Search PubMed⌕ Search

Biomedical subjects

G A Rodan

Publications and source records attributed to G A Rodan.

At least 37 records · Page 2Linked to original sources
PubMed2000

In vivo effects of bisphosphonates on the osteoclast mevalonate pathway.

Estrogen deficiency is a leading cause of osteoporosis associated with increased osteoclastic bone resorption. In vitro studies indicate that the clinically used nitrogen-containing bisphosphonates (N-BPs) such as alendronate (ALN), risedronate (RIS) and ibandronate (IBA) suppress bone resorption via inhibition of the mevalonate pathway enzyme farnesyl diphosphate (FPP) synthase in osteoclasts (Ocs). The object of this study was to test the hypothesis that N-BPs inhibit the mevalonate pathway of Ocs in vivo. The mevalonate pathway enzyme hydroxymethyl-glutaryl-coenzyme A reductase (HMGR), is modulated by feedback inhibition from downstream metabolites. We therefore evaluated the in vivo expression of HMGR in Ocs from animals treated with BP. The N-BPs, ALN, IBA and RIS, selectively suppressed HMGR expression in up to 85% of rat tibia osteoclasts, after 48 hr treatment. Etidronate and clodronate, bisphosphonates that do not inhibit FPP synthase, were without effect. Simvastatin treatment opposed ALN reduction of HMGR expression, suggesting regulation by a metabolite(s) between mevalonate and FPP. These data provide the first in vivo evidence for N-BP effects on the mevalonate pathway in osteoclasts, and strongly support the hypothesis that N-BPs act via this mechanism.

Alendronate↗
PubMed1999

Bisphosphonates act directly on the osteoclast to induce caspase cleavage of mst1 kinase during apoptosis. A link between inhibition of the mevalonate pathway and regulation of an apoptosis-promoting kinase.

Bisphosphonates (BPs) include potent inhibitors of bone resorption used to treat osteoporosis and other bone diseases. BPs directly or indirectly induce apoptosis in osteoclasts, the bone resorbing cells, and this may play a role in inhibition of bone resorption. Little is known about downstream mediators of apoptosis in osteoclasts, which are difficult to culture. Using purified osteoclasts, we examined the effects of alendronate, risedronate, pamidronate, etidronate, and clodronate on apoptosis and signaling kinases. All BPs induce caspase-dependent formation of pyknotic nuclei and cleavage of Mammalian Sterile 20-like (Mst) kinase 1 to form the active 34-kDa species associated with apoptosis. Withdrawal of serum and of macrophage colony stimulating factor, necessary for survival of purified osteoclasts, or treatment with staurosporine also induce apoptosis and caspase cleavage of Mst1. Consistent with their inhibition of the mevalonate pathway, apoptosis and cleavage of Mst1 kinase induced by alendronate, risedronate, and lovastatin, but not clodronate, are blocked by geranylgeraniol, a precursor of geranylgeranyl diphosphate. Together these findings suggest that BPs act directly on the osteoclast to induce apoptosis and that caspase cleavage of Mst1 kinase is part of the apoptotic pathway. For alendronate and risedronate, these events seem to be downstream of inhibition of geranylgeranylation.

Alendronate↗
PubMed1999

Transcription control and neuronal differentiation by agents that activate the LXR nuclear receptor family.

