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Extracellular nucleotides enhance parathyroid hormone-stimulated bone resorption
578
Abstracts
77
Bone Vol. 17, No. 6 December 1995:557-596
79
EXTRACELLULAR NUCLEOTIDES ENHANCE PARATHYROID HORMONE-STIMULATED BONE RESORPTION. W.B.Bowler and LA.Galla~her. Dept. Human Anatomy and Cell Biology, The University of Liverpool, Liverpool, L69 3BX, U.K.
CATHEPSIN O, A NOVEL OSTEOCLAST CYSTEINE PROTEASE, IS EXPRESSED IN GIANT CELLS OF OSTEOARTHRITIC SYNOVIUM. R. A. Dodds. J. R. Connor. F. Drake and Maxine Gowen, SmithKline Beecham Pharmaceuticals, Department of Cellular Biochemistry, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406, USA.
The complex process of bone remeclelling is controlled by many local and systemic factors, some of which have well documented effects on bone forming and or resorbing cells such as parathyroid hormone (PTH). However, the effects of extraceilular nucleotides on bone cells remain relatively unexplored. We have previously demonstrated that extracellular nucleotides interact with at least two purinoceptor subtypes expressed by bone cells, the result of this interaction being an elevation of [Ca2+]i and induction of the immediate early response gene c-los. In addition, we have demonstrated that extraceUular nucleotides can synergize with PTH to elevate c-fos expression in $aos-2 cells. In this study, we investigated whether extracellular nucleotides and PTH can interact to influence the functional behaviour of bone cells. We focused on the important PTH mediated response of bone resorption. Following PTH and nucleotide stimulation, we quantified resorption using the in vitro "bone slice" assay. Nucleotide agonists alone had no significant effect on resorption, while PTH in combination with ATP or UTP both significantly increased bone resorption above vehicle and PTH. Other purinergic agonists tested, ADP and 2-MeThio ATP, had no significant effects on PTH-induced bone resorption. We propose that extracellular nucleotides present in the bone micro-environment as a result of tissue injury, mechanical stress or physiological release, are capable of influencing bone cells and controlling the remodelling process through an interaction with other factors. These data further suggest a major role for nucleotides in controlling bone cell function.
Multinucleated giant cells (MGC) are a documented feature of diseased synovial tissue and inflammatory tissues. Evidence suggests that they are of macrophaga origin and may share the same precursor cell as the osteoclast (Oc), although morphological, functional, and phenotypic differences also support the contention for separate differentation pathways. We recently identified a novel cysteine protease (cathepsin O; Drake et al, J Bone Min Res 9: $177, 1994) that appears to be osteoclast specific. Thus, the aim of this study was to investigate the expression of cathepsin O mRNA, together with cathepsins L, S and B, in the MGCs found in osteoarthritic reparative connective tissue and associated inflammatory synovium and compare their expression with that of Ocs in the osteophytes within the same tissue. Sections were processed for in situ hybridization; serial sections were reacted for TRAP and nonspecific esterase (NSE) activity, selective markers for the Ocs and cells of the macrophagelmonocytelineage respectively. At sites of bone debris removal, TRAP*, NSE MGCs and mononuclear cells (monos) were detected in the intimal layer of the inflammatory synovium and within the adjacent osteogenic connective tissue at sites of intreamembmnous bone formation. They formed in clusters of NSE +, T R A P monos often containing azurophilic granules (a characteristic feature of phagocytes). These phagocytic-like monos expressed cathepsins O, L, B and B mRNA. However, the TRAP*cells expressed only cathepsin O mRNA. As previously described, Ocs andTRAP * monos at sites of osteophytic bone remodelling expressed only cathepsin O mRNA. Our data suggest that MGCs that form by the fusion of tissue macrophages (specific for calcified tissues) may differentiate towards a more Oe phenotype. This includes the expression of the novel cysteina protease, cathepsin O, and the loss of cathepsins L, S, and B expression. In support of this contention, we previously demonstrated that MGCs stained positively with 2 murine anti-human osteoclast monoclonal antibodies, namely 23C6 (vitronectin) and C35 (osteoclast selective). Altematively, the NSE * monos located within the inflammatory connective tissues (derived from cells from the periosteal-chondrosynovial junction) may represent undifferentiated myeloid-monocytic cells that ultimately differentiate into Ocs at sites of osteophytic bone formation. Thus, the fact that MGSs express classical ostaoclast phenotypic markers in a bone environment implies that these cells may in fact be true osteoclasts.
