Drug discovery and target validation.

Recent drug discovery has been driven largely by a genomics-based approach. This revolution in pharmaceutics is based on localized expression of either a novel gene or homologue of a known gene found in cDNA libraries made from normal versus diseased tissue. The choice and quality of cDNA library is critical for the success of this approach. Expression is normally verified at the cellular level by either immunocytochemistry or in situ hybridization. Activity of the recombinant protein in secondary cell-based assays allows highthroughput screens to be formulated to identify small-molecule effectors of this protein. More recently, a proteomics approach has also been incorporated into this process. This technology directly measures proteins whose expression is localized in disease tissue as the basis for cell-based screens to look for either activators or inhibitors, of this activity. The majority of screens are designed to look for inhibitors. Activity of small-molecules found by screening gives rise to pharmacokinetic studies and verification of activity in animal models of the disease. Structure-activity relationship (SAR) optimization of these small-molecules allows for suitable oral bioavailability and pharmacokinetics, resulting in compounds progressing from discovery to development. Based on these strategies, we have developed inhibitors of osteoclast-mediated bone resorption and are currently screening for bone anabolic agents. In addition, we have also developed small-molecule caspase inhibitors which prevent chondrocyte apoptosis and retain cell function in an attempt to find therapeutic agents to either prevent or treat osteoarthritis. These agents may well have utility in the treatment of temporomandibular joint diseases.

Antagonism of oestrogen action in human breast and endometrial cells in vitro: potential novel antitumour agents.

PURPOSE: There is a need to find novel oestrogen receptor (ER) ligands that antagonize oestrogen action in the reproductive tissues and would therefore have therapeutic potential in oestrogen-dependent tumours. We tested novel ER ligands in both breast and endometrial cells to profile agonism/antagonism in these oestrogen target reproductive tissues. METHODS: Novel analogues of the ER antagonist ICI 182,780 were synthesized and tested for their ability to inhibit gene expression dependent on oestrogen response elements (ERE) in human breast (MCF-7) and endometrial (Ishikawa) cell lines. This activity was correlated with inhibition of oestrogen-induced cell proliferation and ER binding. RESULTS: The sulphide analogue (compound 1) and sulphone analogue (compound 2) had no intrinsic ERE-dependent agonism in either breast cancer or endometrial cells in culture. All three compounds dose-dependently inhibited ERE-mediated oestrogen agonism. Moreover, these ER ligands inhibited oestrogen-stimulated proliferation of breast cancer and endometrial cells. ICI 182,780, compound 1 and compound 2 were all able to bind both isoforms of the ER (ER alpha and ER beta). In endometrial cells, the relative binding to ER beta correlated with the ERE-dependent antioestrogenic effect of these ligands, suggesting that in this tissue this receptor is the predominant isoform that determines antioestrogenic activity. CONCLUSIONS: The ability of these analogues of ICI 182,780 to inhibit oestrogen-stimulated transcriptional activity and cell proliferation suggests that these agents, in particular the sulphone analogue, have therapeutic potential in the treatment of breast cancer without exhibiting the unwanted oestrogenic effects in the endometrium.

Potent and selective nonpeptide inhibitors of caspases 3 and 7.

5-Dialkylaminosulfonylisatins have been identified as potent, nonpeptide inhibitors of caspases 3 and 7. The most active compound within this series (34) inhibited caspases 3 and 7 in the 2-6 nM range and exhibited approximately 1000-fold selectivity for caspases 3 and 7 versus a panel of five other caspases (1, 2, 4, 6, and 8) and was at least 20-fold more selective versus caspase 9. Sequence alignments of the active site residues of the caspases strongly suggest that the basis of this selectivity is due to binding in the S2 subsite comprised of residues Tyr204, Trp206, and Phe256 which are unique to caspases 3 and 7. These compounds inhibit apoptosis in three cell-based models: human Jurkat T cells, human chondrocytes, and mouse bone marrow neutrophils.