LXR and PPAR receptors belong to the nuclear receptor superfamily of transcriptional activating factors. Using ligand-dependent transcription assays, we found that 5-tetradecyloxy-2-furancarboxylic acid (TOFA) transactivates chimeric receptors composed of the glucocorticoid receptor DNA binding domain and the ligand binding regions of PPARalpha, PPARbeta (NUC-1) and LXRbeta (NER) receptors. In the same assays, ligands for PPARs (oleic acid, WY-14643 and L-631,033) and LXRs (hydroxycholesterols) maintain their respective receptor selectivity. TOFA and hydroxycholesterols also stimulate transcription from a minimal fibrinogen promoter that is under the control of AP-1 or NF-kappaB transcription factor binding sites. In addition to their effects on transcription, these LXRbeta activators induce neuronal differentiation in rat pheochromocytoma cells. TOFA and the natural LXR agonist, 22 (R)-hydroxycholesterol, stimulate neurite outgrowth in 55 and 28% of cells, respectively. No neurite outgrowth was induced by the related 22(S)-hydroxycholesterol, which does not activate the LXR family. These results suggest that the hydroxycholesterol signaling pathway has a complex effect on transcription that mediates the activity of TOFA and hydroxycholesterol on neuronal differentiation in pheochromocytoma cells.

Animals↗
PubMed1999

Interleukin 1 induces multinucleation and bone-resorbing activity of osteoclasts in the absence of osteoblasts/stromal cells.

Interleukin-1 (IL-1) is one of the most potent bone-resorbing factors involved in bone loss associated with inflammation. We previously reported that IL-1 prolonged the survival of multinucleated osteoclast-like cells (OCLs) formed in cocultures of murine osteoblasts/stromal cells and bone marrow cells via the prevention of spontaneously occurring apoptosis. It was reported that macrophage colony-stimulating factor (M-CSF/CSF-1) prolongs the survival of OCLs without the help of osteoblasts/stromal cells. The present study was conducted to determine whether IL-1 also directly induces the multinucleation and activation of OCLs. Mononuclear osteoclast-like cells (prefusion osteoclasts; pOCs) were purified using the "disintegrin" echistatin from cocultures of murine osteoblastic cells (MB 1.8 cells) and bone marrow cells. Both IL-1 and M-CSF prolonged the survival and induced the multinucleation of pOCs through their respective receptors. However, actin ring formation (a functional marker of osteoclasts) by multinucleated cells was observed in the pOC cultures treated with IL-1, but not those treated with M-CSF. We previously reported that enriched multinucleated OCLs as well as pOCs placed on bone/dentine slices formed few resorption pits, but their pit-forming activity was greatly increased by the addition of osteoblasts/stromal cells. Here, pit-forming activity of both pOCs and enriched OCLs placed on dentine slices was induced by adding IL-1, even in the absence of osteoblasts/stromal cells. M-CSF failed to induce pit-forming activity in pOC and enriched OCL cultures. These results indicate that IL-1 induces the multinucleation and bone-resorbing activity of osteoclasts even in the absence of osteoblasts/stromal cells.

Animals↗
PubMed1999

Stable association of PYK2 and p130(Cas) in osteoclasts and their co-localization in the sealing zone.

Bone resorption is initiated by osteoclast attachment to the mineralized matrix, cytoskeletal reorganization, cellular polarization, and the formation of the sealing zone. The present study examines the interaction between PYK2 and p130(Cas) (Crk-associated substrate), suggested to be part of the signaling pathway initiated by osteoclast adhesion. Using murine osteoclast-like cells (OCLs) and their mononuclear precursors (pOCs), generated in a co-culture of bone marrow and osteoblastic MB1.8 cells, we show that: 1) p130(Cas) is tyrosine-phosphorylated upon adhesion of pOCs to vitronectin or ligation of beta3 integrins; 2) p130(Cas) colocalizes with PYK2 and the cytoskeletal proteins F-actin, vinculin, and paxillin in the podosomal-rich ring-like structures of OCLs plated on glass and in the sealing zone in actively resorbing OCLs on bone; 3) p130(Cas) and PYK2 form a stable complex in pOCs, independent of tyrosine phosphorylation of either molecule, and this complex is present in Src (-/-) OCLs, in which neither protein is phosphorylated or associated with the osteoclast adhesion structure; 4) the association of p130(Cas) and PYK2 is mediated by the SH3 domain of p130(Cas) and the C-terminal domain of PYK2. These findings suggest that p130(Cas) and its association with PYK2 may play an important role in the adhesion-dependent signaling that leads to cytoskeletal reorganization and formation of the sealing zone during osteoclast activation.

