Direct extraction and assay of bone tissue collagenase and its relation to parathyroid-hormone-induced bone resorption.
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ABSTRACT:
SUBMITTER: Eeckhout Y
PROVIDER: S-EPMC1147359 | biostudies-other | 1986 Nov
REPOSITORIES: biostudies-other
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Project description:IntroductionGlucosamine is an amino-monosaccharide and precursor of glycosaminoglycans, major components of joint cartilage. Glucosamine has been clinically introduced for the treatment of osteoarthritis but the data about its protective role in disease are insufficient. The goal of this study was to investigate the effect of long term administration of glucosamine on bone resorption and remodeling.MethodsThe effect of glucosamine on bone resorption and remodeling was studied in a model of collagenase-induced osteoarthritis (CIOA). The levels of macrophage-inflammatory protein (MIP)-1α, protein regulated upon activation, normal T-cell expressed, and secreted (RANTES), soluble receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor (TNF)-α, and interleukin (IL)-6, 4 and 10 in synovial fluid were measured by enzyme-linked immunosorbent assay (ELISA). Cell populations in synovial extracts and the expression of RANKL, of receptors for TNF-α (TNF-αR) and interferon γ (IFN-γR) on clusters of differentiation (CD) three positive T cells were analyzed by flow cytometry. Transforming growth factor (TGF)-β3, bone morphogenetic protein (BMP)-2, phosphorylated protein mothers against decapentaplegic homolog 2 (pSMAD-2), RANKL and Dickkopf-1 protein (DKK-1) positive staining in CIOA joints were determined by immunohistochemistry.ResultsThe administration of glucosamine hydrochloride in CIOA mice inhibited loss of glycosaminoglycans (GAGs) and proteoglycans (PGs) in cartilage, bone erosion and osteophyte formation. It decreased the levels of soluble RANKL and IL-6 and induced IL-10 increase in the CIOA joint fluids. Glucosamine limited the number of CD11b positive Ly6G neutrophils and RANKL positive CD3 T cells in the joint extracts. It suppressed bone resorption via down-regulation of RANKL expression and affected bone remodeling in CIOA by decreasing BMP-2, TGF-β3 and pSMAD-2 expression and up-regulating DKK-1 joint levels.ConclusionsOur data suggest that glucosamine hydrochloride inhibits bone resorption through down-regulation of RANKL expression in the joints, via reduction of the number of RANKL positive CD3 T cells and the level of sRANKL in the joints extracts. These effects of glucosamine appear to be critical for the progression of CIOA and result in limited bone remodeling of the joints.
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Project description:Intermittent PTH administration builds bone mass and prevents fractures, but its mechanism of action is unclear. We genetically deleted the PTH/PTHrP receptor (PTH1R) in mesenchymal stem cells using Prx1Cre and found low bone formation, increased bone resorption, and high bone marrow adipose tissue (BMAT). Bone marrow adipocytes traced to Prx1 and expressed classic adipogenic markers and high receptor activator of nuclear factor kappa B ligand (Rankl) expression. RANKL levels were also elevated in bone marrow supernatant and serum, but undetectable in other adipose depots. By cell sorting, Pref1+RANKL+ marrow progenitors were twice as great in mutant versus control marrow. Intermittent PTH administration to control mice reduced BMAT significantly. A similar finding was noted in male osteoporotic patients. Thus, marrow adipocytes exhibit osteogenic and adipogenic characteristics, are uniquely responsive to PTH, and secrete RANKL. These studies reveal an important mechanism for PTH's therapeutic action through its ability to direct mesenchymal cell fate.
