The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow

The ontogeny of bone marrow and its stromal compartment, which is generated from skeletal stem/progenitor cells, was investigated in vivo and ex vivo in mice expressing constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor (PTH/PTHrP; caPPR) under the control of the 2.3-kb bone-specific mouse Col1A1 promoter/enhancer. The transgene promoted increased bone formation within prospective marrow space, but delayed the transition from bone to bone marrow during growth, the formation of marrow cavities, and the appearance of stromal cell types such as marrow adipocytes and cells supporting hematopoiesis. This phenotype resolved spontaneously over time, leading to the establishment of marrow containing a greatly reduced number of clonogenic stromal cells. Proliferative osteoprogenitors, but not multipotent skeletal stem cells (mesenchymal stem cells), capable of generating a complete heterotopic bone organ upon in vivo transplantation were assayable in the bone marrow of caPPR mice. Thus, PTH/PTHrP signaling is a major regulator of the ontogeny of the bone marrow and its stromal tissue, and of the skeletal stem cell compartment.     

Constitutively active parathyroid hormone receptor signaling in cells in osteoblastic lineage suppresses mechanical unloading-induced bone resorption

Multiple signaling pathways participate in the regulation of bone remodeling, and pathological negative balance in the regulation results in osteoporosis. However, interactions of signaling pathways that act comprehensively in concert to maintain bone mass are not fully understood. We investigated roles of parathyroid hormone receptor (PTH/PTHrP receptor) signaling in osteoblasts in unloading-induced bone loss using transgenic mice. Hind limb unloading by tail suspension reduced bone mass in wild-type mice. In contrast, signaling by constitutively active PTH/PTHrP receptor (caPPR), whose expression was regulated by the osteoblast-specific Col1a1 promoter (Col1a1-caPPR), suppressed unloading-induced reduction in bone mass in these transgenic mice. In Col1a1-caPPR transgenic (Tg) mice, hind limb unloading suppressed bone formation parameters in vivo and mineralized nodule formation in vitro similarly to those observed in wild-type mice. In addition, serum osteocalcin levels and mRNA expression levels of type I collagen, Runx2 and Osterix in bone were suppressed by unloading in both wild-type mice and Tg mice. However, in contrast to unloading-induced enhancement of bone resorption parameters in wild-type mice, Col1a1-caPPR signaling suppressed, rather than enhanced, osteoclast number and osteoclast surface as well as urinary deoxypyridinoline excretion upon unloading. Col1a1-caPPR signaling also suppressed mRNA expression levels of RANK and c-fms in bone upon unloading. Although the M-CSF and monocyte chemoattractant protein 1 (MCP-1) mRNA levels were enhanced in control Tg mice, these levels were suppressed in unloaded Tg mice. These results indicated that constitutive activation of PTH/PTHrP receptor signaling in osteoblastic cells suppresses unloading-induced bone loss specifically through the regulation of osteoclastic activity.     

Mechanisms underlying catabolic and anabolic functions of parathyroid hormone on bone by combination of culture systems of mouse cells

Since bone resorption and formation by continuous and intermittent parathyroid hormone (PTH) treatments involve various types of cells in bone, this study examined the underlying mechanism by combining culture systems using mouse primary calvarial osteoblasts and bone marrow cells. The PTH/PTHrP receptor (PTH1R) expression and the cAMP accumulation in response to PTH were increased in accordance with the differentiation of osteoblasts. Osteoclast formation was strongly induced by continuous PTH treatment in the monolayer co‐culture of osteoblasts and bone marrow cells, which was associated with RANKL expression in differentiated osteoblasts. Bone formation determined by ALP activity and the type I collagen mRNA expression was stimulated by intermittent PTH treatment in the monolayer co‐culture and in the bone marrow cell layer of the separated co‐culture in a double chamber dish, but not in the culture of bone marrow cells alone. The stimulation in the separated co‐culture, accompanied by IGF‐I production by osteoblasts, was abolished when bone marrow cells were derived from knockout mice of insulin‐receptor substrate‐1 (IRS‐1?/?) or when osteoblasts were from PTH1R?/? mice. We conclude that differentiated osteoblasts are most likely the direct target of both continuous and intermittent PTH, while bone marrow cells are likely the effector cells. The osteoblasts stimulated by continuous PTH express RANKL which causes osteoclastogenesis from the precursors in bone marrow via cell‐to‐cell contact, leading to bone resorption; while the osteoblasts stimulated by intermittent PTH secrete IGF‐I which activates IRS‐1 in osteoblast precursors in bone marrow via a paracrine mechanism, leading to bone formation. J. Cell. Biochem. 109: 755–763, 2010. © 2010 Wiley‐Liss, Inc.     

