ME2) is a naturally occurring molecule with no known physiologic function, although at pharmacologic concentrations it has antiproliferative and antiangiogenic activities. We investigated the impact of orally administered 2ME2 on the initiation and development of proliferative synovitis using the anti-collagen monoclonal antibodies (CAIA) model.</p> <p>Methods</p> <p>Severe polyarticular arthritis was induced in Balb/c female mice by administration of 2 mg of a monoclonal antibody cocktail intravenously into the tail vein of mice. Twenty-four hours following monoclonal antibody administration, mice were injected with 25 μg of LPS (<it>E. coli </it>strain 0111:B4) via the intraperitoneal route. Treatment with 2ME2 (100, 75, 50, 25, 10, 1 mg/kg, p.o., daily), or vehicle control began 24 hrs following LPS challenge and continued to day 21. Hind limbs were harvested, sectioned and evaluated for DMARD activity and general histopathology by histomorphometric analysis and immunohistochemistry (vWF staining). In a separate study, different dosing regimens of 2ME2 (100 mg/kg; q.d. <it>vs </it>q.w. <it>vs </it>q.w. × 2) were evaluated. The effect of treatment with 2ME2 on gene expression of inflammatory cytokines and angiogenic growth factors in the joint space was evaluated 5 and 14 days after the induction of arthritis.</p> <p>Results</p> <p>Mice treated with 2ME2 beginning 24 hours post anti-collagen monoclonal antibody injection, showed a dose-dependent inhibition in mean arthritic scores. At study termination (day 21), blinded histomorphometric assessments of sectioned hind limbs demonstrated decreases in synovial inflammation, articular cartilage degradation, pannus formation, osteoclast activity and bone resorption. At the maximal efficacious dosing regimen (100 mg/kg/day), administration of 2ME2 resulted in total inhibition of the study parameters and prevented neovascularization into the joint. Examination of gene expression on dissected hind limbs from mice treated for 5 or 14 days with 2ME2 showed inhibition of inflammatory cytokine message for IL-1β, TNF-α, IL-6 and IL-17, as well as the angiogenic cytokines, VEGF and FGF-2.</p> <p>Conclusion</p> <p>These data demonstrate that in the CAIA mouse model of RA, 2ME2 has disease modifying activity that is at least partially attributable to the inhibition of neovascular development. Further, the data suggests new mechanistic points of intervention for 2ME2 in RA, specifically inhibition of inflammatory mediators and osteoclast activity.</p

Sequential morphological characteristics of murine fetal liver hematopoietic microenvironment in Swiss Webster mice

Embryonic hematopoiesis occurs via dynamic development with cells migrating into various organs. Fetal liver is the main hematopoietic organ responsible for hematopoietic cell expansion during embryologic development. We describe the morphological sequential characteristics of murine fetal liver niches that favor the settlement and migration of hematopoietic cells from 12 days post-coitum (dpc) to 0 day post-partum. Liver sections were stained with hematoxylin and eosin, Lennert’s Giemsa, Sirius Red pH 10.2, Gomori’s Reticulin, and Periodic Acid Schiff/Alcian Blue pH 1.0 and pH 2.5 and were analyzed by bright-field microscopy. Indirect imunohistochemistry for fibronectin, matrix metalloproteinase-1 (MMP-1), and MMP-9 and histochemistry for naphthol AS-D chloroacetate esterase (NCAE) were analyzed by confocal microscopy. The results showed that fibronectin was related to the promotion of hepatocyte and trabecular differentiation; reticular fibers did not appear to participate in fetal hematopoiesis but contributed to the physical support of the liver after 18 dpc. During the immature phase, hepatocytes acted as the fundamental stroma for the erythroid lineage. The appearance of myeloid cells in the liver was related to perivascular and subcapsular collagen, and NCAE preceded MMP-1 expression in neutrophils, an occurrence that appeared to contribute to their liver evasion. Thus, the murine fetal liver during ontogenesis shows two different phases: one immature and mainly endodermic (<14 dpc) and the other more developed (endodermic-mesenchymal; >15 dpc) with the maturation of hepatocytes, a better definition of trabecular pattern, and an increase in the connective tissue in the capsule, portal spaces, and liver parenchyma. The decrease of hepatic hematopoiesis (migration) coincides with hepatic maturation

Matrix metalloproteinases (MMP) and cathepsin K contribute differently to osteoclastic activities

Udgivelsesdato: 2003-Aug-15The best established proteolytic event of osteoclasts is bone matrix solubilization by the cysteine proteinase cathepsin K. Here, however, we draw the attention on osteoclastic activities depending on matrix metalloproteinases (MMPs). We discuss the observations supporting that MMPs contribute significantly to bone matrix solubilization in specific areas of the skeleton and in some developmental and pathological situations. Our discussion takes into account (1) the characteristics of the bone remodeling persisting in the absence of cathepsin K, (2) the ultrastructure of the resorption zone in response to inactivation of MMPs and of cathepsin K in different bone types, (3) bone resorption levels in MMP knockout mice compared to wild-type mice, (4) the identification of MMPs in osteoclasts and surrounding cells, and (5) the effect of different bone pathologies on the serum concentrations of specific collagen fragments believed to discriminate between cathepsin K and MMP cleavage. Next, we provide evidence that MMPs are very critical for osteoclast migration, thereby controlling also the cell-matrix interactions required for cell attachment/detachment. The evidence supporting this role is based on a model of osteoclast recruitment in primitive long bones, an assay of osteoclast invasion through collagen gel, and the effect of proteinase inhibitors/knockouts in these models. Furthermore, we mention observations indicating a role of MMPs in initiation of bone resorption. Finally, we emphasize the many distinct ways MMPs may alter focally the extracellular environment thereby regulating the osteoclast behavior. Although the understanding of MMPs in osteoclast biology is rapidly expanding, it is suspected that important roles remain to be discovered

Matrix metalloproteinase 9 and vascular endothelial growth factor are essential for osteoclast recruitment into developing long bones.

