KMN-159, a novel EP4 receptor selective agonist, stimulates osteoblastic differentiation in cultured whole rat bone marrow.

KMN-159 is the lead compound from a series of novel difluorolactam prostanoid EP4 receptor agonists aimed at inducing local bone formation while avoiding the inherent side effects of systemic EP4 activation. KMN-159 is a potent, selective small molecule possessing pharmacokinetic properties amenable to local administration. Unfractionated rat bone marrow cells (BMCs) were treated once at plating with escalating doses of KMN-159 (1 pM to 10 µM). The resulting elevated alkaline phosphatase (ALP) levels measured 9 days post-dose are consistent with increased osteoblastic differentiation and exposure to KMN-159 at low nanomolar concentrations for as little as 30 min was sufficient to induce complete osteoblast differentiation of the BMCs from both sexes and regardless of age. ALP induction was blocked by an EP4 receptor antagonist but not by EP1 or EP2 receptor antagonists and was not induced by EP2 or EP3 receptor agonists. Addition of BMCs to plates coated with KMN-159 24 days earlier resulted in ALP activation, highlighting the chemical stability of the compound. The expression of phenotype markers such as ALP, type I collagen, and osteocalcin was significantly elevated throughout the osteoblastic differentiation timecourse initiated by KMN-159 stimulation. An increased number of tartrate-resistant acid phosphatase-positive cells was observed KMN-159 or PGE2 treated BMCs but only in the presence of exogenous receptor activator of nuclear factor kappa-Β ligand (RANKL). No change in the number of adipocytes was observed. KMN-159 also increased bone healing in a rat calvarial defect model with a healing rate equivalent to recombinant human bone morphogenetic protein-2. Our studies show that KMN-159 is able to stimulate osteoblastic differentiation with a very short time of exposure, supporting its potential as a therapeutic candidate for augmenting bone mass.

Difluoromethylene at the γ-Lactam α-Position Improves 11-Deoxy-8-aza-PGE1 Series EP4 Receptor Binding and Activity: 11-Deoxy-10,10-difluoro-8-aza-PGE1 Analog (KMN-159) as a Potent EP4 Agonist.

A series of small-molecule full agonists of the prostaglandin E2 type 4 (EP4) receptor have been generated and evaluated for binding affinity and cellular potency. KMN-80 and its gem-difluoro analog KMN-159 possess high selectivity relative to other prostanoid receptors. Difluoro substitution is positioned alpha to the lactam ring carbonyl and results in KMN-159's fivefold increase in potency versus KMN-80. The two analogs exhibit electronic and conformational variations, including altered nitrogen hybridization and lactam ring puckering, that may drive the observed difluoro-associated increased potency within this four-compound series.

Transgenic Expression of Osteoactivin/gpnmb Enhances Bone Formation In Vivo and Osteoprogenitor Differentiation Ex Vivo.

Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased threefold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post-fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV-promoter (OA-Tg). Western blot analysis showed increased OA/gpnmb in OA-Tg osteoblasts, compared to wild-type (WT). In OA-Tg mouse femurs versus WT littermates, micro-CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA-Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over-expressed in osteoclasts in OA-Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX-1 and RANK-L, and decreased osteoclast numbers in OA-Tg, compared to WT mice, indicating decreased bone remodeling in OA-Tg mice. The proliferation rate of OA-Tg osteoblasts in vitro was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA-Tg osteoprogenitors. Quantitative RT-PCR analysis showed increased TGF-ß1 and TGF-ß receptors I and II expression in OA-Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo.

The role of connective tissue growth factor (CTGF/CCN2) in skeletogenesis.