Distinct roles for mitogen-activated protein kinase phosphatase-1 (MKP-1) and ERK-MAPK in PTH1R signaling during osteoblast proliferation and differentiation

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) activate one single receptor (PTH1R) which mediates catabolic and anabolic actions in the bone. Activation of PTH1R modulates multiple intracellular signaling responses. We previously reported that PTH and PTHrP down-regulate pERK1/2 and cyclin D1 in differentiated osteoblasts. In this study we investigate the role of MAPK phosphatase-1 (MKP-1) in PTHrP regulation of ERK1/2 activity in relation to osteoblast proliferation, differentiation and bone formation. Here we show that PTHrP increases MKP-1 expression in differentiated osteoblastic MC3T3-E1 cells, primary cultures of differentiated bone marrow stromal cells (BMSCs) and calvarial osteoblasts. PTHrP had no effect on MKP-1 expression in proliferating osteoblastic cells. Overexpression of MKP-1 in MC-4 cells inhibited osteoblastic cell proliferation. Cell extracts from differentiated MC-4 cells treated with PTHrP inactivate/dephosphorylate pERK1/2 in vitro; immunodepletion of MKP-1 blocked the ability of the extract to dephosphorylate pERK1/2; these data indicate that MKP-1 is involved in PTHrP-induced pERK1/2 dephosphorylation in the differentiated osteoblastic cells. PTHrP regulation of MKP-1 expression is partially dependent on PKA and PKC pathways. Treatment of nude mice, bearing ectopic ossicles, with intermittent PTH for 3 weeks, up-regulated MKP-1 and osteocalcin, a bone formation marker, with an increase in bone formation. These data indicate that PTH and PTHrP increase MKP-1 expression in differentiated osteoblasts; and that MKP-1 induces growth arrest of osteoblasts, via inactivating pERK1/2 and down-regulating cyclin D1; and identify MKP-1 as a possible mediator of the anabolic actions of PTH1R in mature osteoblasts.     

Preactivation of β-catenin in osteoblasts improves the osteoanabolic effect of PTH in type 1 diabetic mice

Type 1 diabetes (T1D) is correlated with osteopenia primarily due to low bone formation. Parathyroid hormone (PTH) is a known anabolic agent for bone, the anabolic effects of which are partially mediated through the Wnt/β-catenin signaling pathway. In the present study, we first determined the utility of intermittent PTH treatment in a streptozotocin-induced T1D mouse model. It was shown that the PTH-induced anabolic effects on bone mass and bone formation were attenuated in T1D mice compared with nondiabetic mice. Further, PTH treatment failed to activate β-catenin signaling in osteoblasts of T1D mice and was unable to improve osteoblast proliferation and differentiation. Next, the Col1–3.2 kb-CreERTM; β-cateninfx(ex3) mice were used to conditionally activate β-catenin in osteoblasts by injecting tamoxifen, and we addressed whether or not preactivation of β-catenin boosted the anabolic action of PTH on T1D-related bone loss. The results demonstrated that pretreatment with activation of osteoblastic β-catenin followed by PTH treatment outperformed PTH or β-catenin activation monotherapy and led to greatly improved bone structure, bone mass, and bone strength in this preclinical model of T1DM. Further analysis demonstrated that osteoblast proliferation and differentiation, as well as osteoprogenitors in the marrow, were all improved in the combination treatment group. These findings indicated a clear advantage of developing β-catenin as a target to improve the efficacy of PTH in the treatment of T1D-related osteopenia.     

