The p27 Pathway Modulates the Regulation of Skeletal Growth and Osteoblastic Bone Formation by Parathyroid Hormone–Related Peptide

Parathyroid hormone–related peptide (PTHrP) 1–84 knock‐in mice (Pthrp KI) develop skeletal growth retardation and defective osteoblastic bone formation. To further examine the mechanisms underlying this phenotype, microarray analyses of differential gene expression profiles were performed in long bone extracts from Pthrp KI mice and their wild‐type (WT) littermates. We found that the expression levels of p27, p16, and p53 were significantly upregulated in Pthrp KI mice relative to WT littermates. To determine whether p27 was involved in the regulation by PTHrP of skeletal growth and development in vivo, we generated compound mutant mice, which were homozygous for both p27 deletion and the Pthrp KI mutation (p27^‐/‐Pthrp KI). We then compared p27^‐/‐Pthrp KI mice with p27^‐/‐, Pthrp KI, and WT littermates. Deletion of p27 in Pthrp KI mice resulted in a longer lifespan, increased body weight, and improvement in skeletal growth. At 2 weeks of age, skeletal parameters, including length of long bones, size of epiphyses, numbers of proliferating cell nuclear antigen (PCNA)‐positive chondrocytes, bone mineral density, trabecular bone volume, osteoblast numbers, and alkaline phosphatase (ALP)‐, type I collagen‐, and osteocalcin‐positive bone areas were increased in p27^‐/‐ mice and reduced in both Pthrp KI and p27^‐/‐Pthrp KI mice compared with WT mice; however, these parameters were increased in p27^‐/‐Pthrp KI mice compared with Pthrp KI mice. As well, protein expression levels of PTHR, IGF‐1, and Bmi‐1, and the numbers of total colony‐forming unit fibroblastic (CFU‐f) and ALP‐positive CFU‐f were similarly increased in p27^‐/‐Pthrp KI mice compared with Pthrp KI mice. Our results demonstrate that deletion of p27 in Pthrp KI mice can partially rescue defects in skeletal growth and osteoblastic bone formation by enhancing endochondral bone formation and osteogenesis. These studies, therefore, indicate that the p27 pathway may function downstream in the action of PTHrP to regulate skeletal growth and development. © 2015 American Society for Bone and Mineral Research.     

Gender‐Specific Differences in the Skeletal Response to Continuous PTH in Mice Lacking the IGF1 Receptor in Mature Osteoblasts

The primary goal of this study was to determine whether the IGF1R in mature osteoblasts and osteocytes was required for the catabolic actions of continuous parathyroid hormone (cPTH). Igf1r was deleted from male and female FVN/B mice by breeding with mice expressing cre recombinase under control of the osteocalcin promoter (^0CNIgfr1^‐/‐). Littermates lacking the cre recombinase served as controls. PTH, 60 μg/kg/d, was administered continuously by Alzet minipumps for 4 weeks. Blood was obtained for indices of calcium metabolism. The femurs were examined by micro‐computed tomography for structure, immunohistochemistry for IGF1R expression, histomorphometry for bone formation rates (BFR), mRNA levels by qPCR, and bone marrow stromal cell cultures (BMSC) for alkaline phosphatase activity (ALP⁺), mineralization, and osteoblast‐induced osteoclastogenesis. Whereas cPTH led to a reduction in trabecular bone volume/tissue volume (BV/TV) and cortical thickness in the control females, no change was found in the control males. Although trabecular BV/TV and cortical thickness were reduced in the ^0CNIgfr1^‐/‐ mice of both sexes, no further reduction after cPTH was found in the females, unlike the reduction in males. BFR was stimulated by cPTH in the controls but blocked by Igf1r deletion in the females. The ^0CNIgfr1^‐/‐ male mice showed a partial response. ALP⁺ and mineralized colony formation were higher in BMSC from control males than from control females. These markers were increased by cPTH in both sexes, but BMSC from male ^0CNIgfr1^‐/‐ also were increased by cPTH, unlike those from female ^0CNIgfr1^‐/‐. cPTH stimulated receptor activator of NF‐κB ligand (RANKL) and decreased osteoprotegerin and alkaline phosphatase expression more in control female bone than in control male bone. Deletion of Igf1r blocked these effects of cPTH in the female but not in the male. However, PTH stimulation of osteoblast‐driven osteoclastogenesis was blocked by deleting Igfr1 in both sexes. We conclude that cPTH is catabolic in female but not male mice. Moreover, IGF1 signaling plays a greater role in the skeletal actions of cPTH in the female mouse than in the male mouse, which may underlie the sex differences in the response to cPTH. © 2015 American Society for Bone and Mineral Research.     

