Osteoblast-specific deficiency of ectonucleotide pyrophosphatase or phosphodiesterase-1 engenders insulin resistance in high-fat diet fed mice.

Supraphysiological levels of the osteoblast-enriched mineralization regulator ectonucleotide pyrophosphatase or phosphodiesterase-1 (NPP1) is associated with type 2 diabetes mellitus. We determined the impact of osteoblast-specific Enpp1 ablation on skeletal structure and metabolic phenotype in mice. Female, but not male, 6-week-old mice lacking osteoblast NPP1 expression (osteoblast-specific knockout [KO]) exhibited increased femoral bone volume or total volume (17.50% vs. 11.67%; p < .01), and reduced trabecular spacing (0.187 vs. 0.157 mm; p < .01) compared with floxed (control) mice. Furthermore, an enhanced ability of isolated osteoblasts from the osteoblast-specific KO to calcify their matrix in vitro compared to fl/fl osteoblasts was observed (p < .05). Male osteoblast-specific KO and fl/fl mice showed comparable glucose and insulin tolerance despite increased levels of insulin-sensitizing under-carboxylated osteocalcin (195% increase; p < .05). However, following high-fat-diet challenge, osteoblast-specific KO mice showed impaired glucose and insulin tolerance compared with fl/fl mice. These data highlight a crucial local role for osteoblast NPP1 in skeletal development and a secondary metabolic impact that predominantly maintains insulin sensitivity.

Focal adhesion kinase promotes BMP2-induced osteogenic differentiation of human urinary stem cells via AMPK and Wnt signaling pathways.

Human urine-derived stem cells (hUSCs) serve as favorable candidates for bone transplants due to their efficient proliferative and multipotent differentiation abilities, as well as the capacity to secrete a variety of vasoactive agents to facilitate tissue engineering. The present study aimed to explore the role of focal adhesion kinase (FAK) in bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation of hUSCs and to investigate the underlying mechanism. The degree of osteogenic differentiation and the correlated signals, following BMP2 overexpression and siRNA-mediated silencing of FAK, were determined in vitro. Moreover, hUSCs induced bone formation in a rat model with cranial defects, in vivo. Our findings revealed that alkaline phosphatase production, calcium deposits, osteocalcin and osteopontin expression, and bone formation were upregulated in vitro and in vivo following BMP2-induced osteogenic differentiation, and AMPK and Wnt signaling pathway activation by FAK could effectively regulate BMP2-enhanced osteogenic differentiation of hUSCs. Taken together, these findings indicated that FAK could mediate BMP2-enhanced osteogenic differentiation of hUSCs through activating adenosine 5'-monophosphate-activated protein kinase and Wnt signaling pathways.

Bone Morphogenetic Protein-9-Stimulated Adipocyte-Derived Mesenchymal Progenitors Entrapped in a Thermoresponsive Nanocomposite Scaffold Facilitate Cranial Defect Repair.

Due to availability and ease of harvest, adipose tissue is a favorable source of progenitor cells in regenerative medicine, but has yet to be optimized for osteogenic differentiation. The purpose of this study was to test cranial bone healing in a surgical defect model utilizing bone morphogenetic protein-9 (BMP-9) transduced immortalized murine adipocyte (iMAD) progenitor cells in a citrate-based, phase-changing, poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN)-gelatin scaffold. Mesenchymal progenitor iMAD cells were transduced with adenovirus expressing either BMP-9 or green fluorescent protein control. Twelve mice underwent craniectomy to achieve a critical-sized cranial defect. The iMAD cells were mixed with the PPCN-gelatin scaffold and injected into the defects. MicroCT imaging was performed in 2-week intervals for 12 weeks to track defect healing. Histologic analysis was performed on skull sections harvested after the final imaging at 12 weeks to assess quality and maturity of newly formed bone. Both the BMP-9 group and control group had similar initial defect sizes (P = 0.21). At each time point, the BMP-9 group demonstrated smaller defect size, higher percentage defect healed, and larger percentage defect change over time. At the end of the 12-week period, the BMP-9 group demonstrated mean defect closure of 27.39%, while the control group showed only a 9.89% defect closure (P < 0.05). The BMP-9-transduced iMADs combined with a PPCN-gelatin scaffold promote in vivo osteogenesis and exhibited significantly greater osteogenesis compared to control. Adipose-derived iMADs are a promising source of mesenchymal stem cells for further studies in regenerative medicine, specifically bone engineering with the aim of potential craniofacial applications.