Direct cleavage of the human DNA fragmentation factor-45 by granzyme B induces caspase-activated DNase release and DNA fragmentation.

The protease granzyme B (GrB) plays a key role in the cytocidal activity during cytotoxic T lymphocyte (CTL)-mediated programmed cell death. Multiple caspases have been identified as direct substrates for GrB, suggesting that the activation of caspases constitutes an important event during CTL-induced cell death. However, recent studies have provided evidence for caspase-independent pathway(s) during CTL-mediated apoptosis. In this study, we demonstrate caspase-independent and direct cleavage of the 45 kDa unit of DNA fragmentation factor (DFF45) by GrB both in vitro and in vivo. Using a novel and selective caspase-3 inhibitor, we show the ability of GrB to process DFF45 directly and mediate DNA fragmentation in the absence of caspase-3 activity. Furthermore, studies with DFF45 mutants reveal that both caspase-3 and GrB share a common cleavage site, which is necessary and sufficient to induce DNA fragmentation in target cells during apoptosis. Together, our data suggest that CTLs possess alternative mechanism(s) for inducing DNA fragmentation without the requirement for caspases.

Visualization of bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro.

Bisphosphonates inhibit osteoclast-mediated bone resorption by mechanisms that have only recently become clear. Whereas nitrogen-containing bisphosphonates affect osteoclast function by preventing protein prenylation (especially geranylgeranylation), non-nitrogen-containing bisphosphonates have a different molecular mechanism of action. In this study, we demonstrate that nitrogen-containing bisphosphonates (risedronate, alendronate, pamidronate, and zoledronic acid) and non-nitrogen-containing bisphosphonates (clodronate and etidronate) cause apoptosis of rabbit osteoclasts, human osteoclastoma-derived osteoclasts, and human osteoclast-like cells generated in cultures of bone marrow in vitro. Osteoclast apoptosis was shown to involve characteristic morphological changes, loss of mitochondrial membrane potential, and the activation of caspase-3-like proteases capable of cleaving peptide substrates with the sequence DEVD. Caspase-3-like activity could be visualized in unfixed, dying osteoclasts and osteoclast-like cells using a cell-permeable, fluorogenic substrate. Bisphosphonate-induced osteoclast apoptosis was dependent on caspase activation, because apoptosis resulting from alendronate, clodronate, or zoledronic acid treatment was suppressed by zVAD-fmk, a broad-range caspase inhibitor, or by SB-281277, a specific isatin sulfonamide inhibitor of caspase-3/-7. Furthermore, caspase-3 (but not caspase-6 or caspase-7) activity could be detected and quantitated in lysates from purified rabbit osteoclasts, whereas the p17 fragment of active caspase-3 could be detected in human osteoclast-like cells by immunofluorescence staining. Caspase-3, therefore, appears to be the major effector caspase activated in osteoclasts by bisphosphonate treatment. Caspase activation and apoptosis induced by nitrogen-containing bisphosphonates are likely to be the consequence of the loss of geranylgeranylated rather than farnesylated proteins, because the ability to cause apoptosis and caspase activation was mimicked by GGTI-298, a specific inhibitor of protein geranylgeranylation, whereas FTI-277, a specific inhibitor of protein farnesylation, had no effect on apoptosis or caspase activity.

Selective inhibitors of apoptotic caspases: implications for novel therapeutic strategies.

Caspases are essential for apoptosis. A crucial question regarding the role(s) of these proteases is whether the selective inhibition of an effector caspase will prevent cell death. We have identified potent, selective non-peptide inhibitors of the effector caspases 3 and 7. Apoptosis can be inhibited and cell functionality maintained using an inhibitor selective for caspases 3 and 7. This has important therapeutic implications and the potential to generate novel anti-apoptotic strategies in diseases that involve dysregulated apoptosis.

Therapeutic potential of oestrogen receptor ligands in development for osteoporosis.