The role of estrogen receptor α in growth plate cartilage for longitudinal bone growth

Estrogens enhance skeletal growth during early sexual maturation, whereas high estradiol levels during late puberty result in growth plate fusion in humans. Although the growth plates do not fuse directly after sexual maturation in rodents, a reduction in growth plate height is seen by treatment with a high dose of estradiol. It is unknown whether the effects of estrogens on skeletal growth are mediated directly via estrogen receptors (ERs) in growth plate cartilage and/or indirectly via other mechanisms such as the growth hormone/insulin‐like growth factor 1 (GH/IGF‐1) axis. To determine the role of ERα in growth plate cartilage for skeletal growth, we developed a mouse model with cartilage‐specific inactivation of ERα. Although mice with total ERα inactivation displayed affected longitudinal bone growth associated with alterations in the GH/IGF‐1 axis, the skeletal growth was normal during sexual maturation in mice with cartilage‐specific ERα inactivation. High‐dose estradiol treatment of adult mice reduced the growth plate height as a consequence of attenuated proliferation of growth plate chondrocytes in control mice but not in cartilage‐specific ERα^?/? mice. Adult cartilage‐specific ERα^?/? mice continued to grow after 4 months of age, whereas growth was limited in control mice, resulting in increased femur length in 1‐year‐old cartilage‐specific ERα^?/? mice compared with control mice. We conclude that during early sexual maturation, ERα in growth plate cartilage is not important for skeletal growth. In contrast, it is essential for high‐dose estradiol to reduce the growth plate height in adult mice and for reduction of longitudinal bone growth in elderly mice. © 2010 American Society for Bone and Mineral Research.     

Defects in mesenchymal stem cell self‐renewal and cell fate determination lead to an osteopenic phenotype in Bmi‐1 null mice

In parathyroid hormone–related protein 1‐84 [PTHrP(1‐84)] knockin mice, expression of the polycomb protein Bmi‐1 is reduced and potentially can mediate the phenotypic alterations observed. We have therefore now examined the skeletal phenotype of Bmi‐1^?/? mice in vivo and also assessed the function of bone marrow mesenchymal stem cells (BM‐MSCs) from Bmi‐1^?/? mice ex vivo in culture. Neonatal Bmi‐1^?/? mice exhibited skeletal growth retardation, with reduced chondrocyte proliferation and increased apoptosis. Osteoblast numbers; gene expression of alkaline phosphatase, type I collagen, and osteocalcin; the mineral apposition rate; trabecular bone volume; and bone mineral density all were reduced significantly; however, the number of bone marrow adipocytes and Ppar‐γ expression were increased. These changes were consistent with the skeletal phenotype observed in the PTHrP(1‐84) knockin mouse. The efficiency of colony‐forming unit fibroblast (CFU‐F) formation in bone marrow cultures was decreased, and the percentage of alkaline phosphatase–positive CFU‐F and Runx2 expression were reduced. In contrast, adipocyte formation and Ppar‐γ expression in cultures were increased, and expression of the polycomb protein sirtuin (Sirt1) was reduced. Reduced proliferation and increased apoptosis of BM‐MSCs were associated with upregulation of senescence‐associated tumor‐suppressor genes, including p16, p19, and p27. Analysis of the skeletal phenotype in Bmi‐1^?/? mice suggests that Bmi‐1 functions downstream of PTHrP. Furthermore, our studies indicate that Bmi‐1 maintains self‐renewal of BM‐MSCs by inhibiting the expression of p27, p16, and p19 and alters the cell fate of BM‐MSCs by enhancing osteoblast differentiation and inhibiting adipocyte differentiation at least in part by stimulating Sirt1 expression. Bmi‐1 therefore plays a critical role in promoting osteogenesis. © 2010 American Society for Bone and Mineral Research     

