The inherent heterogeneity of bone cells complicates the interpretation of microarray studies designed to identify genes highly associated with osteoblast differentiation. To overcome this problem, ...we have utilized Col1a1 promoter-green fluorescent protein transgenic mouse lines to isolate bone cells at distinct stages of osteoprogenitor maturation. Comparison of gene expression patterns from unsorted or isolated sorted bone cell populations at days 7 and 17 of calvarial cultures revealed an increased specificity regarding which genes are selectively expressed in a subset of bone cell types during differentiation. Furthermore, distinctly different patterns of gene expression associated with major signaling pathways (Igf1, Bmp, and Wnt) were observed at different levels of maturation. Some of our data differ from current models of osteoprogenitor cell differentiation and emphasize components of the pathways that were not revealed in studies based on a total cell population. Thus, applying methods to generate more homogeneous populations of cells at a defined level of cellular differentiation from a primary osteogenic culture is feasible and leads to a novel interpretation of the gene expression associated with increasing levels of osteoprogenitor maturation.
Nuclear coactivator-62 kDa/Ski-interacting protein (NCoA62/SKIP) is a putative vitamin D receptor (VDR) and nuclear receptor coactivator protein that is unrelated to other VDR coactivators such as ...those in the steroid receptor coactivator (SRC) family. The mechanism through which NCoA62/SKIP functions in VDR-activated transcription is unknown. In the present study, we identified a nuclear localization sequence in the COOH terminus of NCoA62/SKIP and showed that NCoA62/SKIP was targeted to nuclear matrix subdomains. Chromatin immunoprecipitation studies revealed that endogenous NCoA62/SKIP associated in a 1,25-dihydroxyvitamin D3-dependent manner with VDR target genes in ROS17/2.8 osteosarcoma cells. A cyclic pattern of promoter occupancy by VDR, SRC-1, and NCoA62/SKIP was observed, with NCoA62/SKIP entering these promoter complexes after SRC-1. These studies provide strong support for the proposed role of NCoA62/SKIP as a VDR transcriptional coactivator, and they indicate that key mechanistic differences probably exist between NCoA62/SKIP and SRC coactivators. To explore potential mechanisms, NCoA62/SKIP-interacting proteins were purified from HeLa cell nuclear extracts and identified by mass spectrometry. The identified proteins represent components of the spliceosome as well as other nuclear matrix-associated proteins. Here, we show that a dominant negative inhibitor of NCoA62/SKIP (dnNCoA62/SKIP) interfered with appropriate splicing of transcripts derived from 1,25-dihydroxyvitamin D3-induced expression of a growth hormone minigene cassette. Taken together, these data show that NCoA62/SKIP has properties that are consistent with those of nuclear receptor coactivators and with RNA spliceosome components, thus suggesting a potential role for NCoA62/SKIP in coupling VDR-mediated transcription to RNA splicing.
Once-daily s.c. administration of either human parathyroid hormone (PTH)-(1-84) or recombinant human PTH-(1-34) provides for dramatic increases in bone mass in women with postmenopausal osteoporosis. ...We initiated a program to discover orally bioavailable small molecule equivalents of these peptides. A traditional high-throughput screening approach using cAMP activation of the PTH/PTH-related peptide receptor (PPR) as a readout failed to provide any lead compounds. Accordingly, we designed a new screen for this receptor that used a modified N-terminal fragment of PTH as a probe for small molecule binding to the transmembrane region of the PPR, driven by the assumption that the pharmacological properties (agonist/antagonist) of compounds that bound to this putative signaling domain of the PPR could be altered by chemical modification. We developed DPC-AJ1951, a 14 amino acid peptide that acts as a potent agonist of the PPR, and characterized its activity in ex vivo and in vivo assays of bone resorption. In addition, we studied its ability to initiate gene transcription by using microarray technology. Together, these experiments indicated that the highly modified 14 amino acid peptide induces qualitatively similar biological responses to those produced by PTH-(1-34), albeit with lower potency relative to the parent peptide. Encouraged by these data, we performed a screen of a small compound collection by using DPC-AJ1951 as the ligand. These studies led to the identification of the benzoxazepinone SW106, a previously unrecognized small molecule antagonist for the PPR. The binding of SW106 to the PPR was rationalized by using a homology receptor model.