FGFR3 mutation causes abnormal membranous ossification in achondroplasia.

FGFR3 gain-of-function mutations lead to both chondrodysplasias and craniosynostoses. Achondroplasia (ACH), the most frequent dwarfism, is due to an FGFR3-activating mutation which results in impaired endochondral ossification. The effects of the mutation on membranous ossification are unknown. Fgfr3(Y367C/+) mice mimicking ACH and craniofacial analysis of patients with ACH and FGFR3-related craniosynostoses provide an opportunity to address this issue. Studying the calvaria and skull base, we observed abnormal cartilage and premature fusion of the synchondroses leading to modifications of foramen magnum shape and size in Fgfr3(Y367C/+) mice, ACH and FGFR3-related craniosynostoses patients. Partial premature fusion of the coronal sutures and non-ossified gaps in frontal bones were also present in Fgfr3(Y367C/+) mice and ACH patients. Our data provide strong support that not only endochondral ossification but also membranous ossification is severely affected in ACH. Demonstration of the impact of FGFR3 mutations on craniofacial development should initiate novel pharmacological and surgical therapeutic approaches.

Papp-a2 modulates development of cranial cartilage and angiogenesis in zebrafish embryos.

Pregnancy-associated plasma protein A2 (PAPP-A2, also known as pappalysin-2) is a large metalloproteinase that is known to be required for normal postnatal growth and bone development in mice. We here report the detection of zebrafish papp-a2 mRNA in the chordamesoderm, notochord and lower jaw of zebrafish (Danio rerio) embryos, and that papp-a2-knockdown embryos display broadened axial mesoderm, notochord bends and severely reduced cranial cartilages. Genetic data link these phenotypes to insulin-like growth factor (Igf)-binding protein-3 (Igfbp-3) and bone morphogenetic protein (Bmp) signaling, and biochemical analysis show specific Igfbp-3 proteolysis by Papp-a2, implicating Papp-a2 in the modulation of Bmp signaling by Igfbp-3 proteolysis. Knockdown of papp-a2 additionally resulted in angiogenesis defects, strikingly similar to previous observations in embryos with mutations in components of the Notch system. Accordingly, we find that Notch signaling is modulated by Papp-a2 in vivo, and, furthermore, that human PAPP-A2 is capable of modulating Notch signaling independently of its proteolytic activity in cell culture. Based on these results, we conclude that Papp-a2 modulates Bmp and Notch signaling by independent mechanisms in zebrafish embryos. In conclusion, these data link pappalysin function in zebrafish to two different signaling pathways outside the IGF system.

Osteoclasts are important for bone angiogenesis.

Increased osteoclastogenesis and angiogenesis occur in physiologic and pathologic conditions. However, it is unclear if or how these processes are linked. To test the hypothesis that osteoclasts stimulate angiogenesis, we modulated osteoclast formation in fetal mouse metatarsal explants or in adult mice and determined the effect on angiogenesis. Suppression of osteoclast formation with osteoprotegerin dose-dependently inhibited angiogenesis and osteoclastogenesis in metatarsal explants. Conversely, treatment with parathyroid hormone related protein (PTHrP) increased explant angiogenesis, which was completely blocked by osteoprotegerin. Further, treatment of mice with receptor activator of nuclear factor-kappaB ligand (RANKL) or PTHrP in vivo increased calvarial vessel density and osteoclast number. We next determined whether matrix metalloproteinase-9 (MMP-9), an angiogenic factor predominantly produced by osteoclasts in bone, was important for osteoclast-stimulated angiogenesis. The pro-angiogenic effects of PTHrP or RANKL were absent in metatarsal explants or calvaria in vivo, respectively, from Mmp9(-/-) mice, demonstrating the importance of MMP-9 for osteoclast-stimulated angiogenesis. Lack of MMP-9 decreased osteoclast numbers and abrogated angiogenesis in response to PTHrP or RANKL in explants and in vivo but did not decrease osteoclast differentiation in vitro. Thus, MMP-9 modulates osteoclast-stimulated angiogenesis primarily by affecting osteoclasts, most probably by previously reported migratory effects on osteoclasts. These results clearly demonstrate that osteoclasts stimulate angiogenesis in vivo through MMP-9.