Impaired osteoclast formation in bone marrow cultures of Fgf2 null mice in response to parathyroid hormone

Fibroblast growth factor (FGF)-2 and parathyroid hormone (PTH) are potent inducers of osteoclast (OCL) formation, and PTH increases FGF-2 mRNA and protein expression in osteoblasts. To elucidate the role of endogenous FGF-2 in PTH responses, we examined PTH-induced OCL formation in bone marrow cultures from wild type and mice with a disruption of the Fgf2 gene. FGF-2-induced OCL formation was similar in marrow culture from both genotypes. In contrast, PTH-stimulated OCL formation in bone marrow cultures or co-cultures of osteoblast-spleen cells from Fgf2-/mice was significantly impaired. PTH increased RANKL mRNA expression in osteoblasts cultures from both genotypes. After 6 days of treatment, osteoprotegerin protein in cell supernatants was 40-fold higher in vehicle-treated and 30-fold higher in PTH-treated co-cultures of osteoblast and spleen cells from Fgf2-/mice compared with Fgf2+/+ mice. However, a neutralizing antibody to osteoprotegerin did not rescue reduced OCL formation in response to PTH. Injection of PTH caused hypercalcemia in Fgf2+/+ but not Fgf2-/mice. We conclude that PTH stimulates OCL formation and bone resorption in mice in part by endogenous FGF-2 synthesis by osteoblasts. Because RANKL- and interleukin-11-induced OCL formation was also reduced in bone marrow cultures from Fgf2-/mice, we further conclude that endogenous FGF-2 is necessary for maximal OCL formation by multiple bone resorbing factors.     

A new bone to pick: osteoblasts and the haematopoietic stem-cell niche

Two recent publications highlight the role of bone-forming cells, the osteoblasts, in controlling the development of neighboring haematopoietic stem cells (HSCs).1,2 Using two distinct transgenic mouse models, one using the conditional deletion of the Bone Morphogenetic Protein Receptor 1A (BMPR1A) gene, the other using over-expression of an active PTH/PTHrP receptor (PPR) mutant within osteoblasts, the authors show parallel, concordant increases in the generation of trabecular osteoblasts and the number of HSCs. In situ staining showed that rarely cycling HSCs sporadically attach to endosteal osteoblasts, while in vitro assays indicated that ligation of Jag1 on osteoblasts by Notch1 on HSCs promotes HSC proliferation. These two independent works have revived and revitalized the notion that osteoblasts are a major, defining component of the HSC niche within the bone marrow (BM). This minireview discusses these results in the context of other recent studies of mesenchymal cells within the BM microenvironment, presents one potential unified model of the functional anatomy of the BM HSC niche, and highlights new questions raised by these and other studies of osteoblasts and HSCs.     

Cthrc1 is a positive regulator of osteoblastic bone formation

Background

Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.

Methodology/Principal Findings

We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1-null mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1-null mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.

Conclusions/Significance

Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.     

Control of bone mass and remodeling by PTH receptor signaling in osteocytes

Osteocytes, former osteoblasts buried within bone, are thought to orchestrate skeletal adaptation to mechanical stimuli. However, it remains unknown whether hormones control skeletal homeostasis through actions on osteocytes. Parathyroid hormone (PTH) stimulates bone remodeling and may cause bone loss or bone gain depending on the balance between bone resorption and formation. Herein, we demonstrate that transgenic mice expressing a constitutively active PTH receptor exclusively in osteocytes exhibit increased bone mass and bone remodeling, as well as reduced expression of the osteocyte-derived Wnt antagonist sclerostin, increased Wnt signaling, increased osteoclast and osteoblast number, and decreased osteoblast apoptosis. Deletion of the Wnt co-receptor LDL related receptor 5 (LRP5) attenuates the high bone mass phenotype but not the increase in bone remodeling induced by the transgene. These findings demonstrate that PTH receptor signaling in osteocytes increases bone mass and the rate of bone remodeling through LRP5-dependent and -independent mechanisms, respectively.     