Ablation of Osteopontin Improves the Skeletal Phenotype of Phospho1−/− Mice

PHOSPHO1 and tissue‐nonspecific alkaline phosphatase (TNAP) have nonredundant functions during skeletal mineralization. Although TNAP deficiency (Alpl^?/? mice) leads to hypophosphatasia, caused by accumulation of the mineralization inhibitor inorganic pyrophosphate (PP_i), comparably elevated levels of PP_i in Phospho1^?/? mice do not explain their stunted growth, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis. We have previously shown that elevated PP_i in Alpl^?/? mice is accompanied by elevated osteopontin (OPN), another potent mineralization inhibitor, and that the amount of OPN correlates with the severity of hypophosphatasia in mice. Here we demonstrate that plasma OPN is elevated and OPN expression is upregulated in the skeleton, particularly in the vertebrae, of Phospho1^?/? mice. Liquid chromatography/tandem mass spectrometry showed an increased proportion of phosphorylated OPN (p‐OPN) peptides in Phospho1^?/? mice, suggesting that accumulation of p‐OPN causes the skeletal abnormalities in Phospho1^?/? mice. We also show that ablation of the OPN gene, Spp1, leads to improvements in the skeletal phenotype in Phospho1^?/? as they age. In particular, their scoliosis is ameliorated at 1 month of age and is completely rescued at 3 months of age. There is also improvement in the long bone defects characteristic of Phospho1^?/? mice at 3 months of age. Mineralization assays comparing [Phospho1^?/?; Spp1^?/?], Phospho1^?/?, and Spp1^?/? chondrocytes display corrected mineralization by the double knockout cells. Expression of chondrocyte differentiation markers was also normalized in the [Phospho1^?/?; Spp1^?/?] mice. Thus, although Alpl and Phospho1 deficiencies lead to similar skeletal phenotypes and comparable changes in the expression levels of PP_i and OPN, there is a clear dissociation in the hierarchical roles of these potent inhibitors of mineralization, with elevated PP_i and elevated p‐OPN levels causing the respective skeletal phenotypes in Alpl^?/? and Phospho1^?/? mice. © 2014 American Society for Bone and Mineral Research.     

Elevated serum IGF‐1 levels synergize PTH action on the skeleton only when the tissue IGF‐1 axis is intact

There is growing evidence that insulin‐like growth factor 1 (IGF‐1) and parathyroid hormone (PTH) have synergistic actions on bone and that part of the anabolic effects of PTH is mediated by local production of IGF‐1. In this study we analyzed the skeletal response to PTH in mouse models with manipulated endocrine or autocrine/paracrine IGF‐1. We used mice carrying a hepatic IGF‐1 transgene (HIT), which results in a threefold increase in serum IGF‐1 levels and normal tissue IGF‐1 expression, and Igf1 null mice with blunted IGF‐1 expression in tissues but threefold increases in serum IGF‐1 levels (KO‐HIT). Evaluation of skeletal growth showed that elevations in serum IGF‐1 in mice with Igf1 gene ablation in all tissues except the liver (KO‐HIT) resulted in a restoration of skeletal morphology and mechanical properties by adulthood. Intermittent PTH treatment of adult HIT mice resulted in increases in serum osteocalcin levels, femoral total cross‐sectional area, cortical bone area and cortical bone thickness, as well as bone mechanical properties. We found that the skeletal response of HIT mice to PTH was significantly higher than that of control mice, suggesting synergy between IGF‐1 and PTH on bone. In sharp contrast, although PTH‐treated KO‐HIT mice demonstrated an anabolic response in cortical and trabecular bone compartments compared with vehicle‐treated KO‐HIT mice, their response was identical to that of PTH‐treated control mice. We conclude that (1) in the presence of elevated serum IGF‐1 levels, PTH can exert an anabolic response in bone even in the total absence of tissue IGF‐1, and (2) elevations in serum IGF‐1 levels synergize PTH action on bone only if the tissue IGF‐1 axis is intact. Thus enhancement of PTH anabolic actions depends on tissue IGF‐1. © 2010 American Society for Bone and Mineral Research.     

The calcium‐sensing receptor and 25‐hydroxyvitamin D–1α‐hydroxylase interact to modulate skeletal growth and bone turnover