Critical role of the extracellular signal-regulated kinase-MAPK pathway in osteoblast differentiation and skeletal development.

The extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase (MAPK) pathway provides a major link between the cell surface and nucleus to control proliferation and differentiation. However, its in vivo role in skeletal development is unknown. A transgenic approach was used to establish a role for this pathway in bone. MAPK stimulation achieved by selective expression of constitutively active MAPK/ERK1 (MEK-SP) in osteoblasts accelerated in vitro differentiation of calvarial cells, as well as in vivo bone development, whereas dominant-negative MEK1 was inhibitory. The involvement of the RUNX2 transcription factor in this response was established in two ways: (a) RUNX2 phosphorylation and transcriptional activity were elevated in calvarial osteoblasts from TgMek-sp mice and reduced in cells from TgMek-dn mice, and (b) crossing TgMek-sp mice with Runx2+/- animals partially rescued the hypomorphic clavicles and undemineralized calvaria associated with Runx2 haploinsufficiency, whereas TgMek-dn; Runx2+/- mice had a more severe skeletal phenotype. This work establishes an important in vivo function for the ERK-MAPK pathway in bone that involves stimulation of RUNX2 phosphorylation and transcriptional activity.

Protein tyrosine phosphatase activity in the neural crest is essential for normal heart and skull development.

Mutations within the protein tyrosine phosphatase, SHP2, which is encoded by PTPN11, cause a significant proportion of Noonan syndrome (NS) cases, typically presenting with both cardiac disease and craniofacial abnormalities. Neural crest cells (NCCs) participate in both heart and skull formation, but the role of SHP2 signaling in NCC has not yet been determined. To gain insight into the role of SHP2 in NCC function, we ablated PTPN11 specifically in premigratory NCCs. SHP2-deficient NCCs initially exhibited normal migratory and proliferative patterns, but in the developing heart failed to migrate into the developing outflow tract. The embryos displayed persistent truncus arteriosus and abnormalities of the great vessels. The craniofacial deficits were even more pronounced, with large portions of the face and cranium affected, including the mandible and frontal and nasal bones. The data show that SHP2 activity in the NCC is essential for normal migration and differentiation into the diverse lineages found in the heart and skull and demonstrate the importance of NCC-based normal SHP2 activity in both heart and skull development, providing insight into the syndromic presentation characteristic of NS.

Possible role of matrix metalloproteinases (MMPs) in hyperostosis of intracranial meningiomas.

OBJECT: Although bone invasion and hyperostosis are common phenomena in patients with intracranial meningiomas, the basic pathomechanism is not fully understood. Based on an immunohistochemical study of surgically resected samples with hyperostosis, we postulate a possible mechanism of hyperostosis in patients with intracranial meningiomas. MATERIALS AND METHODS: Forty-six meningiomas were evaluated in this study. Twenty-six meningiomas associated with hyperostosis specimens served as the study group, and 20 meningiomas without any bony changes served as controls. An immunohistochemical staining technique was used to detect the expression of matrix metalloproteinase (MMP)-2, -9, and -13, membrane type (MT)1-MMP, estrogen receptor (ER), and progesterone receptor (PR) in the main tumor and hyperostotic portions of the studied samples. RESULTS: In the non-hyperostosis group, expression of MMP-13, MT1-MMP, and ER was significantly less than in the main tumor portion of hyperostotic meningiomas, while there was no difference in the expression of MMP-2 and -9 and PR in the main tumor between the two groups. In the hyperostosis group, the immunoreactivity of MMP-2 in the hyperostotic portion revealed a higher pattern of expression than the main tumor (p < 0.002). The expression of MMP-9, MT1-MMP, ER, and PR had relatively positive immunoreactivity in the main tumor portion (P < 0.05). CONCLUSIONS: Increased expression of MMP-13 and MT1-MMP in the tumor portion of hyperostosis of meningiomas might contribute to the initiation of osteolysis. Activated MMP-2 in hyperostotic lesions may change the physiological metabolism of the skull bone, thus playing an important role in hyperostosis formation.

Pan-caspase inhibition suppresses polyethylene particle-induced osteolysis.