Mechanisms of bone metastasis formation

Bone is a common metastatic site for many cancers. Tumor cells located in the bone marrow cavity disturb the natural balance (bone remodelling) established between new bone formation performed by osteoblasts and bone resorption carried out by osteoclasts. Tumor cells produce many factors including growth factors and cytokines (PTHrP, ET-1, BMPs, others...) that stimulate either ostoclast activity leading to osteolytic lesions or osteoblast activity generating osteosclerotic bone metastases. Growth factors released from resorbed bone matrix or throughout osteoblastic bone formation sustain tumor growth. Therefore, bone metastases are the site of vicious cycles wherein tumor growth and bone metabolism sustain each other.     

Heparanase is expressed in osteoblastic cells and stimulates bone formation and bone mass

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules. In bone, they are associated with cell surfaces and the extracellular matrix (ECM). The heparan sulfate (HS) chains of HSPGs bind a multitude of bioactive molecules, thereby controlling normal and pathologic processes. The HS-degrading endoglycosidase, heparanase, has been implicated in processes such as inflammation, vascularization associated with wound healing and malignancies, and cancer metastasis. Here we show progressive mRNA expression of the hpa gene (encoding heparanase) in murine bone marrow stromal cells undergoing osteoblastic (bone forming) differentiation and in primary calvarial osteoblasts. Bone marrow stromal cells derived from transgenic mice expressing recombinant human heparanase (rh-heparanase) and MC3T3 E1 osteoblastic cells exposed to soluble rh-heparanase spontaneously undergo osteogenic differentiation. In addition, the transgenic bone marrow stromal cells degrade HS chains. In wild-type (WT) and hpa-transgenic (hpa-tg) mice, heparanase is weakly expressed throughout the bone marrow with a substantial increase in osteoblasts and osteocytes, especially in the hpa-tg mice. Heparanase expression was absent in osteoclasts. Micro-computed tomographic and histomorphometric skeletal analyses in male and female hpa-tg versus WT mice show markedly increased trabecular bone mass, cortical thickness, and bone formation rate, but no difference in osteoclast number. Collectively, our data suggest that proteoglycans tonically suppress osteoblast function and that this inhibition is alleviated by HS degradation with heparanase.     

Fibroblast growth factor 2 stimulation of osteoblast differentiation and bone formation is mediated by modulation of the Wnt signaling pathway

Fibroblast growth factor 2 (FGF2) positively modulates osteoblast differentiation and bone formation. However, the mechanism(s) is not fully understood. Because the Wnt canonical pathway is important for bone homeostasis, this study focuses on modulation of Wnt/β-catenin signaling using Fgf2(-/-) mice (FGF2 all isoforms ablated), both in the absence of endogenous FGF2 and in the presence of exogenous FGF2. This study demonstrates a role of endogenous FGF2 in bone formation through Wnt signaling. Specifically, mRNA expression for the canonical Wnt genes Wnt10b, Lrp6, and β-catenin was decreased significantly in Fgf2(-/-) bone marrow stromal cells during osteoblast differentiation. In addition, a marked reduction of Wnt10b and β-catenin protein expression was observed in Fgf2(-/-) mice. Furthermore, Fgf2(-/-) osteoblasts displayed marked reduction of inactive phosphorylated glycogen synthase kinase-3β, a negative regulator of Wnt/β-catenin pathway as well as a significant decrease of Dkk2 mRNA, which plays a role in terminal osteoblast differentiation. Addition of exogenous FGF2 promoted β-catenin nuclear accumulation and further partially rescued decreased mineralization in Fgf2(-/-) bone marrow stromal cell cultures. Collectively, our findings suggest that FGF2 stimulation of osteoblast differentiation and bone formation is mediated in part by modulating the Wnt pathway.     