We examined parathyroid and skeletal function in 3‐month‐old mice expressing the null mutation for 25‐hydroxyvitamin D–1α‐hydroxylase [1α(OH)ase^?/?] and in mice expressing the null mutation for both the 1α(OH)ase and the calcium‐sensing receptor [Casr^?/?1α(OH)ase^?/?] genes. On a normal diet, all mice were hypocalcemic, with markedly increased parathyroid hormone (PTH), increased trabecular bone volume, increased osteoblast activity, poorly mineralized bone, enlarged and distorted cartilaginous growth plates, and marked growth retardation, especially in the compound mutants. Osteoclast numbers were reduced in the Casr^?/?1α(OH)ase^?/? mice. On a high‐lactose, high‐calcium, high‐phosphorus “rescue” diet, serum calcium and PTH were normal in the 1α(OH)ase^?/? mice but increased in the Casr^?/?1α(OH)ase^?/? mice with reduced serum phosphorus. Growth plate architecture and mineralization were improved in both mutants, but linear growth of the double mutants remained abnormal. Mineralization of bone improved in all mice, but osteoblast activity and trabecular bone volume remained elevated in the Casr^?/?1α(OH)ase^?/? mice. These studies support a role for calcium‐stimulated maturation of the cartilaginous growth plate and mineralization of the growth plate and bone and calcium‐stimulated CaSR‐mediated effects on bone resorption. PTH‐mediated bone resorption may require calcium‐stimulated CaSR‐mediated enhancement of osteoclastic activity. © 2010 American Society for Bone and Mineral Research. © 2010 American Society for Bone and Mineral Research     

IGF‐I Signaling in Osterix‐Expressing Cells Regulates Secondary Ossification Center Formation,Growth Plate Maturation,and Metaphyseal Formation During Postnatal Bone Development

To investigate the role of IGF‐I signaling in osterix (OSX)‐expressing cells in the skeleton, we generated IGF‐I receptor (IGF‐IR) knockout mice (^OSXIGF‐IRKO) (floxed‐IGF‐IR mice × OSX promoter‐driven GFP‐labeled cre‐recombinase [^OSXGFPcre]), and monitored postnatal bone development. At day 2 after birth (P2), ^OSXGFP‐cre was highly expressed in the osteoblasts in the bone surface of the metaphysis and in the prehypertrophic chondrocytes (PHCs) and inner layer of perichondral cells (IPCs). From P7, ^OSXGFP‐cre was highly expressed in PHCs, IPCs, cartilage canals (CCs), and osteoblasts (OBs) in the epiphyseal secondary ossification center (SOC), but was only slightly expressed in the OBs in the metaphysis. Compared with the control mice, the IPC proliferation was decreased in the ^OSXIGF‐IRKOs. In these mice, fewer IPCs invaded into the cartilage, resulting in delayed formation of the CC and SOC. Immunohistochemistry indicated a reduction of vessel number and lower expression of VEGF and ephrin B2 in the IPCs and SOC of ^OSXIGF‐IRKOs. Quantitative real‐time PCR revealed that the mRNA levels of the matrix degradation markers, MMP‐9, 13 and 14, were decreased in the ^OSXIGF‐IRKOs compared with the controls. The ^OSXIGF‐IRKO also showed irregular morphology of the growth plate and less trabecular bone in the tibia and femur from P7 to 7 weeks, accompanied by decreased chondrocyte proliferation, altered chondrocyte differentiation, and decreased osteoblast differentiation. Our data indicate that during postnatal bone development, IGF‐I signaling in OSX‐expressing IPCs promotes IPC proliferation and cartilage matrix degradation and increases ephrin B2 production to stimulate vascular endothelial growth factor (VEGF) expression and vascularization. These processes are required for normal CC formation in the establishment of the SOC. Moreover, IGF‐I signaling in the OSX‐expressing PHC is required for growth plate maturation and osteoblast differentiation in the development of the metaphysis. © 2015 American Society for Bone and Mineral Research.     

Increased Osteopontin Contributes to Inhibition of Bone Mineralization in FGF23‐Deficient Mice