Animals↗
PubMed1999

Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro.

Nitrogen-containing bisphosphonates were shown to cause macrophage apoptosis by inhibiting enzymes in the biosynthetic pathway leading from mevalonate to cholesterol. This study suggests that, in osteoclasts, geranylgeranyl diphosphate, the substrate for prenylation of most GTP binding proteins, is likely to be the crucial intermediate affected by these bisphosphonates. We report that murine osteoclast formation in culture is inhibited by both lovastatin, an inhibitor of hydroxymethylglutaryl CoA reductase, and alendronate. Lovastatin effects are blocked fully by mevalonate and less effectively by geranylgeraniol whereas alendronate effects are blocked partially by mevalonate and more effectively by geranylgeraniol. Alendronate inhibition of bone resorption in mouse calvaria also is blocked by mevalonate whereas clodronate inhibition is not. Furthermore, rabbit osteoclast formation and activity also are inhibited by lovastatin and alendronate. The lovastatin effects are prevented by mevalonate or geranylgeraniol, and alendronate effects are prevented by geranylgeraniol. Farnesol and squalene are without effect. Signaling studies show that lovastatin and alendronate activate in purified osteoclasts a 34-kDa kinase. Lovastatin-mediated activation is blocked by mevalonate and geranylgeraniol whereas alendronate activation is blocked by geranylgeraniol. Together, these findings support the hypothesis that alendronate, acting directly on osteoclasts, inhibits a rate-limiting step in the cholesterol biosynthesis pathway, essential for osteoclast function. This inhibition is prevented by exogenous geranylgeraniol, probably required for prenylation of GTP binding proteins that control cytoskeletal reorganization, vesicular fusion, and apoptosis, processes involved in osteoclast activation and survival.

Alendronate↗
PubMed1999

Retinoic acid effects on an SV-40 large T antigen immortalized adult rat bone cell line.

Clonal cell lines were established from adult rat tibia cells immortalized with SV-40 large T antigen. One clone (TRAB-11), in which retinoic acid (RA) induced alkaline phosphatase (AP) activity, was selected for further study. The TRAB-11 cells express high levels of type I collagen mRNA, type IV collagen, fibronectin, practically no type III collagen, little osteopontin, and no osteocalcin. RA stimulates proliferation of TRAB-11 cells (starting at 10 pM) and survival (starting at 100 pM). TRAB-11 cells synthesize fibroblast growth factor-2 (FGF-2), which has potent autocrine mitogenic effects on these cells and acts synergistically with RA. TRAB-11 cells attach better to type IV collagen than to fibronectin or laminin. Cell attachment to type IV collagen is increased by RA and decreased (65%) by an antibody directed against alpha1beta1 integrin. RA up-regulates steady-state levels of alpha1, mRNA without affecting beta1 mRNA expression. In conclusion, we report the establishment of a clonal cell line from the outgrowth of adult rat tibiae which is highly sensitive to RA in its growth and survival in culture, apparently as a result of integrin-mediated cell interaction with extracellular matrix proteins.

Alkaline Phosphatase↗
PubMed1999

Prostaglandin E2-bisphosphonate conjugates: potential agents for treatment of osteoporosis.

Conjugates of bisphosphonates (potential bone resorption inhibitors) and prostaglandin E2 (a bone formation enhancer) were prepared and evaluated for their ability to bind to bone and to liberate, enzymatically, free PGE2. The conjugate 3, an amide at C-1 of PGE2 proved to be too stable in vivo while conjugate 6, a thioester, was too labile. Several PGE2, C-15 ester-linked conjugates (18, 23, 24 and 31) were prepared and conjugate 23 was found to bind effectively to bone in vitro and in vivo and to liberate PGE2 at an acceptable rate. A 4-week study in a rat model of osteoporosis showed that 23 was better tolerated and more effective as a bone growth stimulant than daily maximum tolerated doses of free PGE2.

