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.

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Using indirect immunofluorescence microscopy and biochemical techniques, we have determined that approximately one-third of the total mitogen-activated protein kinase (MAPK) is associated with the microtubule cytoskeleton in NIH 3T3 mouse fibroblasts. This population of enzyme can be separated from the soluble form that is found distributed throughout the cytosol and is also present in the nucleus after mitogen stimulation. The microtubuleassociated enzyme pool constitutes half of all detectable MAPK activity after mitogenic stimulation. These findings extend the known in vivo associations of MAPK with microtubules to include the entire microtubule cytoskeleton of proliferating cells, and they suggest that a direct association of MAPK with microtubules may be in part responsible for the observed correlations between MAPK activities and cytoskeletal alteration.Mitogen-activated protein kinase (MAPK), also known as the extracellular signal-regulated kinase (ERK), is involved in the transmission of signals between plasma membrane receptors and the nucleus (1, 2). MAPK has been shown to phosphorylate and regulate cytoskeletal components such as the microtubule-associated proteins in vitro, and it was originally named microtubule-associated protein-2 kinase after its substrate, MAP2 (3). Nonetheless, numerous immunocytochemical analyses have failed to show an association of MAPK with microtubules in proliferating cells (4-9). Thus, despite the often misunderstood nature of the original name, it is generally considered that MAPK is not actually associated with the microtubules in systems where mitogenesis occurs. However, MAPK was shown to associate with microtubules in certain rat brain dendrites in situ (10) and to copolymerize with bovine brain microtubules in vitro (11). MAPK was also shown to associate with the microtubule-organizing centers, but not spindle structures, in mouse oocytes (12). These findings raise the possibility that MAPK physically interacts with and regulates microtubule dynamics under certain unique circumstances such as meiosis and dendritic remodeling in the brain.In proliferating cells, significant evidence suggests that MAPK plays a role in cytoskeletal regulation. In addition to MAPs found only in the brain, such as MAP2 and tau, MAPK also phosphorylates cytoskeletal components present in cycling cells such as MAP4 and caldesmon (13,14). MAPs, which bind to and stabilize microtubules, are phosphorylated in response to cell stimulation by a variety of mitogens. The resulting phosphorylation inhibits their capacity to stabilize the microtubules (15). MAPK, which is activated by these mitogens, has been shown to be causative in MAP inhibition in vitro (13,16). MAPK activation is triggered not only by a large number of mitogens but also upon integrin-extracellular matrix association (17, 18), a first step toward cell spreading. Although these findings collectively imply a possible role for MAPK in the regulation of the cytoskeleton, the abovedescribed immunofluorescence evidence sugge...

Multinucleated bone-resorbing osteoclasts (Ocl) are cells of hematopoietic origin that play a major role in osteoporosis pathophysiology. Ocl survival and activity require M-CSF and RANK ligand (RANKL). M-CSF signals to Akt, while RANKL, like TNFa, activates NF-jB. We show here that although these are separate pathways in the Ocl, signaling of all three cytokines converges on mammalian target of rapamycin (mTOR) as part of their antiapoptotic action. Accordingly, rapamycin blocks M-CSF-and RANKL-dependent Ocl survival inducing apoptosis, and suppresses in vitro bone resorption proportional to the reduction in Ocl number. The cytokine signaling intermediates for mTOR/ribosomal protein S6 kinase (S6K) activation include phosphatidylinositol-3 kinase, Akt, Erks and geranylgeranylated proteins. Inhibitors of these intermediates suppress cytokine activation of S6K and induce Ocl apoptosis. mTOR regulates protein translation acting via S6K, 4E-BP1 and S6. We find that inhibition of translation by other mechanisms also induces Ocl apoptosis, demonstrating that Ocl survival is highly sensitive to continuous de novo protein synthesis. This study thus identifies mTOR/S6K as an essential signaling pathway engaged in the stimulation of cell survival in osteoclasts.

Abstract. Wild-type and mutant chicken integrin/31 subunit (/31o) cDNAs were expressed in NIH 3T3 cells and assayed for localization in focal adhesions of cells plated on fibronectin substrates. Focal adhesion localization in stable transfected cells was assayed by indirect immunofluorescent staining with chicken-specific anti-/3L: antibodies. Mutant/31c integrins containing internal deletions of 13 amino acids adjacent to the membrane, A759-771, and 20 centrally located amino acids, A771-790, localized in focal adhesions demonstrating that sequences required for direction to focal adhesion structures were not limited to one region of the cytoplasmic domain. Point mutations revealed three clusters of amino acids which contribute to localization in focal adhesions. These three clusters or signals are: cyto-1 (764-774), cyto-2 (785-788), and cyto-3 (79%800). The l 1-residue cyto-1 signal is only found on integrin/3 subunit sequences, except/34. Four residues within this region, 13764, F768, F771, and E774, could not be altered without reducing focal adhesion staining intensities, and likely form a signal that occupies one side of an tx helix. Mutations involving two cyto-1 residues, K770 and F771, also appeared to affect heterodimer affinity and specificity. Cyto-2 (785-788,), NPIY, is an NPXY signal that forms a tight turn motif. Cyto-2 provides a structural conforrnation, which when perturbed by proline removal or addition, inhibits integrin localization in focal adhesions. Cyto-3 (79%800), NPKY, resembles cyto-2, however, the nonconserved proline residue can be replaced without alteration of the localization phenotype. Cyto-3, therefore, constitutes a unique integrin signal, NXXY. Both serine and tyrosine residues at positions 790 and 788, respectively, which have been implicated in integrin phosphorylation/regulation, were conservatively replaced without detectable effect on focal adhesion localization. However, acidic replacements for these amino acids reduced focal adhesion staining intensities, suggesting that phosphorylation at these sites may negatively regulate integrin function.T H~ integrin superfamily of heterodimeric cell surface receptors associates with extracellular matrix (ECM) I proteins and with cytoskeletal-associated proteins such as talin and c~-actinin (Burridge et al., 1988;Albeda and Buck, 1990). There are at least four integrin families, each defined by a different common/5 subunit, and at least four other/5 subunits that associate with only one or. At least 12 ot subunits have been identified which associate with one or more of the eight/5 subunits. The ot subunits generally confer ECM substrate specificity (Hemler, 1990; A1-beda and Buck, 1990;Springer, 1990), although otv, which associates with several different 13 subunlts, does not follow this rule (Smith et al., 1990;Bodary and McLean, 1990;Vogel et al., 1990).Both ot and/5 integrin subunits have large extracellular, single membrane-spanning, and small cytoplasmic domains. The avian/51 subunit (/31o) is 803 amino acids long Yokichi...

