William A. Grasser

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.

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...

A novel link between the proteasome pathway and the signal transduction pathway of the Bone Morphogenetic Proteins (BMPs)

BackgroundThe intracellular signaling events of the Bone Morphogenetic Proteins (BMPs) involve the R-Smad family members Smad1, Smad5, Smad8 and the Co-Smad, Smad4. Smads are currently considered to be DNA-binding transcriptional modulators and shown to recruit the master transcriptional co-activator CBP/p300 for transcriptional activation. SNIP1 is a recently discovered novel repressor of CBP/p300. Currently, the detailed molecular mechanisms that allow R-Smads and Co-Smad to co-operatively modulate transcription events are not fully understood.ResultsHere we report a novel physical and functional link between Smad1 and the 26S proteasome that contributes to Smad1- and Smad4-mediated transcriptional regulation. Smad1 forms a complex with a proteasome β subunit HsN3 and the ornithine decarboxylase antizyme (Az). The interaction is enhanced upon BMP type I receptor activation and occur prior to the incorporation of HsN3 into the mature 20S proteasome. Furthermore, BMPs trigger the translocation of Smad1, HsN3 and Az into the nucleus, where the novel CBP/p300 repressor protein SNIP1 is further recruited to Smad1/HsN3/Az complex and degraded in a Smad1-, Smad4- and Az-dependent fashion. The degradation of the CBP/p300 repressor SNIP1 is likely an essential step for Smad1-, Smad4-mediated transcriptional activation, since increased SNIP1 expression inhibits BMP-induced gene responses.ConclusionsOur studies thus add two additional important functional partners of Smad1 into the signaling web of BMPs and also suggest a novel mechanism for Smad1 and Smad4 to co-modulate transcription via regulating proteasomal degradation of CBP/p300 repressor SNIP1.

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.

Common mechanism for the estrogen agonist and antagonist activities of droloxifene

The incidence of postmenopausal osteoporosis is increasing as the population ages. Even though estrogen replacement therapy has proven beneficial in reducing the number of skeletal fractures, the known risks and associated side‐effects of estrogen replacement therapy make compliance poor. Recent research has focused on the development of tissue specific estrogen agonist/anatagonists such as droloxifene which can prevent estrogen deficiency‐induced bone loss without causing uterine hypertrophy. Furthermore, droloxifene acts as a full estrogen antagonist on breast tissue and is being evaluated for treatment of advanced breast cancer. In this report we propose a common mechanism of action for droloxifene that underlies its estrogen agonist and antagonist effects in different tissues. Droloxifene and estrogen, which have identical effects on bone in vivo, both induced p53 expression and apoptosis in cells of in vitro rat bone marrow cultures resulting in a decrease in the number of bone‐resorbing osteoclasts. Droloxifene is growth inhibitory in MCF‐7 human breast cancer cells and therefore acts as an antagonist, whereas estrogen is mitogenic to these cells and acts as an agonist. Droloxifene, but not estrogen, induced p53 expression and apoptosis in MCF‐7 cells. These results indicate that the induction of apoptosis by droloxifene may be the common mechanism for both its estrogen agonist effects in bone and its antagonist effects in breast tissue. J. Cell. Biochem. 65:159–171.

Prostate-derived factor and growth and differentiation factor-8: newly discovered members of the TGF-β superfamily

The transforming growth factor-β (TGF-β) superfamily is a large group of structurally related proteins that play various important roles during embryonic development, as well as in adult life. This superfamily in addition to TGF-βs also contains the inhibins, activins, Mullerian inhibiting substance, and bone morphogenetic proteins (BMPs), as well as the various growth and differentiation factors (GDFs). Members of the TGF-β superfamily are highly conserved, secreted molecules whose biologically active C-terminal domains play a variety of roles in embryonic pattern formation, body plan establishment and organogenesis in numerous species fromDrosophilaand C.elegansthrough humans [1, 2, 3]. Animals and humans lacking or having mutations in various TGF-β family members exhibit a wide variety of phenotypes, ranging from early embryonic death due to lack of mesodermal development to viable, but severely compromised animals with a variety of skeletal defects, to human diseases such as fibrodysplasia ossificans progressiva and dentinogenesis imperfecta. Among the TGF-s family members, the BMPs form a large subgroup of proteins, which were originally named on the basis of their ability as components of demineralized bone matrix to induce ectopic bone formation. Subsequently , classical protein chemistry in conjunction with molecular biology resulted in the cloning and expression of a number of BMPs. Their extensive homology to each other, in addition to highly conserved structural features, places them in the TFG-β superfamily[4].