Particle-induced osteolysis is a major cause of aseptic loosening after total joint replacement. Earlier studies demonstrated apoptotic macrophages, giant cells, fibroblasts and T-lymphocytes in capsules and interface membranes of patients with aseptic hip implant loosening. The aim of the current study was to determine in a murine calvarial model of wear particle-induced osteolysis whether inhibition of apoptosis using the pan-caspase inhibitor BOC-D-FMK reduces aseptic loosening. Healthy 12-week-old male C57BL/6J mice were treated with UHMWPE particles and received a daily peritoneal injection of BOK-D-FMK, respectively only buffer at a dose of 3 mg/kg of body weight for 12 days until sacrifice. Bone resorption was measured by histomorphometry, micro CT (computed tomography) and TRAP-5b serum analysis. Apoptosis was measured using caspase-3 cleaved staining. The results demonstrated that UHMWPE particles induced stronger apoptotic reactions in macrophages and osteoblasts and increased bone resorption in non-specifically treated mice, whereas peritoneal application of BOC-D-FMK significantly counteracted these adverse particle-related effects. We think that in particle-induced osteolysis apoptosis is pathologically increased, and that failure to reduce the quantity of apoptotic bodies leads to an up-regulation of proinflammatory cytokines, which may be responsible for the induction of osteolysis. We showed for the first time in vivo that a reduction in apoptosis leads to a significant reduction in particle-induced osteolysis. Clinically, the apoptotic cascade could become an interesting novel therapeutic target to modulate particle-induced osteolysis.

MT1-MMP-dependent, apoptotic remodeling of unmineralized cartilage: a critical process in skeletal growth.

Skeletal tissues develop either by intramembranous ossification, where bone is formed within a soft connective tissue, or by endochondral ossification. The latter proceeds via cartilage anlagen, which through hypertrophy, mineralization, and partial resorption ultimately provides scaffolding for bone formation. Here, we describe a novel and essential mechanism governing remodeling of unmineralized cartilage anlagen into membranous bone, as well as tendons and ligaments. Membrane-type 1 matrix metalloproteinase (MT1-MMP)-dependent dissolution of unmineralized cartilages, coupled with apoptosis of nonhypertrophic chondrocytes, mediates remodeling of these cartilages into other tissues. The MT1-MMP deficiency disrupts this process and uncouples apoptotic demise of chondrocytes and cartilage degradation, resulting in the persistence of "ghost" cartilages with adverse effects on skeletal integrity. Some cells entrapped in these ghost cartilages escape apoptosis, maintain DNA synthesis, and assume phenotypes normally found in the tissues replacing unmineralized cartilages. The coordinated apoptosis and matrix metalloproteinase-directed cartilage dissolution is akin to metamorphosis and may thus represent its evolutionary legacy in mammals.

Stimulation of ectopic bone formation in response to BMP-2 by Rho kinase inhibitor: a pilot study.

The small GTPase Rho and Rho-associated protein kinase (Rho kinase, ROCK) signal participates in a variety of biological functions including vascular contraction, tumor invasion, and penile erection. Evidence also suggests Rho-ROCK is involved in signaling for mesenchymal cellular differentiation. However, whether it is involved in osteoblastic differentiation is unknown. We therefore asked whether Rho-ROCK signaling participates in recombinant human bone morphogenetic protein (rhBMP-2)-induced osteogenesis both in vitro and in vivo. Continuous delivery of a specific ROCK inhibitor (Y-27632) enhanced ectopic bone formation induced by rhBMP-2 impregnated into an atelocollagen carrier in mice without affecting systemic bone metabolism. Treatment with Y-27632 also enhanced the osteoblastic differentiation of cultured murine neonatal calvarial cells. These effects were associated with increased expression of BMP-4 gene. Expression of a dominant negative mutant of ROCK in ST2 cells promoted osteoblastic differentiation, while a constitutively active mutant of ROCK attenuated osteoblastic differentiation and the ROCK inhibitor reversed this phenotype. Thus, ROCK inhibits osteogenesis, and a ROCK inhibitor in combination with the local delivery of rhBMP/collagen composite may be clinically applicable for stimulating bone formation.

Effects of pyrophosphate on desmal and endochondral mineralization and TNAP activity in organoid culture.