Animals↗
PubMed1999

Role of alpha(v)beta(3) integrin in osteoclast migration and formation of the sealing zone.

The alpha(v)beta(3) integrin is abundantly expressed in osteoclasts and has been implicated in the regulation of osteoclast function, especially in cell attachment. However, in vivo studies have shown that echistatin, an RGD-containing disintegrin which binds to alpha(v)beta(3), inhibits bone resorption without changing the number of osteoclasts on the bone surface, suggesting inhibition of osteoclast activity. The objective of this study was to examine how occupancy of alpha(v)beta(3) integrins inhibits osteoclast function, using primary rat osteoclasts and murine pre-fusion osteoclast-like cells formed in a co-culture system. We show that: (1) echistatin inhibits bone resorption in vitro at lower concentrations (IC(50 )= 0.1 nM) than those required to detach osteoclasts from bone (IC(50 ) approximately 1 microM); (2) echistatin (IC(50 )= 0.1 nM) inhibits M-CSF-induced migration and cell spreading of osteoclasts; (3) alpha(v)beta(3) integrins are localized in podosomes at the leading edge of migrating osteoclasts, whereas, with echistatin treatment (0.1 nM), alpha(v)beta(3) disperses randomly throughout the adhesion surface; and (4) when bone resorption is fully inhibited with echistatin, there is visible disruption of the sealing zone (IC(50 )= 13 nM), and alpha(v)beta(3) visualized with confocal microscopy re-distributes from the basolateral membranes to intracellular vesicular structures. Taken together, these findings suggest that alpha(v)beta(3) integrin plays a role in the regulation of two processes required for effective osteoclastic bone resorption: cell migration (IC(50 )= 0.1 nM) and maintenance of the sealing zone (IC(50) approximately 10 nM).

Actins↗
PubMed1999

Preclinical safety profile of alendronate.

Acute toxicity studies showed that the LD50 values of oral alendronate in female animals corresponded to human oral doses of 27,800 mg in rats and 48,300 mg in mice; LD50 values in male animals were even higher. Chronic toxicity studies showed clinically nonrelevant retention of primary spongiosa of bone in areas of endochondral bone formation, serum biochemical changes (reductions in serum concentrations of calcium, phosphate and alkaline phosphatase) and nephrotoxicity. Of the 5 genotoxicity studies performed, 4 showed no evidence of mutagenicity, including those most relevant to human carcinogenic potential. Carcinogenicity studies in rats and mice at maximum tolerated doses showed no increased tumour incidence associated with alendronate treatment. Alendronate had no effect on fertility or reproductive performance in male or female rats receiving oral doses up to 5 mg/kg/day. No adverse developmental effects were noted at doses up to 25 mg/kg/day in rats and 35 mg/kg/day in rabbits. Alendronate had no deleterious effect on bone strength or morphology. The evidence presented in these studies supports the conclusion that alendronate administered to humans at therapeutic doses is a safe drug for the treatment of postmenopausal osteoporosis.

Alendronate↗
PubMed1999

Preclinical evidence of normal bone with alendronate.

This review summarises the results of preclinical studies aimed at elucidating the mode of action of alendronate and assessing its effects on bone quality. Alendronate preferentially localises at bone resorption sites, where the drug inhibits osteoclastic activity. In a variety of estrogen-deficient animal models, alendronate normalised bone turnover, promoted normal mineralisation and increased bone mass and strength. In these studies, bone formed during alendronate therapy was histologically normal and was not associated with spontaneous fractures. Therefore, preclinical studies have established that the antiresorptive activity of alendronate results in the prevention of bone loss and the accretion of normal-quality bone.

Alendronate↗
PubMed1998

PYK2 in osteoclasts is an adhesion kinase, localized in the sealing zone, activated by ligation of alpha(v)beta3 integrin, and phosphorylated by src kinase.