Excessive FGF23 has been identified as a pivotal phosphaturic factor leading to renal phosphate‐wasting and the subsequent development of rickets and osteomalacia. In contrast, loss of FGF23 in mice (Fgf23^?/?) leads to high serum phosphate, calcium, and 1,25‐vitamin D levels, resulting in early lethality attributable to severe ectopic soft‐tissue calcifications and organ failure. Paradoxically, Fgf23^?/? mice exhibit a severe defect in skeletal mineralization despite high levels of systemic mineral ions and abundant ectopic mineralization, an abnormality that remains largely unexplained. Through use of in situ hybridization, immunohistochemistry, and immunogold labeling coupled with electron microscopy of bone samples, we discovered that expression and accumulation of osteopontin (Opn/OPN) was markedly increased in Fgf23^?/? mice. These results were confirmed by qPCR analyses of Fgf23^?/? bones and ELISA measurements of serum OPN. To investigate whether elevated OPN levels were contributing to the bone mineralization defect in Fgf23^?/? mice, we generated Fgf23^?/?/Opn^?/? double‐knockout mice (DKO). Biochemical analyses showed that the hypercalcemia and hyperphosphatemia observed in Fgf23^?/? mice remained unchanged in DKO mice; however, micro‐computed tomography (µCT) and histomorphometric analyses showed a significant improvement in total mineralized bone volume. The severe osteoidosis was markedly reduced and a normal mineral apposition rate was present in DKO mice, indicating that increased OPN levels in Fgf23^?/? mice are at least in part responsible for the osteomalacia. Moreover, the increased OPN levels were significantly decreased upon lowering serum phosphate by feeding a low‐phosphate diet or after deletion of NaPi2a, indicating that phosphate levels contribute in part to the high OPN levels in Fgf23^?/? mice. In summary, our results suggest that increased OPN is an important pathogenic factor mediating the mineralization defect and the alterations in bone metabolism observed in Fgf23^?/? bones. © 2014 American Society for Bone and Mineral Research.     

B cells assist allograft rejection in the deficiency of protein kinase c‐theta

We have previously shown that mice deficient in protein kinase C theta (PKCθ) have the ability to reject cardiac allografts, but are susceptible to tolerance induction. Here we tested role of B cells in assisting alloimmune responses in the absence of PKCθ. Mouse cardiac allograft transplantations were performed from Balb/c (H‐2d) to PKCθ knockout (PKCθ^?/?), PKCθ and B cell double‐knockout (PBDK, H‐2b) mice and wild‐type (WT) C57BL/6 (H‐2b) mice. PBDK mice spontaneously accepted the allografts with the inhibition of NF‐κB activation in the donor cardiac allograft. Anti‐B cell antibody (rituximab) significantly delayed allograft rejection in PKCθ^?/?, but not in WT mice. Co‐transfer of PKCθ^?/? T plus PKCθ^?/? B cells or primed sera triggered allograft rejection in Rag1^?/? mice, and only major histocompatibility complex class II‐enriched B cells, but not class I‐enriched B cells, were able to promote rejection. This, together with the inability of PKCθ^?/? and CD28^?/? double‐deficient (PCDK) mice to acutely reject allografts, suggested that an effective cognate interaction between PKCθ^?/? T and B cells for acute rejection is CD28 molecule dependent. We conclude that T–B cell interactions synergize with PKCθ^?/? T cells to mediate acute allograft rejection.     

Fracture healing is accelerated in the absence of the adaptive immune system

Fracture healing is a unique biologic process starting with an initial inflammatory response. As in other regenerative processes, bone and the immune system interact closely during fracture healing. This project was aimed at further elucidating how the host immune system participates in fracture healing. A standard closed femoral fracture was created in wild‐type (WT) and recombination activating gene 1 knockout (RAG1^?/?) mice lacking the adaptive immune system. Healing was investigated using micro–computed tomography (µCT), biomechanical testing, and histologic and mRNA expression analyses. Biomechanical testing demonstrated a significantly higher torsional moment on days 14 and 21 in the RAG1^?/? mice compared to the WT group. µCT evaluation of RAG1^?/? specimens showed earlier mineralization and remodeling. Histologically, endochondral ossification and remodeling were accelerated in the RAG1^?/? compared with the WT mice. Histomorphometric analysis on day 7 showed a significantly higher fraction of bone and a significantly lower fraction of cartilage in the callus of the RAG1^?/? mice than in the WT mice. Endochondral ossification was accelerated in the RAG1^?/? mice. Lymphocytes were present during the physiologic repair process, with high numbers in the hematoma on day 3 and during formation of the hard callus on day 14 in the WT mice. Expression of inflammatory cytokines was reduced in the RAG1^?/? mice. In contrast, expression of anti‐inflammatory interleukin 10 (IL‐10) was strongly upregulated in RAG1^?/? mice, indicating protective effects. This study revealed an unexpected phenotype of enhanced fracture healing in RAG1^?/? mice, suggesting detrimental functions of lymphocytes on fracture healing. The shift from proinflammatory to anti‐inflammatory cytokines suggests that immunomodulatory intervention strategies that maximise the regenerative and minimize the destructive effects of inflammation may lead to enhanced fracture repair. © 2011 American Society for Bone and Mineral Research.     