Expression and activity of osteoblast-targeted Cre recombinase transgenes in murine skeletal tissues

The Cre/loxP recombination system can be used to circumvent many of the limitations of generalized gene ablation in mice. Here we present the development and characterization of transgenic mice in which Cre recombinase has been targeted to cells of the osteoblast lineage with 2.3 kb (Col 2.3-Cre) and 3.6 kb (Col 3.6-Cre) fragments of the rat Col1a1 promoter. Cre mRNA was detected in calvaria and long bone of adult Col 2.3-Cre and Col 3.6-Cre mice, as well as in tendon and skin of Col 3.6-Cre mice. To obtain a historical marking of the temporal and spatial pattern of Cre-mediated gene rearrangement, Col-Cre mice were bred with ROSA26 (R26R) mice in which Cre-mediated excision of a floxed cassette results in LacZ expression. In Col 2.3-Cre;R26R and Col 3.6-Cre;R26R progeny, calvarial and long bone osteoblasts showed intense beta-gal staining at embryonic day 18 and postnatal day 5. The spatial pattern of beta-gal staining was more restricted in bone and in bone marrow stromal cultures established from Col 2.3-Cre;R26R mice. Similar differences in the spatial patterns of expression were seen in transgenic bone carrying Col1a1-GFP visual reporters. Our data suggest that Col 2.3-Cre and Col 3.6-Cre transgenic mice may be useful for conditional gene targeting in vivo or for obtaining osteoblast populations for in vitro culture in which a gene of interest has been inactivated.     

PTHrP, PTH, and the PTH/PTHrP receptor in endochondral bone development

Endochondral bone development is a fascinating story of proliferation, maturation, and death. An understanding of this process at the molecular level is emerging. In particular, significant advances have been made in understanding the role of parathyroid-hormone-related peptide (PTHrP), parathyroid hormone (PTH), and the PTH/PTHrP receptor in endochondral bone development. Mutations of the PTH/PTHrP receptor have been identified in Jansen metaphyseal chondrodysplasia, Blomstrand's lethal chondrodysplasia, and enchondromatosis. Furthermore, genetic manipulations of the PTHrP, PTH, and the PTH/PTHrP receptor genes, respectively, have demonstrated the critical role of these proteins in regulating both the switch between proliferation and differentiation of chondrocytes, and their replacement by bone cells. A future area of investigation will be the identification of downstream effectors of PTH, PTHrP, and PTH/PTHrP receptor activities. Furthermore, it will be of critical importance to study how these proteins cooperate and integrate with other molecules that are essential for growth plate development.     

Recql4 haploinsufficiency in mice leads to defects in osteoblast progenitors: Implications for low bone mass phenotype

The cellular and molecular mechanisms that underlie skeletal abnormalities in defective Recql4-related syndromes are poorly understood. Our objective in this study was to explore the function of Recql4 in osteoblast biology both in vitro and in vivo. Immunohistochemistry on adult mouse bone showed Recql4 protein localization in active osteoblasts around growth plate, but not in fully differentiated osteocytes. Consistent with this finding, Recql4 gene expression was high in proliferating mouse osteoblastic MC3T3.E1 cells and decreased as cells progressively lost their proliferation activity during differentiation. Recql4 overexpression in osteoblastic cells exhibited higher proliferation activity, while its depletion impeded cell growth. In addition, bone marrow stromal cells from male Recql4+/- mice had fewer progenitor cells, including osteoprogenitors, indicated by reduced total fibroblast colony forming units (CFU-f) and alkaline phosphatase-positive CFU-f colonies concomitant with reduced bone mass. These findings provide evidence that Recql4 functions as a regulatory protein during osteoprogenitor proliferation, a critical cellular event during skeleton development.