Accelerated bone loss secondary to loss of ovarian function at menopause is well recognised as a major risk factor for osteoporotic fractures in postmenopausal women. Postmenopausal bone loss can be prevented or arrested by oestrogen replacement therapy (ERT). It has also been reported that ERT protects against cardiovascular disease by improving the serum lipid profile, however there are mixed reports concerning these benefits. Unopposed ERT causes an unacceptable increase in the risk of endometrial cancer and proliferative effects in mammary tissue resulting in an increased risk of breast cancer. While this can be counteracted by combining ERT with a low-dose of a progestin, withdrawal bleeding and the continuing uncertainty about the effect of oestrogen on the risk of breast cancer contribute to poor compliance for long-term use. Because of the known and suspected risks of oestrogen therapy it has been estimated that in the US < 40% of women on ERT will continue treatment beyond one year. An ideal therapy would retain the desirable skeletal and cardiovascular effects of oestrogen, lack oestrogenic activity on the endometrium and reduce the incidence of breast cancer. The concept of selective oestrogen receptor modulation (SERM) has been demonstrated for a number of compounds including tamoxifen, raloxifene, droloxifene, GW-5638 and levormeloxifene. However, the clinical utility of these agents will depend on the profile of tissue-specific effects and the extent to which they are translated into in vivo efficacy. A SERM is defined as a compound that has oestrogen agonism on one or more of the desired target tissues, such as bone or liver, and has antagonism and/or minimal agonism (i.e., clinically insignificant) in reproductive tissue, such as the breast or uterus. Although tamoxifen acts as a SERM, it is also associated with an increased incidence (4% gynaecological symptoms greater than placebo control) of endometrial cancer. Indeed, there have been a number of mechanistic-based studies to explain the increased incidence of endometrial carcinomas in tamoxifen treated patients, which provide an in vitro insight into the adverse clinical observations in vivo. Attempts to improve on the pharmacological profile of tamoxifen have resulted in compounds that differ in their oestrogen agonist/antagonist characteristics, including the pure oestrogen antagonists. This suggests that it may be possible to develop a molecule with a desired profile of tissue-specific agonist/antagonist activities by establishing bone and cardiovascular protective effects but having no effects (or even behaving as an antagonist) in the reproductive tissues.

Role of caspases in human renal proximal tubular epithelial cell apoptosis.

In the present study, we have used an in vitro model of apoptosis using primary human renal proximal tubular epithelial (RPTE) cells to investigate the mechanisms involved in renal cell apoptosis. Treatment of RPTE cells with okadaic acid for 24-48 h induced apoptosis in a concentration-dependent manner. Apoptosis was accompanied by the activation of the p38 mitogen-activated protein kinase (MAPK) pathway followed by the activation of caspase-9, -3, and -7. The induction of caspase activity correlated with the proteolytic cleavage of beta-catenin, suggesting that beta-catenin is a caspase substrate. The caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), resulted in a dose-dependent inhibition of apoptosis and beta-catenin cleavage. These data suggest that okadaic acid-induced apoptosis is p38 MAPK and caspase-dependent and that proteolytic cleavage of beta-catenin by caspases is likely to be a downstream molecular event associated with the morphological and cytoskeletal changes induced during apoptosis.

The selective oestrogen receptor modulators idoxifene and raloxifene have fundamentally different cell-specific oestrogen-response element (ERE)-dependent/independent mechanisms in vitro.

Idoxifene and raloxifene are selective oestrogen receptor modulators (SERMs) that by definition exhibit tissue-specific agonist or antagonist properties via interactions with the oestrogen receptor (ER). Idoxifene acts as an oestrogen agonist in osteoblastic cells via an ER/ERE-mediated mechanism. In contrast, raloxifene is an antagonist via the ERE in osteoblastic cells. Like the pure antagonist ICI 182,780, raloxifene inhibited the potent agonist activity of both 17beta-oestradiol and idoxifene through the ERE whereas idoxifene had no effect on the agonist activity of 17beta-oestradiol via the ERE. In breast cancer cells, both raloxifene and idoxifene were potent antagonists of ERE-mediated 17beta-oestradiol action suggesting an ERE-dependent mechanism of action for both ligands in these cells. Therefore, these SERMs exhibit cell-specific ERE-dependent and -independent mechanisms of action.

Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis.

A bovine cartilage explant system was used to evaluate the effects of injurious compression on chondrocyte apoptosis and matrix biochemical and biomechanical properties within intact cartilage. Disks of newborn bovine articular cartilage were compressed in vitro to various peak stress levels and chondrocyte apoptotic cell death, tissue biomechanical properties, tissue swelling, glycosaminoglycan loss, and nitrite levels were quantified. Chondrocyte apoptosis occurred at peak stresses as low as 4.5 MPa and increased with peak stress in a dose-dependent manner. This increase in apoptosis was maximal by 24 h after the termination of the loading protocol. At high peak stresses (>20 MPa), greater than 50% of cells apoptosed. When measured in uniaxial confined compression, the equilibrium and dynamic stiffness of explants decreased with the severity of injurious load, although this trend was not significant until 24-MPa peak stress. In contrast, the equilibrium and dynamic stiffness measured in radially unconfined compression decreased significantly after injurious stresses of 12 and 7 MPa, respectively. Together, these results suggested that injurious compression caused a degradation of the collagen fibril network in the 7- to 12-MPa range. Consistent with this hypothesis, injurious compression caused a dose-dependent increase in tissue swelling, significant by 13-MPa peak stress. Glycosaminoglycans were also released from the cartilage in a dose-dependent manner, significant by 6- to 13-MPa peak stress. Nitrite levels were significantly increased above controls at 20-MPa peak stress. Together, these data suggest that injurious compression can stimulate cell death as well as a range of biomechanical and biochemical alterations to the matrix and, possibly, chondrocyte nitric oxide expression. Interestingly, chondrocyte programmed cell death appears to take place at stresses lower than those required to stimulate cartilage matrix degradation and biomechanical changes. While chondrocyte apoptosis may therefore be one of the earliest responses to tissue injury, it is currently unclear whether this initial cellular response subsequently drives cartilage matrix degradation and changes in the biomechanical properties of the tissue.

Distinct mechanisms of action of selective estrogen receptor modulators in breast and osteoblastic cells.

Raloxifene and idoxifene are selective estrogen receptor modulators (SERMs) that exhibit tissue-specific agonist or antagonist properties via interactions with the estrogen receptor (ER). Both compounds are similarly osteoprotective in the ovariectomized rat in vivo as assessed by measurement of bone mineral density, urinary pyridinium cross-links, and serum osteocalcin, suggesting a similar mechanism of action. However, we have identified a fundamental difference in this mechanism via the estrogen response element (ERE) in osteoblast-like cells. With the use of ERE-luciferase reporter constructs, raloxifene, like the complete ER-antagonist ICI-182780, acts as an antagonist via the ERE in osteoblastic cells. In contrast, idoxifene, like 17beta-estrogen itself and 4-OH-tamoxifen, acts as an agonist in osteoblastic cells via an ER/ERE-mediated mechanism. Both ICI-182780 and raloxifene inhibited the ERE-dependent agonist activity of 17beta-estradiol and idoxifene in osteoblastic cells. In contrast, in breast cells, raloxifene, idoxifene, 4-OH-tamoxifen, and ICI-182780 had no agonist activity and, indeed, raloxifene and idoxifene were potent antagonists of ERE-mediated 17beta-estradiol action, indicating an ERE-dependent mode of action in these cells. Although these SERMs exhibit a similar antagonist activity profile in breast cells, they can be distinguished mechanistically in osteoblastic cells.

Inhibition of caspase-3-like activity prevents apoptosis while retaining functionality of human chondrocytes in vitro.