Glutamate Receptor Interacting Protein 1 Regulates CD4+ CTLA‐4 Expression and Transplant Rejection

PDZ domains are common 80‐ to 90‐amino‐acid regions named after the first three proteins discovered to share these domains: postsynaptic density 95, discs large, and zonula occludens. PDZ domain‐containing proteins typically interact with the C‐terminus of membrane receptors. Glutamate receptor interacting protein 1 (GRIP1), a seven–PDZ domain protein scaffold, regulates glutamate receptor surface expression and trafficking in neurons. We have found that human and mouse T cells also express GRIP1. T cell–specific GRIP1^?/? mice >11 weeks old had prolonged cardiac allograft survival. Compared with wild‐type T cells, in vitro stimulated GRIP1^?/? T cells had decreased expression of activation markers and increased apoptotic surface marker expression. Surface expression of the strong T cell inhibitory molecule cytotoxic T lymphocyte antigen‐4 (CTLA‐4) was increased on GRIP1^?/? T cells from mice >11 weeks old. CTLA‐4 increases with T cell stimulation and its surface expression on GRIP1^?/? T cells remained high after stimulation was removed, indicating a possible internalization defect in GRIP1‐deficient T cells. CTLA‐4–blocking antibody treatment following heart transplantation led to complete rejection in T cell GRIP1^?/? mice, indicating that increased CTLA‐4 surface expression contributed to the extended graft survival. Our data indicate that GRIP1 regulates T cell activation by regulating CTLA‐4 surface expression.     

SDF‐1/CXCR4 Axis in Tie2‐Lineage Cells Including Endothelial Progenitor Cells Contributes to Bone Fracture Healing

CXC chemokine receptor 4 (CXCR4) is a specific receptor for stromal‐derived‐factor 1 (SDF‐1). SDF‐1/CXCR4 interaction is reported to play an important role in vascular development. On the other hand, the therapeutic potential of endothelial progenitor cells (EPCs) in fracture healing has been demonstrated with mechanistic insight of vasculogenesis/angiogenesis and osteogenesis enhancement at sites of fracture. The purpose of this study was to investigate the influence of the SDF‐1/CXCR4 pathway in Tie2‐lineage cells (including EPCs) in bone formation. We created CXCR4 gene conditional knockout mice using the Cre/loxP system and set two groups of mice: Tie2‐Cre^ER CXCR4 knockout mice (CXCR4^?/?) and wild‐type mice (WT). We report here that in vitro, EPCs derived from of CXCR4^?/? mouse bone marrow demonstrated severe reduction of migration activity and EPC colony‐forming activity when compared with those derived from WT mouse bone marrow. In vivo, radiological and morphological examinations showed fracture healing delayed in the CXCR4^?/? group and the relative callus area at weeks 2 and 3 was significantly smaller in CXCR4^?/? group mice. Quantitative analysis of capillary density at perifracture sites also showed a significant decrease in the CXCR4^?/? group. Especially, CXCR4^?/?group mice demonstrated significant early reduction of blood flow recovery at fracture sites compared with the WT group in laser Doppler perfusion imaging analysis. Real‐time RT‐PCR analysis showed that the gene expressions of angiogenic markers (CD31, VE‐cadherin, vascular endothelial growth factor [VEGF]) and osteogenic markers (osteocalcin, collagen 1A1, bone morphogenetic protein 2 [BMP2]) were lower in the CXCR4^?/? group. In the gain‐of‐function study, the fracture in the SDF‐1 intraperitoneally injected WT group healed significantly faster with enough callus formation compared with the SDF‐1 injected CXCR4^?/? group. We demonstrated that an EPC SDF‐1/CXCR4 axis plays an important role in bone fracture healing using Tie2‐Cre^ER CXCR4 conditional knockout mice. © 2014 American Society for Bone and Mineral Research.     

Cyclin‐Dependent Kinase Inhibitor‐1‐Deficient Mice are Susceptible to Osteoarthritis Associated with Enhanced Inflammation