Connective tissue growth factor (CTGF) is a 38 kDa, cysteine rich, extracellular matrix protein composed of 4 domains or modules. CTGF has been shown to regulate a diverse array of cellular functions and has been implicated in more complex biological processes such as angiogenesis, chondrogenesis, and osteogenesis. A role for CTGF in the development and maintenance of skeletal tissues first came to light in studies demonstrating its expression in cartilage and bone cells, which was dramatically increased during skeletal repair or regeneration. The physiological significance of CTGF in skeletogenesis was confirmed in CTGF-null mice, which exhibited multiple skeletal dysmorphisms as a result of impaired growth plate chondrogenesis, angiogenesis, and bone formation/mineralization. Given the emerging importance of CTGF in osteogenesis and chondrogenesis, this review will focus on its expression in skeletal tissues, its effects on osteoblast and chondrocyte differentiation and function, and the skeletal implications of ablation or over-expression of CTGF in knockout or transgenic mouse models, respectively. In addition, this review will examine the role of integrin-mediated signaling and the regulation of CTGF expression as it relates to skeletogenesis. We will emphasize CTGF studies in bone or bone cells, and will identify opportunities for future investigations concerning CTGF and chondrogenesis/osteogenesis.

Temporal and spatial expression of osteoactivin during fracture repair.

We previously identified osteoactivin (OA) as a novel secreted osteogenic factor with high expression in developing long bones and calvaria, and that stimulates osteoblast differentiation and matrix mineralization in vitro. In this study, we report on OA mRNA and protein expression in intact long bone and growth plate, and in fracture calluses collected at several time points up to 21 days post-fracture (PF). OA mRNA and protein were highly expressed in osteoblasts localized in the metaphysis of intact tibia, and in hypertrophic chondrocytes localized in growth plate, findings assessed by in situ hybridization and immunohistochemistry, respectively. Using a rat fracture model, Northern blot analysis showed that expression of OA mRNA was significantly higher in day-3 and day-10 PF calluses than in intact rat femurs. Using in situ hybridization, we examined OA mRNA expression during fracture healing and found that OA was temporally regulated, with positive signals seen as early as day-3 PF, reaching a maximal intensity at day-10 PF, and finally declining at day-21 PF. At day-5 PF, which correlates with chondrogenesis, OA mRNA levels were significantly higher in the soft callus than in intact femurs. Similarly, we detected high OA protein immunoexpression throughout the reparative phase of the hard callus compared to intact femurs. Interestingly, the secreted OA protein was also detected within the newly made cartilage matrix and osteoid tissue. Taken together, these results suggest the possibility that OA plays an important role in bone formation and serves as a positive regulator of fracture healing.

Functional roles of osteoactivin in normal and disease processes.

Osteoactivin (OA) protein was discovered in bone cells a decade ago. Recent literature suggests that osteoactivin is crucial for the differentiation and functioning of different cell types, including bone-forming osteoblasts and bone-resorbing osteoclast cells. Here, we review the literature to date on various regulatory functions of osteoactivin, as well as its discovery, structure, expression, and function in different tissues and cells. The transcriptional regulation of osteoactivin and its mechanism of action in normal and diseased conditions with special emphasis on bone are also covered in this review. In addition, we touch on the therapeutic potential of osteoactivin in cancer and bone diseases.

Discovery of CP-533536: an EP2 receptor selective prostaglandin E2 (PGE2) agonist that induces local bone formation.

Sulfonamides, exemplified by 3a, were identified as highly selective EP(2) agonists. Lead optimization led to the identification of CP-533536, 7f, a potent and selective EP(2) agonist. CP-533536 demonstrated the ability to heal fractures when administered locally as a single dose in rat models of fracture healing.

Osteoactivin, an anabolic factor that regulates osteoblast differentiation and function.