Glucokinase (GK) is a key regulator of glucose homeostasis, and its small-molecule activators represent a promising opportunity for the treatment of type 2 diabetes. Several GK activators have been ...advanced into clinical trials and have demonstrated promising efficacy; however, hypoglycemia represents a key risk for this mechanism. In an effort to mitigate this hypoglycemia risk while maintaining the efficacy of the GK mechanism, we have investigated a series of amino heteroaryl phosphonate benzamides as ''partial" GK activators. The structure-activity relationship studies starting from a "full GK activator"
, which culminated in the discovery of the "partial GK activator"
(BMS-820132), are discussed. The synthesis and
and
preclinical pharmacology profiles of
and its pharmacokinetics (PK) are described. Based on its promising
efficacy and preclinical ADME and safety profiles,
was advanced into human clinical trials.
Glucokinase (GK) is a key regulator of glucose homeostasis, and its small-molecule activators represent a promising opportunity for the treatment of type 2 diabetes. Several GK activators have been ...advanced into clinical trials and have demonstrated promising efficacy; however, hypoglycemia represents a key risk for this mechanism. In an effort to mitigate this hypoglycemia risk while maintaining the efficacy of the GK mechanism, we have investigated a series of amino heteroaryl phosphonate benzamides as ‘‘partial” GK activators. The structure–activity relationship studies starting from a “full GK activator” 11, which culminated in the discovery of the “partial GK activator” 31 (BMS-820132), are discussed. The synthesis and in vitro and in vivo preclinical pharmacology profiles of 31 and its pharmacokinetics (PK) are described. Based on its promising in vivo efficacy and preclinical ADME and safety profiles, 31 was advanced into human clinical trials.
There is considerable divergence in the sequences of steroid receptor response elements, including the vitamin D response elements (VDREs). Two major VDRE-containing and thus 1,25-dihydroxyvitamin ...D3(1,25-(OH)2D3)-regulated genes are the two non-collagenous, osteoblast-derived bone matrix proteins osteocalcin and osteopontin. We observed a stronger induction of osteopontin than osteocalcin mRNA expression by 1,25-(OH)2D3. Subsequently, we have shown that vitamin D receptor/retinoid X receptor α (VDR/RXRα) heterodimers bind more tightly to the osteopontin VDRE than to the osteocalcin VDRE. Studies using point mutants revealed that the internal dinucleotide at positions 3 and 4 of the proximal steroid half-element are most important for modulating the strength of receptor binding. In addition, studies with VDRE-driven luciferase reporter gene constructs revealed that the central dinucleotide influences the transactivation potential of VDR/RXRα with the same order of magnitude as that observed in the DNA binding studies. The synthetic vitamin D analog KH1060 is a more potent stimulator of transcription and inducer of VDRE binding of VDR/RXR in the presence of nuclear factors isolated from ROS 17/2.8 osteoblast-like cells than the natural ligand 1,25-(OH)2D3. Interestingly, however, KH1060 is comparable or even less potent than 1,25-(OH)2D3 in stimulating VDRE binding ofin vitro synthesized VDR/RXRα. Thus, the extent of 1,25-(OH)2D3- and KH1060-dependent binding of VDR/RXRα is specified by a central dinucleotide in the VDRE, and the ligand-induced effects on DNA binding are in part controlled by the cellular context of nuclear proteins.
Statins, which are inhibitors of 3‐hydroxy‐3‐glutaryl‐coenzyme A (HMG‐CoA) reductase, decrease the hepatic biosynthesis of cholesterol by blocking the mevalonate pathway. Nitrogen‐containing ...bisphosphonate drugs also inhibit the mevalonate pathway, preventing the production of the isoprenoids, which consequently results in the inhibition of osteoclast formation and osteoclast function. Therefore, we hypothesized that statins could affect bone metabolism in vivo through effects on osteoclastic bone resorption. In vitro, cerivastatin inhibited the parathyroid hormone (PTH)‐stimulated bone resorption. Using a panel of 40 statin analogs, which showed variable effects on HMG‐CoA reductase activity, we found that the ability of compounds to inhibit bone resorption is directly related to HMG‐CoA reductase activity. However, in the thyro‐parathyrodectomy (TPTX) model for bone resorption in the rat in vivo, cerivastatin did not prevent experimentally induced increases in bone resorption. The lack of effect of cerivastatin in this model is not related to a limited penetration of the target tissue (bone marrow), because a significant effect on HMG‐CoA reductase activity was demonstrated in the total rat bone marrow cell extracts of rats posttreatment in vivo. Furthermore, cerivastatin inhibited protein prenylation in osteoclasts isolated from the rabbit bone marrow of rabbits after treatment in vivo. In contrast to other studies, none of the statins tested showed anabolic effects in parietal bone explant cultures. Taken together, we conclude that statins inhibit bone resorption in vitro, which correlates directly with the potency of the compounds for inhibition of HMG‐CoA reductase activity. However, cerivastatin does not affect bone resorption in the rat TPTX model in vivo.