MST1 is a member of theIn mammalian cells, Sterile-20 (Ste20)-related kinases participate in the regulation of the cytoskeleton that controls cell morphology and motility, and in the regulation of apoptosis (1). These kinases share a conserved catalytic (kinase) domain at the amino terminus and a C-terminal regulatory region of great structural diversity, which interacts with signaling molecules that regulate the cytoskeleton. Currently, four closely related MST kinases have been described (5-9). Most Ste20 group kinases activate mitogen-activated protein kinase (MAPK) 1 cascades in the signaling pathways between the cellular membrane and the nuclear compartment (2). In yeast, the mating pheromone receptor Ste20p phosphorylates and activates a MAPK kinase kinase, Ste11p, raising the possibility that mammalian homologs of Ste20p (e.g. MST1 kinase) also function as MAPK kinase kinase kinases (3, 4). MST1 was shown to act upstream of MAPK kinases that regulate p38 and JNK activities, probably acting via the MAPK kinase kinase MEKK1 (10).There is substantial evidence that MST1 promotes apoptosis, although its role in this process may vary in different cell types. Overexpression of MST1 can induce apoptosis and nuclear condensation in BJAB, 293T and COS-1 cells (4, 14, 15), whereas MST1 promotes nuclear condensation without apparent chromosomal cleavage in HeLa cells (13).A consistent feature of MST1 in all tested cell types is its proteolytic cleavage by caspase, in response to apoptotic stimuli, to a 34 -36-kDa product (hereafter referred to as 36-kDa MST1). Cleavage increases MST1 kinase activity severalfold (4, 16 -19) and influences its subcellular localization (13, 15). Recently, a second caspase cleavage site was identified in the human MST1 sequence that is absent in mouse MST1 and in MST2 from several species (10). Mutation of this site has no impact on accumulation of the 36-kDa species.Expression of a kinase-dead MST1 mutant (K59R) can partially or fully suppress apoptosis or chromatin condensation in HL-60 and 293T cells treated with chemical apoptotic stimuli (15,18). This protective effect is associated with suppression of both JNK activity and DNA fragmentation. However, the K59R mutant fails to suppress apoptosis in BJAB and HeLa cells treated with Fas ligand or TNF-␣ (4, 16). The basis for the cell type-and stimulus-specific differences in MST function has not yet been elucidated.Interestingly, like PAK proteins (11, 12), MST1 (full-length or cleaved) can have a profound effect on cell shape (cell rounding and detachment) independent of caspase activation and prior to nuclear condensation (13). This action and the high basal activity of MST1 kinase suggest a possible function unrelated to apoptosis.Stress-inducing agents such as staurosporine and sodium arsenite (9) can increase MST1 kinase activity; however, no physiological activator of MST1 has been identified, and little is known about the endogenous activation of MST1. Recently, it was proposed that in addition to caspase cleavage, MST1 phosphor...

Abstract. Chicken integrin B1 eDNA and its sitedirected mutants were cloned into a mammalian expression vector and introduced into mouse NIH 3T3 cells. Stable transfectants expressing the chicken/31 subunit or its site-directed mutants were identified by immunostaining with antibodies specific for the chicken integrin/31 subunit. The chicken/~1 proteins were expressed predominately in the endoplasmic reticulum of transfectants and to a lesser degree in the plasma membrane. Immunoblots and immunoprecipitations, using anti-chicken integrin antibodies, revealed three different sizes of the chicken subunit (90, 95, and 120 kD) and a mouse 140-kD cz subunit. Immunoprecipitations of the cell surface receptors showed only two peptides, an 120-kD/31 and an 140-kD ot subunit. Antibodies perturbing mouse and chicken integrin-specific cell adhesions were used to demonstrate that the chimeric receptors functioned in adhesion to both laminin and fibronectin. Immunofluorescent staining with antibodies specific for either the chicken or mouse receptors showed that both the wild type and the chimeric receptors localized in focal contacts. Several mutations in the cytoplasmic domain were synthesized and used in the transfection experiments. In one mutant the tyrosine (Tyr 788) in the consensus sequence for phosphorylation was replaced by a phenylalanine. In another the lysine (Lys 757) at the end of the membrane spanning region was replaced by a leucine. Both of these mutants formed dimers with mouse ~ subunits, participated in adhesion, localized in focal contacts, and displayed biological properties indistinguishable from the wild-type transfection. In contrast, mutants containing deletions greater than 5-15 amino acids nearest the carboxyl end in the cytoplasmic domain neither promoted adhesion nor localized in focal contacts. They did, however, form heterodimers that were expressed on the cell surface.

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