Apoptosis was induced in a human chondrocyte cell line, T/C 28a4, by treatment with various stimuli, including camptothecin, tumor necrosis factor-alpha, staurosporine, okadaic acid, and reduced serum conditions. All stimuli induced a cytosolic DEVDase activity, coincident with apoptosis. Caspase activities in the lysates were characterized and quantitated with peptide cleavage profiles. To confirm that the results were not related to the immortalized nature of the cell line, primary human chondrocytes also were shown to undergo apoptosis under similar conditions, which resulted in increased cytosolic DEVDase activity. There was little or no caspase-1 (interleukin-1beta-converting enzyme) or caspase-8-like activity in the apoptotic cells. In all cases, the irreversible nonselective caspase inhibitor, Z-VAD-FMK, and the caspase-3-selective inhibitor, Ac-DMQD-CHO, inhibited DEVDase activity and apoptosis, whereas the caspase-1-selective inhibitor, Ac-YVAD-CHO, had no effect. Human chondrocytes were stably and transiently transfected with a type-II collagen gene (COL2A1) regulatory sequence driving a luciferase reporter as a specific marker of chondrocyte gene expression. Treatment of the cells with camptothecin or tumor necrosis factor-alpha plus cycloheximide significantly inhibited COL2A1 transcriptional activity. Significantly, cotreatment with Z-VAD-FMK or Ac-DMQD-CHO maintained COL2A1-reporter gene activity, indicating that the prevention of apoptosis by caspase-3 inhibition was sufficient to maintain cell functionality as assessed by the retention of type-II collagen promoter activity.

Is there a therapeutic opportunity to either prevent or treat osteopenic disorders by inhibiting marrow adipogenesis?

Bone marrow stromal cells can undergo adipogenesis or osteoblastogenesis in vitro and in vivo. This review explores the stromal cell's differentiation plasticity in the context of osteoporosis and other metabolic bone disorders. Attention is focused on the apparent reciprocal relationship that is postulated to exist between the adipocyte and osteoblast phenotypes. The signal transduction pathways implicated in this process are evaluated as potential targets for therapeutic intervention and drug design.

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality.

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

Differential expression of egr-1 in osteoarthritic compared to normal adult human articular cartilage.

OBJECTIVE: The purpose of this study was to identify genes that are differentially expressed in normal versus osteoarthritic human articular cartilage as either potential novel therapeutic targets or diagnostic markers of this disease. DESIGN: mRNA was isolated from histologically normal and osteoarthritic adult human articular cartilage. The Differential Display technique was employed which identified differentially expressed genes in the normal and diseased tissue. Northern and reverse Northern hybridization were used to confirm the gene expression pattern. Immunohistochemistry and in-situ hybridization were used to localize expression of Egr-1 protein and mRNA respectively in cartilage. RESULTS: A transcription factor, early growth response protein-1 (Egr-1) was found to be down-regulated more than six-fold in multiple human OA cartilage samples when compared to normal tissue. Immunohistochemistry indicated that Egr-1 was expressed throughout normal adult cartilage, in deep-, mid- and superficial-zones. In contrast, in OA cartilage there was expression of Egr-1 mRNA and protein only in the chondrocytes undergoing cloning. CONCLUSIONS: Egr-1 is differentially expressed in OA versus normal cartilage and because of its role in transcriptional activation and repression and regulation of proliferation, differentiation and apoptosis, Egr-1 may play an important role in the pathogenesis of OA. Up-regulation of Egr-1 may therefore provide a novel therapeutic approach for either the prevention or treatment of OA.

Identification and cloning of a human urea transporter HUT11, which is downregulated during adipogenesis of explant cultures of human bone.