During endochondral and desmal osteogenesis, mineralization of bone and cartilage matrix requires an appropriate solubility product of calcium and phosphate, collagen as a nucleator and deactivation of inhibitors, in order to prevent heterotopic calcification. In the 1960s, Fleisch and coworkers detected pyrophosphate (PPi) as an inhibitor of hydroxyapatite crystal growth, which should be removed by cleavage to tissue non-specific alkaline phosphatase (TNAP) activity. This theory had been established by basic experiments performed with collagen gels and demineralized matrices. In order to investigate the effect of PPi on matrix mineralization in bone and cartilage, calcium content and TNAP activity were measured in organoid cultures of mouse calvarial osteoblasts and limb bud cartilage after treatment with PPi and/or levamisole. In organoid cultures, bone and cartilage develop in a clear histotypical manner. PPi did not induce mineralization. Beta-glycerophosphate (beta-GP) and inorganic phosphate (Pi) induced mineralization which could be significantly reduced by PPi. Levamisole, an inhibitor of TNAP, also reduced mineralization; the combination with PPi was additive. TNAP activity was increased after treatment with PPi and levamisole in both osteoblast and cartilage cultures. Mineralization induced by beta-GP and Pi decreased TNAP activity in the osteoblast but not in cartilage organoid culture. In this culture system, PPi reduced mineralization as predicted by Fleisch's theory. Indications of cleavage of PPi were indirectly found because inhibition of hydrolysis of PPi by levamisole further reduced mineralization, probably due to the higher amounts of PPi available for binding to hydroxyapatite.

Erk pathways negatively regulate matrix mineralization.

Skeletal mineralization is an important step regulating the mechanical properties of the calcified tissues, but molecular events underlying mineralization still remain elusive. We examined the role of extracellular signal-regulated kinase (Erk) pathways in matrix mineralization of osteogenic cells both in vitro and in vivo. Matrix mineralization by preosteocytic MLO-A5 cells and osteoblastic MC3T3-E1 cells was increased by either PD98059 Mek inhibitor treatment or adenovirus vector-mediated dominant negative Ras (Ras(DN)) expression and was suppressed by Erk activation by platelet-derived growth factor (PDGF) treatment or constitutively active Mek1 (Mek(CA)) expression. Administration of adenovirus vectors carrying Ras(DN) gene onto the calvaria of 1-day-old mice increased the mineralization of the tissues, while that of the Mek(CA) adenovirus suppressed it. These results suggest that the Erk pathway is a negative regulator of the matrix mineralization both in vitro and in vivo.

Bioactive Glass Particles in Two-Dimensional and Three-Dimensional Osteogenic Cell Cultures.

This study aimed to investigate the influence of a three-dimensional cell culture model and bioactive glass (BG) particles on the expression of osteoblastic phenotypes in rat calvaria osteogenic cells culture. Cells were seeded on two-dimensional (2D) and three-dimensional (3D) collagen with BG particles for up to 14 days. Cell viability and alkaline phosphatase (ALP) activity was performed. Cell morphology and immunolabeling of noncollagenous bone matrix proteins were assessed by epifluorescence and confocal microscopy. The expressions of osteogenic markers were analyzed using RT-PCR. Mineralized bone-like nodule formation was visualized by microscopy and calcium content was assessed quantitatively by alizarin red assay. Experimental cultures produced a growing cell viability rate up to 14 days. Although ALP activity at 7 days was higher on BG cultures, cells on 3D and 3D+BG had an activity decrease of ALP at 14 days. Three-dimensional conditions favored the immunolabeling for OPN and BSP and the expression of ALP and COL I mRNAs. BG particles influenced positively the OC and OPN mRNAs expression and calcified nodule formation in vitro. The results indicated that the 3D cultures and BG particles contribute to the expression of osteoblastic phenotype and to differentiated and mineralized matrix formation.

Osteoclastic bone degradation and the role of different cysteine proteinases and matrix metalloproteinases: differences between calvaria and long bone.