Osteoactivin (OA) is a novel glycoprotein that is highly expressed during osteoblast differentiation. Using Western blot analysis, our data show that OA protein has two isoforms, one is transmembranous and the other is secreted into the conditioned medium of primary osteoblasts cultures. Fractionation of osteoblast cell compartments showed that the mature, glycosylated OA isoform of 115 kDa is found in the membranous fraction. Both OA isoforms (secreted and transmembrane) are found in the cytoplasmic fraction of osteoblasts. Overexpression of EGFP-tagged OA in osteoblasts showed that OA protein accumulates into vesicles for transportation to the cell membrane. We examined OA protein production in primary osteoblast cultures and found that OA is maximally expressed during the third week of culture (last stage of osteoblast differentiation). Glycosylation studies showed that OA isoform of 115 kDa is highly glycosylated. We also showed that retinoic acid (RA) stimulates the mannosylation of OA protein. In contrast, tunicamycin (TM) strongly inhibited N-glycans incorporation into OA protein. The functional role of the secreted OA isoform was revealed when cultures treated with anti-OA antibody, showed decreased osteoblast differentiation compared to untreated control cultures. Gain-of-function in osteoblasts using the pBABE viral system showed that OA overexpression in osteoblast stimulated their differentiation and function. The availability of a naturally occurring mutant mouse with a truncated OA protein provided further evidence that OA is an important factor for terminal osteoblast differentiation and mineralization. Using bone marrow mesenchymal cells derived from OA mutant and wild-type mice and testing their ability to differentiate into osteoblasts showed that differentiation of OA mutant osteoblasts was significantly reduced compared to wild-type osteoblasts. Collectively, our data suggest that OA acts as a positive regulator of osteoblastogenesis.

Isolation and culture of rodent osteoprogenitor cells.

Osteoblasts are the cells responsible for formation of new bone throughout life. Rats are one of the most widely studied mammalian species in skeletal biology and serve as useful models for many aspects of human skeletal physiology. The availability of genetically modified mice as research tools has greatly enabled our understanding of how specific genes contribute to the process of skeletogenesis. In order to explore the impact of biochemical, genetic, or pharmacological manipulation on bone formation, various osteogenic cell culture systems have been developed. Two of the most widely accepted rodent osteogenic culture models, using osteoprogenitor cells isolated from calvaria or bone marrow, are described in this chapter.

The role of osteoactivin-derived peptides in osteoblast differentiation.

BACKGROUND: In our previous studies, we found that osteoactivin (OA) plays an important role in the regulation of osteoblast differentiation in vitro. Our studies also suggested that the region of OA protein that contains an RGD motif might play a vital role in the function of OA in osteoblast differentiation. In this study, we examined the functional role of OA-derived peptide containing the RGD motif (OA-D) in osteoblast differentiation. MATERIAL/METHODS: For this purpose, we designed another peptide, termed OA-E, that has sequence similar to OA-D but with glutamic acid (E) instead of aspartic acid (D). The effect of OA-E peptide on osteoblast differentiation was examined. Interestingly, OA-E peptide induced osteoblast differentiation in a manner similar to OA-D peptide. These data suggested that the effect of OA-derived peptides is RGD independent and it could be dependent on other features in the amino acid sequence of these peptides. RESULTS: OA-D peptide treatment markedly induced osteoblast differentiation markers in vitro compared to cultures treated with negative control peptide (NCP). Interestingly, OA-E peptide induced osteoblast differentiation in a manner similar to OA-D peptide. These data suggested that the effect of OA-derived peptides is RGD independent and it could be dependent on other features in the amino acid sequence of these peptides. Since phosphorylation of amino acid residues in proteins and peptides plays a major role in biological systems, the phosphorylation pattern of amino acid sequences of OA-derived peptides and OA protein family members were examined using bioinformatic analysis tools. We found that OA-derived peptides and OA protein family members have serine residue, close to c-terminus and might be phosphorylated with casein kinase II. Casein kinase II is known to phosphorylate many osteoblast-related proteins that regulate osteoblast development and differentiation such as osteopontin and vitronectin. CONCLUSIONS: Collectively, these data showed that both OA-D and OA-E peptides significantly induced osteoblast differentiation in vitro and that effect is RGD independent.

