Thomas A. Owen

An EP2 receptor-selective prostaglandin E2 agonist induces bone healing

The morbidity and mortality associated with impaired/delayed fracture healing remain high. Our objective was to identify a small nonpeptidyl molecule with the ability to promote fracture healing and prevent malunions. Prostaglandin E2 (PGE2) causes significant increases in bone mass and bone strength when administered systemically or locally to the skeleton. However, due to side effects, PGE2 is an unacceptable therapeutic option for fracture healing. PGE2 mediates its tissue-specific pharmacological activity via four different G protein-coupled receptor subtypes, EP1, -2, -3, and -4. The anabolic action of PGE2 in bone has been linked to an elevated level of cAMP, thereby implicating the EP2 and/or EP4 receptor subtypes in bone formation. We identified an EP2 selective agonist, CP-533,536, which has the ability to heal canine long bone segmental and fracture model defects without the objectionable side effects of PGE2, suggesting that the EP2 receptor subtype is a major contributor to PGE2s local bone anabolic activity. The potent bone anabolic activity of CP-533,536 offers a therapeutic alternative for the treatment of fractures and bone defects in patients.

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.

Effects of CP-336,156, a New, Nonsteroidal Estrogen Agonist/Antagonist, on Bone, Serum Cholesterol, Uterus, and Body Composition in Rat Models.

We have discovered a new, nonsteroidal, potent estrogen agonist/antagonist, CP-336,156. CP-336,156 binds selectively and with high affinity to the human estrogen receptor-α with a half-inhibition concentration of 1.5 nm, which is similar to that seen with estradiol (4.8 nm). When given orally to immature (3-week-old) female Sprague-Dawley rats for 3 days at doses of 0.1, 1.0, 10, or 100 μg/kg·day, unlike 17α-ethynyl estradiol, CP-336,156 had no effect on uterine wet or dry weight. Similarly, no uterine hypertrophy was observed in aged (17-month-old) female rats treated (po) with CP-336,156 at 10 or 100 μg/kg·day for 28 days. We also found that CP-336,156 decreased total serum cholesterol and fat body mass and had no effect on lean body mass in these aged female rats. In 5-month-old ovariectomized (OVX) Sprague-Dawley female rats, CP-336,156 completely prevented OVX-induced increases in body weight gain, total serum cholesterol, and serum osteocalcin at doses between 10 and 1000 μg/kg·day after 4 weeks. At...

Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts.

Osteoblast development is a complex process involving the expression of specific growth factors and regulatory proteins that control cell proliferation, differentiation, and maturation. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between mutant op and normal littermates. Total RNA isolated from long bone and calvaria was used as a template for mRNA differential display. One of many cDNAs that were selectively expressed in either normal or mutant bone was cloned and sequenced and found to share some homology to the human nmb and Pmel 17 genes. This novel cDNA was named osteoactivin. Osteoactivin has an open reading frame of 1716 bp that encodes a protein of 572 amino acids with a predicted molecular weight of 63.8 kD. Protein sequence analysis revealed the presence of a signal peptide and a cleavage site at position 23. The protein also has thirteen predicted N‐linked glycosylation sites and a potential RGD integrin recognition site at position 556. Northern blot analysis confirmed that osteoactivin was 3‐ to 4‐fold overexpressed in op versus normal bone. RT‐PCR analysis showed that osteoactivin is most highly expressed in bone compared with any of the other non‐osseous tissues examined. In situ hybridization analysis of osteoactivin in normal bone revealed that it is primarily expressed in osteoblasts actively engaged in bone matrix production and mineralization. In primary rat osteoblast cultures, osteoactivin showed a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization and correlated with the expression of alkaline phosphatase and osteocalcin. Our findings show that osteoactivin expression in bone is osteoblast‐specific and suggest that it may play an important role in osteoblast differentiation and matrix mineralization. Furthermore, osteoactivin overexpression in op mutant bone may be secondary to the uncoupling of bone resorption and formation resulting in abnormalities in osteoblast gene expression and function. J. Cell. Biochem. 84: 12–26, 2002.

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

CP‐533,536, a newly discovered, non‐prostanoid EP2 receptor‐selective PGE2 agonist, stimulates local bone formation and enhances fracture healing in rat models.

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.

Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-β1 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 β1 (TGF‐β1) 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‐β1. 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‐β1 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‐β1‐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‐β1 was also found to be mediated by CTGF. Immunofluorescence of developing mouse vertebrae showed that CTGF, TGF‐β1 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‐β1‐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‐β1 induced MC condensation. J. Cell. Physiol. 210: 398–410, 2007.

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. J. Cell. Physiol. 210: 26–37, 2007.

Regulation of BMP-7 expression by retinoic acid and prostaglandin E2

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor‐beta (TGF‐β) gene superfamily of growth and differentiation factors. Members of the BMP family were originally cloned and characterized by their ability to induce ectopic bone formation. Of the various BMPs cloned, the bone inductive ability of BMP‐7 (OP‐1) and BMP‐2 has been well characterized. Both BMP‐7 and ‐2 have been shown to have clinical utility in the healing of non‐union fractures. However, in spite of the various advances in BMP research, the physiological regulation of BMPs is not well understood. Here we studied the expression of BMP‐7 by cloning a 4.6‐kB fragment of the human BMP‐7 promoter (hBMP‐7p) and placing it upstream of a luciferase reporter. The promoter reporter construct was stably transfected into different cell backgrounds and its regulation by various factors was investigated. We show that retinoic acid (RA) treatment results in an upregulation of the hBMP‐7p reporter activity. This regulation of the hBMP‐7p was further confirmed by Northern blot, PCR, and Western blot analyses, which showed an increase in both BMP‐7 mRNA and protein expression upon treatment with RA. We further show that RA specifically upregulates expression of osteocalcin via activation of BMP‐7 mRNA and protein in vitro. Similarly, prostaglandin E2 (PGE2) treatment increases BMP‐7 mRNA and protein levels, but does not transcriptionally activate the hBMP‐7p. Additionally, in vivo expression of BMP‐7 in bone was increased upon PGE2 treatment. In conclusion, RA and PGE2 upregulate BMP‐7 protein expression both in vitro and in vivo. J. Cell. Physiol. 190: 207–217, 2002.

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

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.