Osteoarthritis (OA) is a multifactorial disease, and recent data suggested that cell cycle–related proteins play a role in OA pathology. Cyclin‐dependent kinase (CDK) inhibitor 1 (p21) regulates activation of other CDKs, and recently, we reported that p21 deficiency induced susceptibility to OA induced by destabilization of the medial meniscus (DMM) surgery through STAT3‐signaling activation. However, the mechanisms associated with why p21 deficiency led to susceptibility to OA by the STAT3 pathway remain unknown. Therefore, we focused on joint inflammation to determine the mechanisms associated with p21 function during in vitro and in vivo OA progression. p21‐knockout (p21^?/?) mice were used to develop an in vivo OA model, and C57BL/6 (p21^+/+) mice with the same background as the p21^?/? mice were used as controls. Morphogenic changes were measured using micro‐CT, IL‐1β serum levels were detected by ELISA, and histological or immunohistological analyses were performed. Our results indicated that p21‐deficient DMM‐model mice exhibited significant subchondral bone destruction and cartilage degradation compared with wild‐type mice. Immunohistochemistry results revealed p21^?/? mice susceptibility to OA changes accompanied by macrophage infiltration and enhanced MMP‐3 and MMP‐13 expression through IL‐1β‐induced NF‐κB signaling. p21^?/? mice also showed subchondral bone destruction according to micro‐CT analysis, and cathepsin K staining revealed increased numbers of osteoclasts. Furthermore, p21^?/? mice displayed increased serum IL‐1β levels, and isolated chondrocytes from p21^?/? mice indicated elevated MMP‐3 and MMP‐13 expression with phosphorylation of IκB kinase complex in response to IL‐1β stimulation, whereas treatment with a specific p‐IκB kinase inhibitor attenuated MMP‐3 and MMP‐13 expression. Our results indicated that p21‐deficient DMM mice were susceptible to alterations in OA phenotype, including enhanced osteoclast expression, macrophage infiltration, and MMP expression through IL‐1β‐induced NF‐κB signaling, suggesting that p21 regulation may constitute a possible therapeutic strategy for OA treatment. © 2017 American Society for Bone and Mineral Research.     

Depletion of annexin A5, annexin A6, and collagen X causes no gross changes in matrix vesicle–mediated mineralization,but lack of collagen X affects hematopoiesis and the Th1/Th2 response

Numerous biochemical studies have pointed to an essential role of annexin A5 (AnxA5), annexin A6 (AnxA6), and collagen X in matrix vesicle–mediated biomineralization during endochondral ossification and in osteoarthritis. By binding to the extracellular matrix protein collagen X and matrix vesicles, annexins were proposed to anchor matrix vesicles in the extracellular space of hypertrophic chondrocytes to initiate the calcification of cartilage. However, mineralization appears to be normal in mice lacking AnxA5 and AnxA6, whereas collagen X–deficient mice show only subtle alterations in the growth plate organization. We hypothesized that the simultaneous lack of AnxA5, AnxA6, and collagen X in vivo induces more pronounced changes in the growth plate development and the initiation of mineralization. In this study, we generated and analyzed mice deficient for AnxA5, AnxA6, and collagen X. Surprisingly, mice were viable, fertile, and showed no obvious abnormalities. Assessment of growth plate development indicated that the hypertrophic zone was expanded in Col10a1^?/? and AnxA5^?/?AnxA6^?/?Col10a1^?/? newborns, whereas endochondral ossification and mineralization were not affected in 13‐day‐ and 1‐month‐old mutants. In peripheral quantitative computed tomography, no changes in the degree of biomineralization were found in femora of 1‐month‐ and 1‐year‐old mutants even though the diaphyseal circumference was reduced in Col10a1^?/? and AnxA5^?/?AnxA6^?/?Col10a1^?/? mice. The percentage of naive immature IgM⁺/IgM⁺ B cells and peripheral T‐helper cells were increased in Col10a1^?/? and AnxA5^?/?AnxA6^?/?Col10a1^?/? mutants, and activated splenic T cells isolated from Col10a1^?/? mice secreted elevated levels of IL‐4 and GM‐CSF. Hence, collagen X is needed for hematopoiesis during endochondral ossification and for the immune response, but the interaction of annexin A5, annexin A6, and collagen X is not essential for physiological calcification of growth plate cartilage. Therefore, annexins and collagen X may rather fulfill functions in growth plate cartilage not directly linked to the mineralization process. © 2012 American Society for Bone and Mineral Research.     