Proline-rich tyrosine kinase 2 regulates osteoprogenitor cells and bone formation, and offers an anabolic treatment approach for osteoporosis.

Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2-/- mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2-/- mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.

Osteoactivin acts as downstream mediator of BMP-2 effects on osteoblast function.

Our laboratory previously showed that osteoactivin (OA) is a novel, osteoblast-related glycoprotein that plays a role in osteoblast differentiation and function. The purpose of this study was to examine the regulation of OA expression by BMP-2 and the role OA plays as a downstream mediator of BMP-2 effects in osteoblast function. Using primary osteoblast cultures, we tested different doses of BMP-2 on the regulation of OA expression during osteoblast development. To test whether Smad-1 signaling is responsible for BMP-2 regulation of OA expression, osteoblast cultures were transfected with Smad1 siRNA, treated with 50 ng/ml of BMP-2 and analyzed by Western blot. BMP-2 treatment increased OA mRNA and protein expression in a dose-dependent manner and this upregulation was blocked in Smad1 siRNA transfected cultures. We next examined whether the role of OA as a downstream mediator of BMP-2 effects on osteoblast differentiation and matrix mineralization. Osteoblast cultures were transfected with OA antisense oligonucleotides and treated with 50 ng/ml of BMP-2. Cultures transfected with OA antisense oligonucleotides and treated with BMP-2 showed a reduction of OA expression associated with a significant reduction in early and late differentiation markers induced by BMP-2. Therefore, OA acts, at least in part, as a downstream mediator of BMP-2 effects on osteoblast differentiation and matrix mineralization. Our findings suggest that BMP-2 regulates OA expression through the Smad1 signaling pathway. Our data also emphasize that OA protein acts as a downstream mediator of BMP-2 effects on osteoblast differentiation and function.

Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-beta1 to induce mesenchymal cell condensation.

Mesenchymal cell (MC) condensation or the aggregation of MCs precedes chondrocyte differentiation and is required for subsequent cartilage formation during endochondral ossification. In this study, we used micromass cultures of C3H10T1/2 cells as an in vitro model system for studying MC condensation and the events important for this process. Transforming growth factor beta1 (TGF-beta1) served as the initiator of MC condensation in our model system and we were interested in determining whether CTGF functions as a downstream mediator of TGF-beta1. CTGF is a matricellular protein that has been found to be expressed in MC condensations and in the perichondrium. Micromass cultures of C3H10T1/2 cells condensed under TGF-beta1 stimulation concomitant with dramatic up-regulation of CTGF mRNA and protein levels. CTGF silencing by either CTGF siRNA or CTGF antisense oligonucleotide approaches showed that TGF-beta1-induced condensation was CTGF dependent. Furthermore, silencing of CTGF expression resulted in significant reductions in cell proliferation and migration, events that are crucial during MC condensation. In addition, up-regulation of Fibronectin (FN) and suppression of Sox9 expression by TGF-beta1 was also found to be mediated by CTGF. Immunofluorescence of developing mouse vertebrae showed that CTGF, TGF-beta1 and FN were co-expressed in condensations of MCs, while Sox9 expression was low at this stage. During subsequent chondrogenesis, Sox9 expression was high in chondrocytes while CTGF expression was limited to the perichondrium. Thus, CTGF is an essential downstream mediator of TGF-beta1-induced MC condensation through its effects on cell proliferation and migration. CTGF is also involved in up-regulating FN and suppressing Sox9 expression during TGF-beta1 induced MC condensation.

A nonprostanoid EP4 receptor selective prostaglandin E2 agonist restores bone mass and strength in aged, ovariectomized rats.