Bipotential cells in human trabecular bone explant cultures that express osteoblast characteristics are able to undergo adipogenesis in the presence of 3-isobutyl-1-methylxanthine plus dexamethasone (Nuttall et al. [1998] J Bone Miner Res 13:371-382). The initial studies of these bipotential cells in explant cultures have been extended to examine differential gene expression during osteoblast/adipocyte transdifferentiation. Using differential display, we have identified a gene expressed in trabecular bone explant cultures that is downregulated as these cells differentiate from an osteoblast to an adipocyte phenotype. Homology searching identified this gene as the human urea transporter HUT11. The expression and downregulation of HUT11 have been observed in multiple patient bone explant cultures. The size of the bone explant-derived HUT11 mRNA is approximately 4.4 kb, which is identical to the largest splice variant reported. In this article, we report the cloning and sequencing of this gene from primary human osteoblasts. In addition, we report tissue distribution for the bone explant-derived form of HUT11 mRNA and show a reciprocal relationship between the expression of HUT11 and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma 2, which is a marker of adipocyte differentiation. Because the control of osteoblast/adipocyte transdifferentiation is unknown, selective downregulation of HUT11 during adipogenesis suggests that HUT11 expression may be a marker of the switch from an osteoblast to an adipocyte phenotype. Understanding the role of HUT11 in osteoblasts may provide insights into the mechanism controlling osteoblast and adipocyte differentiation.

Identification and cloning of a connective tissue growth factor-like cDNA from human osteoblasts encoding a novel regulator of osteoblast functions.

We have identified and cloned a novel connective tissue growth factor-like (CTGF-L) cDNA from primary human osteoblast cells encoding a 250-amino acid single chain polypeptide. Murine CTGF-L cDNA, encoding a polypeptide of 251 amino acids, was obtained from a murine lung cDNA library. CTGF-L protein bears significant identity ( approximately 60%) to the CCN (CTGF, Cef10/Cyr61, Nov) family of proteins. CTGF-L is composed of three distinct domains, an insulin-like growth factor binding domain, a von Willebrand Factor type C motif, and a thrombospondin type I repeat. However, unlike CTGF, CTGF-L lacks the C-terminal domain implicated in dimerization and heparin binding. CTGF-L mRNA ( approximately 1.3 kilobases) is expressed in primary human osteoblasts, fibroblasts, ovary, testes, and heart, and a approximately 26-kDa protein is secreted from primary human osteoblasts and fibroblasts. In situ hybridization indicates high expression in osteoblasts forming bone, discrete alkaline phosphatase positive bone marrow cells, and chondrocytes. Specific binding of 125I-labeled insulin-like growth factors to CTGF-L was demonstrated by ligand Western blotting and cross-linking experiments. Recombinant human CTGF-L promotes the adhesion of osteoblast cells and inhibits the binding of fibrinogen to integrin receptors. In addition, recombinant human CTGF-L inhibits osteocalcin production in rat osteoblast-like Ros 17/2.8 cells. Taken together, these results suggest that CTGF-L may play an important role in modulating bone turnover.

Idoxifene: a novel selective estrogen receptor modulator prevents bone loss and lowers cholesterol levels in ovariectomized rats and decreases uterine weight in intact rats.

Idoxifene, a novel selective estrogen receptor modulator, was tested for its effects on bone loss, serum cholesterol, and uterine wet weight and histology in the ovariectomized (Ovx) rat. Idoxifene (0.5 mg/kg x day) completely prevented loss of both lumbar and proximal tibial bone mineral density (BMD). In an intervention study, idoxifene (0.5 and 2.5 mg/kg x day) completely prevented further loss of both lumbar and proximal tibial BMD during a 2-month treatment period commencing 1 month after surgery, when significant loss of BMD had occurred in the Ovx control group. Idoxifene reduced total serum cholesterol, which was maximal at 0.5 mg/kg x day. Idoxifene alone displayed minimal uterotrophic activity in Ovx rats and inhibited the agonist activity of estrogen in intact rats. Histologically, myometrial and endometrial atrophy were observed in both idoxifene and vehicle-treated Ovx rats. In this report, we also provide molecular-based evidence to support the observations in vivo of a novel selective estrogen receptor modulator (SERM) mechanism of action in bone and endometrial cells. Idoxifene is an agonist through the estrogen response element (ERE) and exhibits similar postreceptor effects to estrogen in bone-forming osteoblasts. Idoxifene also stimulates osteoclast apoptosis, and these pleiotropic effects ultimately could contribute to the maintenance of bone homeostasis. However, idoxifene differs from estrogen in a tissue-specific manner. In human endometrial cells, where estrogen is a potent agonist through the ERE, idoxifene has negligible agonist activity. Moreover, idoxifene was able to block estrogen induced gene expression in endometrial cells, which is in agreement with the observation in the intact rat study. In the uterus, idoxifene has a pharmacologically favorable profile, lacking agonist and therefore growth-promoting activity. Together with its cholesterol lowering effect and lack of uterotrophic activity, these data suggest that idoxifene may be effective in the prevention of osteoporosis and other postmenopausal diseases without producing unwanted estrogenic effects on the endometrium.

Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype: implications for osteopenic disorders.

The decrease in bone volume associated with osteoporosis and age-related osteopenia is accompanied by increased marrow adipose tissue formation. Reversal of this process may provide a novel therapeutic approach for osteopenic disorders. We have shown that cells cultured from human trabecular bone are not only osteogenic, but are able also to undergo adipocyte differentiation under defined culture conditions. Osteoblast differentiation was induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and adipocyte differentiation by dexamethasone (dex) plus 3-isobutyl-1-methylxanthine (IBMX) treatment. Adipogenesis was characterized by lineage-specific enzyme and gene activities, alpha-glycerophosphate-3-dehydrogenase activity, fatty acid binding protein, aP2 and lipoprotein lipase expression. Osteoblastogenesis was assessed by osteoblast characteristic 1,25(OH)2D3 induction of alkaline phosphatase activity and osteoblast-specific 1,25(OH)2D3-induced osteocalcin synthesis and release. We provide evidence for a common pluripotent mesenchymal stem cell that is able either to undergo adipogenesis or osteoblastogenesis, using clonal cell lines derived from human trabecular bone cell cultures. Adipogenesis can be induced also by long chain fatty acids and the thiazolidinedione troglitazone. Dex plus IBMX-induced adipogenesis can be inhibited by interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta. Interestingly, and in contrast to extramedullary adipocyte differentiation as shown by mouse 3T3L-1 and a human liposarcoma SW872 cell line, trabecular bone adipogenesis was unaffected by insulin. Also, the formation of fully differentiated adipocytes from trabecular bone cells after troglitazone treatment and long chain fatty acids was dependent on increased expression of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma2 caused by dex plus IBMX. Specific inhibition of marrow adipogenesis and promotion of osteoblastogenesis of a common precursor cell may provide a novel therapeutic approach to the treatment of osteopenic disorders.

Human osteoclastoma-derived stromal cells: correlation of the ability to form mineralized nodules in vitro with formation of bone in vivo.

It has been suggested that the stromal element of human osteoclastomas contains osteoblastic cells. In this study, we demonstrate that osteoclast-depleted, passaged stromal cells express alkaline phosphatase and osteocalcin in vitro and form mineralized nodules under appropriate culture conditions. In addition, we describe a model in which severe combined immunodeficient (SCID) mice were used to support the differentiation of these putative human osteoblast progenitors in vivo. Lesions formed from human stromal cells were identified using the OKa blood group antigen and human procollagen type I antibodies. By 21 days, the lesion was a complete bone unit: a fully mineralized cortex, remodeling trabeculae, and a highly cellular marrow space. Stromal cells derived from six out of seven osteoclastomas produced identical lesions. Further studies have demonstrated that the capacity of the osteoclastoma-derived stromal cells to form bone in vivo and in vitro is passage dependent; early passages were osteogenic in both model systems, while later passages were not. In conclusion, we have developed a model in which the osteogenic nature of cells can be confirmed in vivo. Furthermore, human osteoclastoma-derived stromal cells provide a source of these osteogenic cells to study human osteoblast differentiation, both in vivo and in vitro.