There are two major defects in type 2 diabetes: 1) insulin resistance and 2) insulin deficiency due to loss of beta-cell function. Here we demonstrated that treatment with muraglitazar (a dual ...peroxisome proliferator-activated receptor alpha/gamma activator), when initiated before or after the onset of diabetes in mice, is effective against both defects. In study 1, prediabetic db/db mice were treated for 12 weeks. The control mice developed diabetes, as evidenced by hyperglycemia, hyperinsulinemia, reduced insulin levels in the pancreas, blunted insulin response to glucose, and impaired glucose tolerance. The muraglitazar-treated mice had normal plasma glucose, and insulin levels, equivalent or higher pancreatic insulin content than normal mice, showed a robust insulin response to glucose and exhibited greater glucose tolerance. In study 2, diabetic db/db mice were treated for 4 weeks. The control mice displayed increased glucose levels, severe loss of islets, and their isolated islets secreted reduced amounts of insulin in response to glucose and exendin-4 compared with baseline. In muraglitazar-treated mice, glucose levels were reduced to normal. These mice showed reduced loss of islets, and their isolated islets secreted insulin at levels comparable to baseline. Thus, muraglitazar treatment decreased both insulin resistance and preserved beta-cell function. As a result, muraglitazar treatment, when initiated before the onset of diabetes, prevented development of diabetes and, when initiated after the onset of diabetes, prevented worsening of diabetes in db/db mice.
In the present study the involvement of protein kinase C in the action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on osteoblast-like
cells and in the stimulation of in vitro bone resorption by ...1,25(OH)2D3 was examined. Incubation for 24 h with 1,25(OH)2D3
potently stimulated osteocalcin synthesis by ROS 17/2.8 cells. This stimulation was inhibited (30-70% inhibition) by 25 microM
of the protein kinase C (PKC) inhibitors 1-O-hexadecyl-2-O-methyl-rac-glycerol (AMG) and sphingosine without affecting basal
osteocalcin synthesis. 1,25(OH)2D3-stimulated osteocalcin secretion by nontransformed isolated fetal rat osteoblasts was also
inhibited (30-55%) by AMG. Also, AMG inhibited 10(-9) M 1,25(OH)2D3-induced up-regulation of vitamin D receptor in ROS 17/2.8
cells. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) did not cause an increase in osteocalcin secretion, while
only a small increase in cellular content of osteocalcin in ROS 17/2.8 cells was observed. Addition of PMA together with 1,25(OH)2D3
did not change the response to 1,25(OH)2D3. The PKC inhibitors were not toxic for the cells. 1,25(OH)2D3 did not stimulate
diacylglycerol production in ROS 17/2.8 cells up to 5 min after administration. However, 4- and 24-h incubation with 10 nM
1,25(OH)2D3 increased phorbol ester binding in ROS 17/2.8 cells. 1,25(OH)2D3 potently stimulated bone resorption after 3 and
6 days of culture in fetal mouse long bones and calvaria. Both the PKC inhibitors AMG (25 microM) and staurosporine (50 nM)
strongly inhibited (60-86% inhibition) 1,25(OH)2D3-stimulated bone resorption without affecting basal 45Ca release. These
effects were not due to a cytotoxic effect of both PKC inhibitors. Nor is it likely that the effects of AMG and staurosporine
are due to inhibition of cell proliferation as hydroxyurea did not affect 1,25(OH)2D3-stimulated bone resorption. The inhibition
of 1,25(OH)2D3-stimulated bone resorption by PKC inhibitors suggests that besides osteocalcin synthesis PKC is also involved
in other responses of 1,25(OH)2D3 in bone. 1,25(OH)2D3 does not directly activate PKC via an increase in diacylglycerol production
but more likely via an increase in PKC. Together, the present study demonstrates a functional involvement of PKC in the action
of 1,25(OH)2D3 in bone and bone cells which may have consequences for the development of 1,25(OH)2D3 analogs, e.g. with less
hypercalcemic and relatively more antiproliferative activity.
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