Bone development requires the recruitment of osteoclast precursors from surrounding mesenchyme, thereby allowing the key events of bone growth such as marrow cavity formation, capillary invasion, and matrix remodeling. We demonstrate that mice deficient in gelatinase B/matrix metalloproteinase (MMP)-9 exhibit a delay in osteoclast recruitment. Histological analysis and specialized invasion and bone resorption models show that MMP-9 is specifically required for the invasion of osteoclasts and endothelial cells into the discontinuously mineralized hypertrophic cartilage that fills the core of the diaphysis. However, MMPs other than MMP-9 are required for the passage of the cells through unmineralized type I collagen of the nascent bone collar, and play a role in resorption of mineralized matrix. MMP-9 stimulates the solubilization of unmineralized cartilage by MMP-13, a collagenase highly expressed in hypertrophic cartilage before osteoclast invasion. Hypertrophic cartilage also expresses vascular endothelial growth factor (VEGF), which binds to extracellular matrix and is made bioavailable by MMP-9 (Bergers, G., R. Brekken, G. McMahon, T.H. Vu, T. Itoh, K. Tamaki, K. Tanzawa, P. Thorpe, S. Itohara, Z. Werb, and D. Hanahan. 2000. Nat. Cell Biol. 2:737-744). We show that VEGF is a chemoattractant for osteoclasts. Moreover, invasion of osteoclasts into the hypertrophic cartilage requires VEGF because it is inhibited by blocking VEGF function. These observations identify specific actions of MMP-9 and VEGF that are critical for early bone development

The N-Terminal Cleavage of Chondromodulin-I in Growth-Plate Cartilage at the Hypertrophic and Calcified Zones during Bone Development

Chondromodulin-I (ChM-I) is a 20–25 kDa anti-angiogenic glycoprotein in cartilage matrix. In the present study, we identified a novel 14-kDa species of ChM-I by immunoblotting, and purified it by immunoprecipitation with a newly raised monoclonal antibody against ChM-I. The N-terminal amino acid sequencing indicated that it was an N-terminal truncated form of ChM-I generated by the proteolytic cleavage at Asp(37)-Asp(38). This 14-kDa ChM-I was shown by the modified Boyden chamber assay to have very little inhibitory activity on the VEGF-A-induced migration of vascular endothelial cells in contrast to the intact 20–25 kDa form of ChM-I (ID(50) = 8 nM). Immunohistochemistry suggested that 20–25 kDa ChM-I was exclusively localized in the avascular zones, i.e. the resting, proliferating, and prehypertrophic zones, of the cartilaginous molds of developing long bone, whereas the 14-kDa form of ChM-I was found in hypertrophic and calcified zones. Immunoblotting demonstrated that mature growth-plate chondrocytes isolated from rat costal cartilage actively secrete ChM-I almost exclusively as the intact 20–25 kDa form into the medium in primary culture. Taken together, our results suggest that intact 20–25 kDa ChM-I is stored as a component of extracellular matrix in the avascular cartilage zones, but it is inactivated by a single N-terminal proteolytic cleavage in the hypertrophic zone of growth-plate cartilage

Osteocyte-induced angiogenesis via VEGF-MAPK-dependent pathways in endothelial cells

Recently, it has been suggested osteocytes control the activities of bone formation (osteoblasts) and resorption (osteoclast), indicating their important regulatory role in bone remodelling. However, to date, the role of osteocytes in controlling bone vascularisation remains unknown. Our aim was to investigate the interaction between endothelial cells and osteocytes and to explore the possible molecular mechanisms during angiogenesis. To model osteocyte/endothelial cell interactions, we co-cultured osteocyte cell line (MLOY4) with endothelial cell line (HUVECs). Co-cultures were performed in 1:1 mixture of osteocytes and endothelial cells or by using the conditioned media (CM) transfer method. Real-time cell migration of HUVECs was measured with the transwell migration assay and xCELLigence system. Expression levels of angiogenesis- related genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of vascular endothelial growth factor (VEGF) and mitogen-activated phosphorylated kinase (MAPK) signaling were monitored by western blotting using relevant antibodies and inhibitors. During the bone formation, it was noted that osteocyte dendritic processes were closely connected to the blood vessels. The CM generated from MLOY4 cells-activated proliferation, migration, tube-like structure formation, and upregulation of angiogenic genes in endothelial cells suggesting that secretory factor(s) from osteocytes could be responsible for angiogenesis. Furthermore, we identified that VEGF secreted from MLOY4-activated VEGFR2–MAPK–ERK-signaling pathways in HUVECs. Inhibiting VEGF and/or MAPK–ERK pathways abrogated osteocyte-mediated angiogenesis in HUVEC cells. Our data suggest an important role of osteocytes in regulating angiogenesis