78
80
METALLOPROTEINASES EXPRESSION BY HUMAN OSTEOCLAST-LIKE CELL LINES. S. Colucci, M. Grano, C. Moil, M. Mastrugiacomo, A. Zambonin Zallone. Institute of Human Anatomy University of Bari, Italy. Bone resorption involves the simultaneous removal of both mineral and organic components of the matrix. Mineral phase is solubilized by H+ secretion in the subosteoclastic lacuna, while organic components, mainly type I collagen, are degraded by protease digestion, involving both cysteine and metallo-proteinases (MMPs). Two matrix MMPs, MMP-2 (72-kDa collagenase) and MMP-9 (92kDa) have been recently described in osteoclasts and may have a role in bone resorption and/or in osteoclast migration• The aim of this study was to better investigate the expression and to understand the mechanism of regulation of MMPs in osteoclasts. The study has been performed on human osteoclast-like cell lines from Giant Cell Tumors of bone (GCTs) obtained and extensively characterized in our laboratory. Immunofluorescence studies first confirmed that GCTs were strongly positive for MMP-2 and MMP-9. The enzymes were present in intracellular granules differently organized along the secretory pathway (i.e. in the Golgi or crowded at the cell periphery). Tissue inhibitors ofMMPs (TIMPs) were also investigated. Cells were positive for TIMP-I and 2. that were visible or along the cell membrane in narrow filaments, or in membrane-bound granules that remained on the substratum if the cell was detached. Zymography was used to identify the gelatin degrading species secreted in serum-free culture media by GCTs in control conditions or in the presence of 107retinoic acid or 10U/ml Hepatocyte Growth Factor (HGF). 0.5mg/ml of gelatin were incorporated into non-reducing SDS-polyacrylamide 8% gels. Results indicated the expression of MMP-2 and MMP-9 bands in control media, strongly enhanced in the presence of retinoic acid and HGF. Moreover unidentified bands of high molecular weight, very poorly evident in the control, were clearly identifiable in media obtained from cells treated with retinoic acid or HGF, indicating a modulation of GCTs basal secretory activity in the presence of stimulatory factors.
FORMATION OF BONE BY HUMAN OSTEOCLASTOMA-DERIVED STROMAL CELLS IN THE SEVERE COMBINED IMMUNODEFICIENT (SCID) MOUSE MODEL IS PREGEDED BY MOUSE OSTEOCLAST DIFFERENTIATION. R.A. Dodds. I.E. James. D.L. Olivera. E. Lee-Rvkaczewski. A. Gohlke and M. Gowen. Department of Cellular Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA. The formation of human bone from osteoclastoma-derived stromal cells injected into SCID mice is passage dependent; passage 4 cells (P#4) formed bone while passage 8 (P#8) cells did not. Furthermore, the resorbing osteoclasts (Ocs) in the P#4 generated bone lesions were not of human origin. The aim of this study was to phenotype the infiltrating hematopoietic mouse cells detected in the lesions derived from both P#4 (osteogenic) and P#8 (non-osteogenic) stromal cells. Ocs depleted P#4 and P#8 stromal cells (106) were injected intramuscularly into contralateral hind limbs of SCID mice. The lesions were allowed to develop for up to 42 days and cryostat sections collected from the 7, 14, 21 and 42 day time-points. To investigate the differentiation of cells of the Ocs and macrophage (rn~) lineage, sections were processed for TRAP and NSE activity. Serial sections were screened •with monoclonal antibodies reactive with human (OKa blood group antigen, procollagen-1 and Ocs selective) and mouse (MOMA-2 and ER-MP 20 mS -reactive) antigens. In P#4 lesions large but discrete centers of mouse mono- and multinucleeted Ocs were observed at 7 days. A mantle of osteoblasts was observed around these centers in occasional 7 day lesions. By 14 days this mantle contained discrete zones of bone formation; osteoblasts forming trabeculae were also observed among the Ocs. The osteoblasts were reective with the human OKa and type I procollagen-reactive antibodies. By 21 days a fully remodeling bone was evident with abundant resorbing multinucleated Ocs; bone remodelling was still apparent at 42 days. At all time points the Ocs were unreactive with the mouse rn~ markers. In P#8 lesions negligible numbers of Ocs or osteoblasts were detected at any time-point. However, an increase in rn~ number was observed over the timecourse. Although no bone developed in any lesion there were discrete spicules of mineralization associated with NSE positive giant cells. These cells were negative for TRAP, unreactive with the human Oc-reactive antibodies but were positive with the mouse mS markers. We propose that the P#4 as opposed to P#8 stroma contain osteoblastic cells that are responsible for recruiting and inducing differentiation of mouse Ocs. Whether this coupling initiates the osteogenic process remains to be determined. The mouse Ocs are ultimately involved in the resorption of the developing human bone. This system highlights the role of stromal cells in controlling hematopoietic cell recruitment and/or differentiation.