Axin2 regulates chondrocyte maturation and axial skeletal development

Axis inhibition proteins 1 and 2 (Axin1 and Axin2) are scaffolding proteins that modulate at least two signaling pathways that are crucial in skeletogenesis: the Wnt/β‐catenin and TGF‐β signaling pathways. To determine whether Axin2 is important in skeletogenesis, we examined the skeletal phenotype of Axin2‐null mice in a wild‐type or Axin1^+/? background. Animals with disrupted Axin2 expression displayed a runt phenotype when compared to heterozygous littermates. Whole‐mount and tissue β‐galactosidase staining of Axin2^LacZ/LacZ mice revealed that Axin2 is expressed in cartilage tissue, and histological sections from knockout animals showed shorter hypertrophic zones in the growth plate. Primary chondrocytes were isolated from Axin2‐null and wild‐type mice, cultured, and assayed for type X collagen gene expression. While type II collagen levels were depressed in cells from Axin2‐deficient animals, type X collagen gene expression was enhanced. There was no difference in BrdU incorporation between null and heterozygous mice, suggesting that loss of Axin2 does not alter chondrocyte proliferation. Taken together, these findings reveal that disruption of Axin2 expression results in accelerated chondrocyte maturation. In the presence of a heterozygous deficiency of Axin1, Axin2 was also shown to play a critical role in craniofacial and axial skeleton development. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:89–95, 2010     

NAD(P)H‐quinone oxidoreductase 1 silencing aggravates hormone‐induced prostatic hyperplasia in mice

NAD(P)H‐quinone oxidoreductase 1 (NQO1) is a highly inducible flavoprotein known to involve in various cellular defence mechanisms. In this study, we explored whether NQO1 deletion affects hormone‐induced prostatic hyperplasia. Testosterone propionate (3 mg/kg, IP) was injected into wild‐type (WT) and NOQ1 knockout C57BL/6 mice (NQO1^?/?) for 14 consecutive days, and the samples were collected for biological and histochemical studies. The testosterone‐treated NQO1^?/? showed about 140% higher prostate weight than the testosterone‐treated WT, with enhanced connective tissue and hyperplastic glands formations. However, increased dihydrotestosterone level after testosterone treatment was not significantly different between the WT and NQO1^?/?. In contrast, the enhanced nuclear expression of proliferating cell nuclear antigen in NQO1^?/? prostate confirmed aggravated prostatic hyperplasia in NQO1^?/?. Moreover, the expression of heat shock protein (HSP) 90‐α was markedly increased in the NQO1^?/?, and this was supported by increased testosterone‐induced nuclear androgen receptor expression in NQO1‐silenced LNCaP cells. Testosterone‐induced prostate‐specific antigen expression was not reversed in NOQ1‐silenced cells after finasteride treatment. Although the exact role of NQO1 in prostatic hyperplasia remains unclear, the hyperplasia exacerbation due to NQO1 deletion might be independent of type 2 5α‐reductase and might be related to enhanced androgen receptor affinity due to enhanced HSP90‐α expression.     

Skeletal recovery after weaning does not require PTHrP

Mice lose 20% to 25% of trabecular bone mineral content (BMC) during lactation and restore it after weaning through unknown mechanisms. We found that tibial Pthrp mRNA expression was upregulated fivefold by 7 days after weaning versus end of lactation in wild‐type (WT) mice. To determine whether parathyroid hormone–related protein (PTHrP) stimulates bone formation after weaning, we studied a conditional knockout in which PTHrP is deleted from preosteoblasts and osteoblasts by collagen I promoter–driven Cre (Cre^ColI). These mice are osteopenic as adults but have normal serum calcium, calcitriol, and parathyroid hormone (PTH). Pairs of Pthrp^flox/flox;Cre^ColI (null) and WT;Cre^ColI (WT) females were mated and studied through pregnancy, lactation, and 3 weeks of postweaning recovery. By end of lactation, both genotypes lost lumbar spine BMC: WT declined by 20.6% ± 3.3%, and null decreased by 22.5% ± 3.5% (p < .0001 versus baseline; p = NS between genotypes). During postweaning recovery, both restored BMC to baseline: WT to –3.6% ± 3.7% and null to 0.3% ± 3.7% (p = NS versus baseline or between genotypes). Similar loss and full recovery of BMC were seen at the whole body and hind limb. Histomorphometry confirmed that nulls had lower bone mass at baseline and that this was equal to the value achieved after weaning. Osteocalcin, propeptide of type 1 collagen (P1NP), and deoxypyridinoline increased equally during recovery in WT and null mice; PTH decreased and calcitriol increased equally; serum calcium was unchanged. Urine calcium increased during recovery but remained no different between genotypes. Although osteoblast‐derived PTHrP is required to maintain adult bone mass and Pthrp mRNA upregulates in bone after weaning, it is not required for recovery of bone mass after lactation. The factors that stimulate postweaning bone formation remain unknown. © 2011 American Society for Bone and Mineral Research.