Osteoclast activation is initiated by adhesion to the bone surface, followed by cytoskeletal rearrangement, the formation of the sealing zone, and a polarized ruffled membrane. This study shows that PYK2/CAKbeta/RAFTK, a cytoplasmic kinase related to the focal adhesion kinase, is highly expressed in rat osteoclasts in vivo. Using murine osteoclast-like cells (OCLs) or their mononuclear precursors (pOCs), generated in a coculture of bone marrow and osteoblastic MB1.8 cells, we show: (a) tyrosine phosphorylation of PYK2 upon ligation of beta3 integrins or adhesion of pOCs to serum, vitronectin, osteopontin, or fibronectin but not to laminin or collagen; (b) coimmunoprecipitation of PYK2 and c-Src from OCLs; (c) PYK2 binding to the SH2 domains of Src; (d) marked reduction in tyrosine phosphorylation and kinase activity of PYK2 in OCLs derived from Src (-/-) mice, which do not form actin rings and do not resorb bone; (e) PYK2 phosphorylation by exogeneous c-Src; (f) translocation of PYK2 to the Triton X-100 insoluble cytoskeletal fraction upon adhesion; (g) localization of PYK2 in podosomes and the ring-like structures in OCLs plated on glass and in the sealing zone in OCLs plated on bone; and (h) activation of PYK2, in the presence of MB1.8 cells, parallels the formation of sealing zones and pit resorption in vitro and is reduced by echistatin or calcitonin and cytochalasin D. Taken together, these findings suggest that Src-dependent tyrosine phosphorylation of PYK2 is involved in the adhesion-induced formation of the sealing zone, required for osteoclastic bone resorption.

Animals↗
PubMed1998

Integrin-mediated signaling in the regulation of osteoclast adhesion and activation.

Integrins are heterodimeric membrane receptors that mediate cell-extracellular matrix (ECM), and cell-cell interactions. Integrins provide a physical link between the ECM and the cell cytoskeleton, and transduce signals which lead to elevation of cytosolic pH and calcium levels, changes in phospholipid metabolism and ultimately regulate gene expression. Osteoclast bone resorption is a complicated multistep process, that starts with matrix recognition, osteoclast attachment, polarization and formation of the sealing zone on the bone, followed by the directional secretion of acids and lysosomal enzymes to the resorbing surface. Osteoclasts exhibit high expression of the alpha v beta 3 integrin, which binds to a variety of RGD-containing proteins including vitronectin, osteopontin and bone sialoprotein. RGD-containing peptides, RGD-mimetics and blocking antibodies to alpha v beta 3 integrins were shown to inhibit bone resorption in vitro and in vivo, suggesting that this integrin plays an important role in regulating osteoclast activity. Furthermore, RGD-containing peptides and proteins modulate osteoclastic cytosolic calcium levels. Phosphatidyl inositol 3-kinase and c-Src were co-immunoprecipitated with alpha v beta 3 integrins in these cells. In addition, c-Cbl was found to be a substrate of c-Src in osteoclasts. More recently, ligand-engagement or clustering of alpha v beta 3 integrins in osteoclasts induced tyrosine phosphorylation of PYK2, a member of the focal adhesion kinase family, and of p130cas, a substrate of v-Src and v-Crk. Both PYK2 and p130cas were also found in the sealing zone of actively resorbing osteoclasts. How these signaling molecules interact with each other in mediating the alpha v beta 3 rate limiting effect on bone resorption is not well understood. They emerged however as key players in linking the adhesion of osteoclasts to the bone matrix, to cytoskeletal organization, and to the polarization and activation of these cells for bone resorption.

Animals↗
PubMed1998

Tyrosine phosphorylation of p130Cas is involved in actin organization in osteoclasts.