UNLABELLED: Osteoclastic bone degradation involves the activity of cathepsin K. We found that in addition to this enzyme other, yet unknown, cysteine proteinases participate in digestion. The results support the notion that osteoclasts from different bone sites use different enzymes to degrade the collagenous bone matrix. INTRODUCTION: The osteoclast resorbs bone by lowering the pH in the resorption lacuna, which is followed by secretion of proteolytic enzymes. One of the enzymes taken to be essential in resorption is the cysteine proteinase, cathepsin K. Some immunolabeling and enzyme inhibitor data, however, suggest that other cysteine proteinases and/or proteolytic enzymes belonging to the group of matrix metalloproteinases (MMPs) may participate in the degradation. In this study, we investigated whether, in addition to cathepsin K, other enzymes participate in osteoclastic bone degradation. MATERIALS AND METHODS: In bones obtained from mice deficient for cathepsin K, B, or L or a combination of K and L, the bone-resorbing activity of osteoclasts was analyzed at the electron microscopic level. In addition, bone explants were cultured in the presence of different selective cysteine proteinase inhibitors and an MMP inhibitor, and the effect on resorption was assessed. Because previous studies showed differences in resorption by calvarial osteoclasts compared with those present in long bones, in all experiments, the two types of bone were compared. Finally, bone extracts were analyzed for the level of activity of cysteine proteinases and the effect of inhibitors hereupon. RESULTS: The analyses of the cathepsin-deficient bone explants showed that, in addition to cathepsin K, calvarial osteoclasts use other cysteine proteinases to degrade bone matrix. It was also shown that, in the absence of cathepsin K, long bone osteoclasts use MMPs for resorption. Cathepsin L proved to be involved in the MMP-mediated resorption of bone by calvarial osteoclasts; in the absence of this cathepsin, calvarial osteoclasts do not use MMPs for resorption. Selective inhibitors of cathepsin K and other cysteine proteinases showed a stronger effect on calvarial resorption than on long bone resorption. CONCLUSIONS: Our findings suggest that (1) cathepsin K-deficient long bone osteoclasts compensate the lack of this enzyme by using MMPs in the resorption of bone matrix; (2) cathepsin L is involved in MMP-mediated resorption by calvarial osteoclasts; (3) in addition to cathepsin K, other, yet unknown, cysteine proteinases are likely to participate in skull bone degradation; and finally, (4) the data provide strong additional support for the existence of functionally different bone-site specific osteoclasts.

Parathyroid hormone induces mitogen-activated kinase phosphatase 1 in murine osteoblasts primarily through cAMP-protein kinase A signaling.

BACKGROUND: Parathyroid hormone (PTH) regulates osteoblast function by binding to the PTH receptor 1 (PTHR1) to activate downstream signaling to induce expression of primary response genes (PRGs), which affect various aspects of the osteoblast phenotype. We previously identified PTH-induced PRGs in MC3T3-E1 cells, including mitogen-activated protein kinase (MAPK) phosphatase 1 (mkp1), which dephosphorylates members of the MAPK family. The aim of this study was to explore the molecular mechanisms of PTH's induction of mkp1 in primary mouse osteoblasts. METHODS: Northern and Western analyses were used to determine mkp1 mRNA and protein expression. In vivo experiments were also performed to determine PTH's effect on mkp1 in mouse calvariae and long bones. RESULTS: A total of 10 nM PTH and PTH-related protein (PTHrP) maximally induced mkp1 mRNA levels after 1 hour in osteoblasts. PTH also increased mkp1 protein expression, and induced mkp1 mRNA independent of new protein synthesis. PTHR1 triggers protein kinase A (PKA), PKC, and calcium pathways. Although PKA and PKC agonists induced mkp1 mRNA levels, only cyclic adenosine 3':5'-monophosphate (cAMP)-PKA inhibition blocked PTH-induced mkp1 mRNA levels. These data suggest that PTH-induced mkp1 mRNA levels are primarily mediated through the cAMP-PKA pathway. Further, prostaglandin E2 (PGE2), which activates cAMP-PKA and PKC, induced mkp1 mRNA to a greater extent than PGF2alpha and fluprostenol, which activate PKC signaling only. Finally, PTH maximally induced mkp1 mRNA levels in mouse calvariae and long bones in vivo at 0.5 hours. CONCLUSIONS: mkp1's in vitro and in vivo induction in PTH-target tissues suggests its involvement in some of the effects of PTH on osteoblast function. mkp1 may be an important target gene in the anabolic effect of PTH on osteoblasts.

Peptidyl proline hydroxylase in adult, developing, and neoplastic rat tissues.