UNLABELLED: CP432 is a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 agonist. CP432 stimulates trabecular and cortical bone formation and restores bone mass and bone strength in aged ovariectomized rats with established osteopenia. INTRODUCTION: The purpose of this study was to determine whether a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 (PGE2) agonist, CP432, could produce bone anabolic effects in aged, ovariectomized (OVX) rats with established osteopenia. MATERIALS AND METHODS: CP432 at 0.3, 1, or 3 mg/kg/day was given for 6 weeks by subcutaneous injection to 12-month-old rats that had been OVX for 8.5 months. The effects on bone mass, bone formation, bone resorption, and bone strength were determined. RESULTS: Total femoral BMD increased significantly in OVX rats treated with CP432 at all doses. CP432 completely restored trabecular bone volume of the third lumbar vertebral body accompanied with a dose-dependent decrease in osteoclast number and osteoclast surface and a dose-dependent increase in mineralizing surface, mineral apposition rate, and bone formation rate-tissue reference in OVX rats. CP432 at 1 and 3 mg/kg/day significantly increased total tissue area, cortical bone area, and periosteal and endocortical bone formation in the tibial shafts compared with both sham and OVX controls. CP432 at all doses significantly and dose-dependently increased ultimate strength in the fifth lumber vertebral body compared with both sham and OVX controls. At 1 and 3 mg/kg/day, CP432 significantly increased maximal load in a three-point bending test of femoral shaft compared with both sham and OVX controls. CONCLUSIONS: CP432 completely restored trabecular and cortical bone mass and strength in established osteopenic, aged OVX rats by stimulating bone formation and inhibiting bone resorption on trabecular and cortical surfaces.

Discovery of highly selective EP4 receptor agonists that stimulate new bone formation and restore bone mass in ovariectomized rats.

Heptanoic acid lactams, exemplified by 2, were identified as highly selective EP4 agonists via high throughput screening. Lead optimization led to the identification of lactams with a 30-fold increase in EP4 potency in vitro. Compounds demonstrated robust bone anabolic effects when administered in vivo in rat models of osteoporosis.

Estrogen receptor beta selective ligands: discovery and SAR of novel heterocyclic ligands.

A series of ligands with varying heterocyclic cores and substituents that display a range of selectivity's (up to >100x) for ER-beta over ER-alpha are reported.

Tetrahydroisoquinolines as subtype selective estrogen agonists/antagonists.

Two series of 6-hydroxy and 7-hydroxy tetrahydroisoquinolines were prepared. Evaluating a range of C-1, C-4, and N-substituents led to the discovery of ER alpha and ER beta selective analogs.

Anti-osteoactivin antibody inhibits osteoblast differentiation and function in vitro.

Osteoactivin (OA) is a novel protein identified by mRNA differential display using bone from osteopetrotic versus normal rats. Bioinformatic analysis showed that OA cDNA has an open reading frame of 1716 bp encoding a protein of 572 aa, the first 21 aa constitute a signal peptide. OA sequence analysis also demonstrated 13 putative N-glycosylation sites suggestive of a heavily glycosylated protein. In this study, we localized OA protein in primary osteoblast culture by immunofluorescent staining and Western blot analysis. Primary osteoblast cultures pass through three stages: proliferation from day 1 to 7, matrix formation from day 7 to 14, and matrix mineralization from day 14 to 21. OA protein was detected at all stages examined, with maximal expression at 3 weeks when osteoblasts are terminally differentiated. Using the Chariot transfection reagent as a vehicle to deliver anti-OA antibody into the cells, we demonstrated that anti-OA antibody significantly inhibited osteoblast differentiation markers, including alkaline phosphatase activity, nodule formation, osteocalcin production, and calcium deposition, without affecting cell proliferation or viability. These data suggest that OA is an osteoblast-related protein that plays an important role in the regulation of osteoblast differentiation and function.

A novel, non-prostanoid EP2 receptor-selective prostaglandin E2 agonist stimulates local bone formation and enhances fracture healing.