Integrin-mediated interaction with the extracellular matrix plays a critical role in the function of osteoclasts, the bone-resorbing cells. This study examines the role of p130Cas (Crk-associated substrate (Cas)) in actin organization in osteoclasts. Multinucleated osteoclast-like cells (OCLs) were obtained in a co-culture of murine bone marrow cells and primary osteoblasts. After plating on culture dishes, OCLs formed a ringlike structure consisting of F-actin dots at cell periphery (actin ring). The percentage of OCLs with actin rings and its diameter increased with time and cell spreading. Tyrosine phosphorylation of a protein (p130) increased with actin ring formation. Treatment with cytochalasin D disrupted actin rings and reduced tyrosine phosphorylation of p130. Using specific antibodies, p130 was identified as Cas. By immunocytochemistry, Cas was localized to the peripheral regions of OCLs and its distribution overlapped that of F-actin. In OCLs derived from Src(-/-) mice, in which osteoclast activity is severely compromised, tyrosine phosphorylation of Cas was markedly reduced. Moreover, Cas was diffusely distributed in the cytoplasm and actin ring formation is not observed. These findings suggest that Src-dependent tyrosine phosphorylation of Cas is involved in the adhesion-induced actin organization associated with osteoclast activation.

Actins↗
PubMed1998

Sphingosine kinase mediates cyclic AMP suppression of apoptosis in rat periosteal cells.

Prostaglandin E stimulates bone formation in humans and animals, and increases intracellular cAMP in osteoblastic cells. We found that cAMP inhibits apoptosis in osteoblastic cells, and examined the mechanism of this effect. We report that the cAMP elevating agent, forskolin, increases cell number in the rat periosteal cell line (RP-11), by suppressing apoptosis in a cell type-specific manner. In RP-11, forskolin transiently up-regulates extracellular signal-regulated kinase activity, a known suppressor of apoptosis. PD98059, a selective inhibitor of the extracellular signal-regulated kinase pathway, only partially reverses the antiapoptotic effect of forskolin, which suggests an additional mechanism for cAMP action. We found that forskolin stimulates cytosolic sphingosine kinase (SPK) activity in these cells; in two other osteoblastic cell lines, however, forskolin does not suppress apoptosis. In contrast to the partial opposing effect of PD98059 to forskolin action, N, N-dimethylsphingosine, a specific inhibitor of SPK, completely reverses the antiapoptotic effect of forskolin, and has no effect on apoptosis in the absence of forskolin. These findings show for the first time that cAMP activates SPK in a cell-type-specific manner, and suggest that cAMP suppression of apoptosis in RP-11 periosteal cells is mediated by its stimulation of SPK.

Animals↗
PubMed1998

Mechanisms of action of bisphosphonates.

Bisphosphonates (BPs) are pyrophosphate analogs in which the oxygen bridge has been replaced by carbon and diverse carbon side chains have generated a large family of compounds. Several are potent inhibitors of bone destruction (resorption) and are in clinical use for the treatment and prevention of osteoporosis, Paget's disease, hypercalcemia caused by malignancy, tumor metastases in bone, and other bone ailments. Selective action on bone is based on the binding of the BP moiety to the bone mineral. The molecular mode of action of BPs, which may differ from compound to compound, is unknown. However, at the tissue level, all BPs inhibit bone destruction and lead to an increase in bone mineral density by decreasing bone resorption and bone turnover. At the cellular level, the ultimate target of BP action is the osteoclast, the bone resorbing cell. In vitro evidence shows BP inhibition of osteoclast formation, via action on osteoblasts, and there is in vitro and in vivo evidence for BP inhibition of osteoclast activity. There is in vivo and in vitro evidence for increased apoptosis. The relative contribution of these various effects on the therapeutic action of BPs remains to be established. At the molecular level, it is not known if BPs act on a single or multiple targets. Enzymes in the cholesterol biosynthesis pathway and protein tyrosine phosphatases were shown to be inhibited by BPs.

Animals↗