A sensitive assay system, optimally supplemented with tritiated protocollagen substrate and cofactors, is described which is suitable for determining the peptidyl proline hydroxylase (PPH) content of a wide spectrum of rat tissues. In most tissues, less than 50 percent of the total activity was soluble; the particulate portion of the activity (concentrated in the mitochondrial and microsomal fractions) was doubled by pretreatment with Triton X-100. Among normal adult tissues, lung had the highest total PPH activity (2.4 times that of liver) and small intestine had the lowest (25 percent that of liver). In brain and lactating mammary gland, the activity was similar to that in skin (60 percent of that in liver). Fetal tissues contained 3 to 8 times more PPH than the corresponding adult tissues, and a much lower portion of the total activity was soluble. In four tissues studied in detail (lung, liver, kidney, and brain), the total PPH declined rapidly during the last few days of gestation; brain attained its low adult value before term, whereas the other three tissues continued to decrease in the course of postnatal development. An injection of cortisol to fetal rats enhanced the decline of PPH in lung, liver, and skull. These experiments suggest that during normal differentiation the decline in collagen synthesis is initiated by fetal glucocorticoid secretion which is maximal on the 19th gestational day. PPH activity appears to be a sensitive indicator of neoplastic growth. In renal, mammary, muscle, and hepatic tumors, the PPH activities were 4 to 10 times higher than in the cognate adult tissue. Even in well-differentiated, slowgrowing tumors, the activity was considerably higher than in any normal, mature, or immature tissue, with the exception of the skull and lung of the 19-day-old fetus.

Differential regulation of MMP-13 (collagenase-3) and MMP-3 (stromelysin-1) in mouse calvariae.

Bone resorption in mice involves the degradation of extracellular matrix. Whereas several proteases seem to be implicated in this process, it becomes increasingly clear that matrix metalloproteinases (MMPs), amongst them especially MMP-13 and MMP-3, play an essential role. We have purified MMP-13 and MMP-3 from mouse calvariae-conditioned media by differential fractionation and analyzed their collagenolytic, caseinolytic, gelatinolytic and proteoglycanolytic activities. It could be shown that in mouse calvariae-conditioned media most of the measured enzyme activities were due to MMP-13, although zymographies revealed that MMP-3, MMP-2, MMP-9 as well as TIMPs were present too. MMP-13 and MMP-3 proteins were detected and their enzyme activities were neutralized by specific polyclonal antisera. Furthermore, it was demonstrated that in cultures of mouse calvariae the production of MMP-13 was induced by the potent MMP-stimulator heparin and by parathyroid hormone (PTH), whereas the levels of MMP-3 remained unchanged. Although PTH-induced bone resorption was inhibited by calcitonin treatment, MMP-13 mRNA and protein expression were not significantly altered by this hormone. Together with previous observations, these results indicate that PTH regulates bone resorption through MMP-13, but not by MMP-3, and that its reversion by calcitonin involves neither of the two enzymes.

Pharmaceutical inhibition of glycogen synthetase kinase 3 beta suppresses wear debris-induced osteolysis.

Aseptic loosening is associated with the development of wear debris-induced peri-implant osteolytic bone disease caused by an increased osteoclastic bone resorption and decreased osteoblastic bone formation. However, no effective measures for the prevention and treatment of peri-implant osteolysis currently exist. The aim of this study was to determine whether lithium chloride (LiCl), a selective inhibitor of glycogen synthetase kinase 3 beta (GSK-3ß), mitigates wear debris-induced osteolysis in a murine calvarial model of osteolysis. GSK-3ß is activated by titanium (Ti) particles, and implantation of Ti particles on the calvarial surface in C57BL/6 mice resulted in osteolysis caused by an increase in the number of osteoclasts and a decrease in the number of osteoblasts. Mice implanted with Ti particles were gavage-fed LiCl (50 or 200 mg kg(-1)d(-1)), 6 days per week for 2 weeks. The LiCl treatment significantly inhibited GSK-3ß activity and increased ß-catenin and axin-2 expression in a dose-dependent manner, dramatically mitigating the Ti particle-induced suppression of osteoblast numbers and the expression of bone formation markers. Finally, we demonstrated that inhibition of GSK-3ß suppresses osteoclast differentiation and reduces the severity of Ti particle-induced osteolysis. The results of this study indicate that Ti particle-induced osteolysis is partly dependent on GSK-3ß and, therefore, the canonical Wnt signaling pathway. This suggests that selective inhibitors of GSK-3ß such as LiCl may help prevent and treat wear debris-induced osteolysis.