Abstracts
77
Bone Vol. 17, No. 6 December 1995:557-596
79
EXTRACELLULAR NUCLEOTIDES ENHANCE PARATHYROID HORMONE-STIMULATED BONE RESORPTION. W.B.Bowler and LA.Galla~her. Dept. Human Anatomy and Cell Biology, The University of Liverpool, Liverpool, L69 3BX, U.K.
CATHEPSIN O, A NOVEL OSTEOCLAST CYSTEINE PROTEASE, IS EXPRESSED IN GIANT CELLS OF OSTEOARTHRITIC SYNOVIUM. R. A. Dodds. J. R. Connor. F. Drake and Maxine Gowen, SmithKline Beecham Pharmaceuticals, Department of Cellular Biochemistry, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406, USA.
The complex process of bone remeclelling is controlled by many local and systemic factors, some of which have well documented effects on bone forming and or resorbing cells such as parathyroid hormone (PTH). However, the effects of extraceilular nucleotides on bone cells remain relatively unexplored. We have previously demonstrated that extracellular nucleotides interact with at least two purinoceptor subtypes expressed by bone cells, the result of this interaction being an elevation of [Ca2+]i and induction of the immediate early response gene c-los. In addition, we have demonstrated that extraceUular nucleotides can synergize with PTH to elevate c-fos expression in $aos-2 cells. In this study, we investigated whether extracellular nucleotides and PTH can interact to influence the functional behaviour of bone cells. We focused on the important PTH mediated response of bone resorption. Following PTH and nucleotide stimulation, we quantified resorption using the in vitro "bone slice" assay. Nucleotide agonists alone had no significant effect on resorption, while PTH in combination with ATP or UTP both significantly increased bone resorption above vehicle and PTH. Other purinergic agonists tested, ADP and 2-MeThio ATP, had no significant effects on PTH-induced bone resorption. We propose that extracellular nucleotides present in the bone micro-environment as a result of tissue injury, mechanical stress or physiological release, are capable of influencing bone cells and controlling the remodelling process through an interaction with other factors. These data further suggest a major role for nucleotides in controlling bone cell function.
Multinucleated giant cells (MGC) are a documented feature of diseased synovial tissue and inflammatory tissues. Evidence suggests that they are of macrophaga origin and may share the same precursor cell as the osteoclast (Oc), although morphological, functional, and phenotypic differences also support the contention for separate differentation pathways. We recently identified a novel cysteine protease (cathepsin O; Drake et al, J Bone Min Res 9: $177, 1994) that appears to be osteoclast specific. Thus, the aim of this study was to investigate the expression of cathepsin O mRNA, together with cathepsins L, S and B, in the MGCs found in osteoarthritic reparative connective tissue and associated inflammatory synovium and compare their expression with that of Ocs in the osteophytes within the same tissue. Sections were processed for in situ hybridization; serial sections were reacted for TRAP and nonspecific esterase (NSE) activity, selective markers for the Ocs and cells of the macrophagelmonocytelineage respectively. At sites of bone debris removal, TRAP*, NSE MGCs and mononuclear cells (monos) were detected in the intimal layer of the inflammatory synovium and within the adjacent osteogenic connective tissue at sites of intreamembmnous bone formation. They formed in clusters of NSE +, T R A P monos often containing azurophilic granules (a characteristic feature of phagocytes). These phagocytic-like monos expressed cathepsins O, L, B and B mRNA. However, the TRAP*cells expressed only cathepsin O mRNA. As previously described, Ocs andTRAP * monos at sites of osteophytic bone remodelling expressed only cathepsin O mRNA. Our data suggest that MGCs that form by the fusion of tissue macrophages (specific for calcified tissues) may differentiate towards a more Oe phenotype. This includes the expression of the novel cysteina protease, cathepsin O, and the loss of cathepsins L, S, and B expression. In support of this contention, we previously demonstrated that MGCs stained positively with 2 murine anti-human osteoclast monoclonal antibodies, namely 23C6 (vitronectin) and C35 (osteoclast selective). Altematively, the NSE * monos located within the inflammatory connective tissues (derived from cells from the periosteal-chondrosynovial junction) may represent undifferentiated myeloid-monocytic cells that ultimately differentiate into Ocs at sites of osteophytic bone formation. Thus, the fact that MGSs express classical ostaoclast phenotypic markers in a bone environment implies that these cells may in fact be true osteoclasts.