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Project description:Parathyroid hormone (PTH), an important regulator of calcium homeostasis, targets most of its complex actions in bone to cells of the osteoblast lineage. Furthermore, PTH is known to stimulate osteoclastogenesis indirectly through activation of osteoblastic cells. To assess the role of the PTH/PTH-related protein receptor (PPR) in mediating the diverse actions of PTH on bone in vivo, we generated mice that express, in cells of the osteoblastic lineage, one of the constitutively active receptors described in Jansen's metaphyseal chondrodysplasia. In these transgenic mice, osteoblastic function was increased in the trabecular and endosteal compartments, whereas it was decreased in the periosteum. In trabecular bone of the transgenic mice, there was an increase in osteoblast precursors, as well as in mature osteoblasts. Osteoblastic expression of the constitutively active PPR induced a dramatic increase in osteoclast number in both trabecular and compact bone in transgenic animals. The net effect of these actions was a substantial increase in trabecular bone volume and a decrease in cortical bone thickness of the long bones. These findings, for the first time to our knowledge, identify the PPR as a crucial mediator of both bone-forming and bone-resorbing actions of PTH, and they underline the complexity and heterogeneity of the osteoblast population and/or their regulatory microenvironment.
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Project description:Bone metastasis is a frequent complication of breast cancer that is often accelerated by TGF-β signaling; however, little is known about how the TGF-β pathway is regulated during bone metastasis. Here we report that deleted in liver cancer 1 (DLC1) is an important regulator of TGF-β responses and osteolytic metastasis of breast cancer cells. In murine models, breast cancer cells lacking DLC1 expression exhibited enhanced capabilities of bone metastasis. Knockdown of DLC1 in cancer cells promoted bone metastasis, leading to manifested osteolysis and accelerated death in mice, while DLC1 overexpression suppressed bone metastasis. Activation of Rho-ROCK signaling in the absence of DLC1 mediated SMAD3 linker region phosphorylation and TGF-β-induced expression of parathyroid hormone-like hormone (PTHLH), leading to osteoclast maturation for osteolytic colonization. Furthermore, pharmacological inhibition of Rho-ROCK effectively reduced PTHLH production and breast cancer bone metastasis in vitro and in vivo. Evaluation of clinical breast tumor samples revealed that reduced DLC1 expression was linked to elevated PTHLH expression and organ-specific metastasis to bone. Overall, our findings define a stroma-dependent paradigm of Rho signaling in cancer and implicate Rho-TGF-β crosstalk in osteolytic bone metastasis.
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Project description:Postmenopausal osteoporosis is often treated with bisphosphonates (eg, alendronate, [ALN]), but oversuppression of bone turnover by long-term bisphosphonate treatment may decrease bone tissue heterogeneity. Thus, alternate treatment strategies after long-term bisphosphonates are of great clinical interest. The objective of the current study was to determine the effect of intermittent parathyroid hormone (PTH) following 12 weeks of ALN (a bisphosphonate) treatment in 6-month-old, ovariectomized (OVX) rats on bone microarchitecture, bone remodeling dynamics, and bone mechanical properties at multiple length scales. By using in vivo ?CT and 3D in vivo dynamic bone histomorphometry techniques, we demonstrated the efficacy of PTH following ALN therapy for stimulating new bone formation, and increasing trabecular thickness and bone volume fraction. In healthy bone, resorption and formation are coupled and balanced to sustain bone mass. OVX results in resorption outpacing formation, and subsequent bone loss and reduction in bone tissue modulus and tissue heterogeneity. We showed that ALN treatment effectively reduced bone resorption activity and regained the balance with bone formation, preventing additional bone loss. However, ALN treatment also resulted in significant reductions in the heterogeneity of bone tissue mineral density and tissue modulus. On the other hand, PTH treatment was able to shift the bone remodeling balance in favor of formation, with or without a prior treatment with ALN. Moreover, by altering the tissue mineralization, PTH alleviated the reduction in heterogeneity of tissue material properties induced by prolonged ALN treatment. Furthermore, switching to PTH treatment from ALN improved bone's postyield mechanical properties at both the whole bone and apparent level compared to ALN alone. The current findings suggest that intermittent PTH treatment should be considered as a viable treatment option for patients with prior treatment with bisphosphonates. © 2017 American Society for Bone and Mineral Research.