UNLABELLED: CP-533,536, a newly discovered, non-prostanoid EP2 receptor-selective PGE2 agonist, stimulates local bone formation and enhances fracture healing in rat models. INTRODUCTION: There is a significant medical need for agents that can stimulate local bone formation and enhance fracture healing. We tested the effects of CP-533,536, a newly discovered, non-prostanoid EP2 receptor-selective prostaglandin E2 (PGE2) agonist, in stimulating local bone formation and enhancing fracture healing in rat models. MATERIALS AND METHODS: In the first model, a single injection of CP-533,536 at doses of 0.3, 1, or 3 mg/kg to the proximal tibial metaphysis of 6-week-old male rats was given on day 1, and the local bone anabolic effect was determined on day 7. We then tested the effects of this compound in inducing bone formation on rat periosteum of the femur. A single dose of 0.3 mg of CP-533,536 incorporated in a poly-(D,L-lactide-co-glycolide) (PLGH) matrix was injected onto the periosteum of the femur in 3-week-old male rats, and local bone formation was determined on day 14. Finally, the ability of CP-533,536 in PLGH matrix in enhancing fracture healing was tested using the rat femoral fracture model. CP-533,536 in PLGH matrix at doses of 0.05, 0.5, or 5 mg was delivered to the local fracture site on the same day of fracture, and its efficacy was evaluated on day 21. RESULTS AND CONCLUSIONS: A single injection of CP-533,536 at doses of 0.3, 1, or 3 mg/kg to the proximal tibial metaphysis dose-dependently stimulated local lamellar bone formation on trabecular, endocortical, and periosteal surfaces, and thus increased bone mineral content and bone strength at the injected site. Similarly, a single injection of 0.3 mg of CP-533,536 incorporated in PLGH matrix onto the periosteum of the femur induced significantly local bone formation. In the rat femoral fracture model, CP-533,536 in PLGH matrix at doses of 0.05, 0.5, and 5 mg dose-dependently increased callus size, density, and strength compared with PLGH matrix alone. These results show that CP-533,536 stimulates new bone formation on trabecular, endocortical, and periosteal surfaces and enhances fracture healing. These data reveal that EP2 receptor-selective agonists provide therapeutic potential for local bone augmentation, bone repair, and bone healing in humans.

Expression of connective tissue growth factor in bone: its role in osteoblast proliferation and differentiation in vitro and bone formation in vivo.

Connective tissue growth factor (CTGF) is a secreted, extracellular matrix-associated signaling protein that regulates diverse cellular functions. In vivo, CTGF is expressed in many tissues with highest levels in the kidney and brain. The purpose of this study was twofold; first, to localize CTGF in normal bone in vivo during growth and repair, and second, to examine CTGF expression and function in primary osteoblast cultures in vitro and test its effect on bone formation in vivo. Northern and Western blot analyses confirmed that CTGF is expressed in normal long bones during the period of growth or modeling. In situ hybridization and immunohistochemical analysis demonstrated intense staining for CTGF mRNA and protein in osteoblasts lining metaphyseal trabeculae. Examination of CTGF expression in the fracture callus demonstrated that it was primarily localized in osteoblasts lining active, osteogenic surfaces. In primary osteoblast cultures, CTGF mRNA levels demonstrated a bimodal pattern of expression, being high during the peak of the proliferative period, abating as the cells became confluent, and increasing to peak levels and remaining high during mineralization. This pattern suggests that CTGF may play a role in osteoblast proliferation and differentiation as previously demonstrated for fibroblasts and chondrocytes. Treatment of primary osteoblast cultures with anti-CTGF neutralizing antibody caused a dose-dependent inhibition of nodule formation and mineralization. Treatment of primary osteoblast cultures with recombinant CTGF (rCTGF) caused an increase in cell proliferation, alkaline phosphatase activity, and calcium deposition, thereby establishing a functional connection between CTGF and osteoblast differentiation. In vivo delivery of rCTGF into the femoral marrow cavity induced osteogenesis that was associated with increased angiogenesis. This study clearly shows that CTGF is important for osteoblast development and function both in vitro and in vivo.