78
80
METALLOPROTEINASES EXPRESSION BY HUMAN OSTEOCLAST-LIKE CELL LINES. S. Colucci, M. Grano, C. Moil, M. Mastrugiacomo, A. Zambonin Zallone. Institute of Human Anatomy University of Bari, Italy. Bone resorption involves the simultaneous removal of both mineral and organic components of the matrix. Mineral phase is solubilized by H+ secretion in the subosteoclastic lacuna, while organic components, mainly type I collagen, are degraded by protease digestion, involving both cysteine and metallo-proteinases (MMPs). Two matrix MMPs, MMP-2 (72-kDa collagenase) and MMP-9 (92kDa) have been recently described in osteoclasts and may have a role in bone resorption and/or in osteoclast migration• The aim of this study was to better investigate the expression and to understand the mechanism of regulation of MMPs in osteoclasts. The study has been performed on human osteoclast-like cell lines from Giant Cell Tumors of bone (GCTs) obtained and extensively characterized in our laboratory. Immunofluorescence studies first confirmed that GCTs were strongly positive for MMP-2 and MMP-9. The enzymes were present in intracellular granules differently organized along the secretory pathway (i.e. in the Golgi or crowded at the cell periphery). Tissue inhibitors ofMMPs (TIMPs) were also investigated. Cells were positive for TIMP-I and 2. that were visible or along the cell membrane in narrow filaments, or in membrane-bound granules that remained on the substratum if the cell was detached. Zymography was used to identify the gelatin degrading species secreted in serum-free culture media by GCTs in control conditions or in the presence of 107retinoic acid or 10U/ml Hepatocyte Growth Factor (HGF). 0.5mg/ml of gelatin were incorporated into non-reducing SDS-polyacrylamide 8% gels. Results indicated the expression of MMP-2 and MMP-9 bands in control media, strongly enhanced in the presence of retinoic acid and HGF. Moreover unidentified bands of high molecular weight, very poorly evident in the control, were clearly identifiable in media obtained from cells treated with retinoic acid or HGF, indicating a modulation of GCTs basal secretory activity in the presence of stimulatory factors.
FORMATION OF BONE BY HUMAN OSTEOCLASTOMA-DERIVED STROMAL CELLS IN THE SEVERE COMBINED IMMUNODEFICIENT (SCID) MOUSE MODEL IS PREGEDED BY MOUSE OSTEOCLAST DIFFERENTIATION. R.A. Dodds. I.E. James. D.L. Olivera. E. Lee-Rvkaczewski. A. Gohlke and M. Gowen. Department of Cellular Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406, USA. The formation of human bone from osteoclastoma-derived stromal cells injected into SCID mice is passage dependent; passage 4 cells (P#4) formed bone while passage 8 (P#8) cells did not. Furthermore, the resorbing osteoclasts (Ocs) in the P#4 generated bone lesions were not of human origin. The aim of this study was to phenotype the infiltrating hematopoietic mouse cells detected in the lesions derived from both P#4 (osteogenic) and P#8 (non-osteogenic) stromal cells. Ocs depleted P#4 and P#8 stromal cells (106) were injected intramuscularly into contralateral hind limbs of SCID mice. The lesions were allowed to develop for up to 42 days and cryostat sections collected from the 7, 14, 21 and 42 day time-points. To investigate the differentiation of cells of the Ocs and macrophage (rn~) lineage, sections were processed for TRAP and NSE activity. Serial sections were screened •with monoclonal antibodies reactive with human (OKa blood group antigen, procollagen-1 and Ocs selective) and mouse (MOMA-2 and ER-MP 20 mS -reactive) antigens. In P#4 lesions large but discrete centers of mouse mono- and multinucleeted Ocs were observed at 7 days. A mantle of osteoblasts was observed around these centers in occasional 7 day lesions. By 14 days this mantle contained discrete zones of bone formation; osteoblasts forming trabeculae were also observed among the Ocs. The osteoblasts were reective with the human OKa and type I procollagen-reactive antibodies. By 21 days a fully remodeling bone was evident with abundant resorbing multinucleated Ocs; bone remodelling was still apparent at 42 days. At all time points the Ocs were unreactive with the mouse rn~ markers. In P#8 lesions negligible numbers of Ocs or osteoblasts were detected at any time-point. However, an increase in rn~ number was observed over the timecourse. Although no bone developed in any lesion there were discrete spicules of mineralization associated with NSE positive giant cells. These cells were negative for TRAP, unreactive with the human Oc-reactive antibodies but were positive with the mouse mS markers. We propose that the P#4 as opposed to P#8 stroma contain osteoblastic cells that are responsible for recruiting and inducing differentiation of mouse Ocs. Whether this coupling initiates the osteogenic process remains to be determined. The mouse Ocs are ultimately involved in the resorption of the developing human bone. This system highlights the role of stromal cells in controlling hematopoietic cell recruitment and/or differentiation.