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Project description:Parathyroid hormone (PTH) is a potent pharmacologic inducer of new bone formation, but no physiologic anabolic effect of PTH on adult bone has been described. We investigated the role of PTH in fetal skeletal development by comparing newborn mice lacking either PTH, PTH-related peptide (PTHrP), or both peptides. PTH-deficient mice were dysmorphic but viable, whereas mice lacking PTHrP died at birth with dyschondroplasia. PTH-deficient mice uniquely demonstrated diminished cartilage matrix mineralization, decreased neovascularization with reduced expression of angiopoietin-1, and reduced metaphyseal osteoblasts and trabecular bone. Compound mutants displayed the combined cartilaginous and osseous defects of both single mutants. These results indicate that coordinated action of both PTH and PTHrP are required to achieve normal fetal skeletal morphogenesis, and they demonstrate an essential function for PTH at the cartilage-bone interface. The effect of PTH on fetal osteoblasts may be relevant to its postnatal anabolic effects on trabecular bone.
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Project description:Mice in which the genes encoding the parathyroid hormone (PTH)-related peptide (PTHrP) or the PTH/PTHrP receptor have been ablated by homologous recombination show skeletal dysplasia due to accelerated endochondral bone formation, and die at birth or in utero, respectively. Skeletal abnormalities due to decelerated chondrocyte maturation are observed in transgenic mice where PTHrP expression is targeted to the growth plate, and in patients with Jansen metaphyseal chondrodysplasia, a rare genetic disorder caused by constitutively active PTH/PTHrP receptors. These and other findings thus indicate that PTHrP and its receptor are essential for chondrocyte differentiation. To further explore the role of the PTH/PTHrP receptor in this process, we generated transgenic mice in which expression of a constitutively active receptor, HKrk-H223R, was targeted to the growth plate by the rat alpha1 (II) collagen promoter. Two major goals were pursued: (i) to investigate how constitutively active PTH/PTHrP receptors affect the program of chondrocyte maturation; and (ii) to determine whether expression of the mutant receptor would correct the severe growth plate abnormalities of PTHrP-ablated mice (PTHrP-/-). The targeted expression of constitutively active PTH/PTHrP receptors led to delayed mineralization, decelerated conversion of proliferative chondrocytes into hypertrophic cells in skeletal segments that are formed by the endochondral process, and prolonged presence of hypertrophic chondrocytes with delay of vascular invasion. Furthermore, it corrected at birth the growth plate abnormalities of PTHrP-/- mice and allowed their prolonged survival. "Rescued" animals lacked tooth eruption and showed premature epiphyseal closure, indicating that both processes involve PTHrP. These findings suggest that rescued PTHrP-/- mice may gain considerable importance for studying the diverse, possibly tissue-specific role(s) of PTHrP in postnatal development.
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Project description:Radiation therapy for soft tissue sarcoma or tumor metastases is frequently associated with damage to the underlying bone. Using a mouse model of limited field hindlimb irradiation, we assessed the ability of parathyroid hormone (1-34) fragment (PTH) delivery to prevent radiation-associated bone damage, including loss of mechanical strength, trabecular architecture, cortical bone volume, and mineral density. Female BALB/cJ mice received four consecutive doses of 5 Gy to a single hindlimb, accompanied by daily injections of either PTH or saline (vehicle) for 8 weeks, and were followed for 26 weeks. Treatment with PTH maintained the mechanical strength of irradiated femurs in axial compression for the first eight weeks of the study, and the apparent strength of irradiated femurs in PTH-treated mice was greater than that of naïve bones during this time. PTH similarly protected against radiation-accelerated resorption of trabecular bone and transient decrease in mid-diaphyseal cortical bone volume, although this benefit was maintained only for the duration of PTH delivery. Overall, PTH conferred protection against radiation-induced fragility and morphologic changes by increasing the quantity of bone, but only during the period of administration. Following cessation of PTH delivery, bone strength and trabecular volume fraction rapidly decreased. These data suggest that PTH does not negate the longer-term potential for osteoclastic bone resorption, and therefore, finite-duration treatment with PTH alone may not be sufficient to prevent late onset radiotherapy-induced bone fragility.
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