The surprising discovery that nitrogen-containing bisphosphonates (N-BPs) act via inhibition of the mevalonate-to-cholesterol pathway raised the possibility that esophageal irritation by N-BPs is mechanism-based. We used normal human epidermal keratinocytes (NHEKs) to model N-BP effects on stratified squamous epithelium of the esophagus. The N-BPs alendronate and risedronate inhibited NHEK growth in a dose-dependent manner without inducing apoptosis. N-BPs (30 microM) caused accumulation of cells in S phase and increased binucleation (inhibited cytokinesis). Consistent with N-BP inhibition of isoprenylation, geranylgeraniol or farnesol prevented accumulation in S phase. Binucleation was also induced by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor lovastatin and by the squalene synthase inhibitor zaragozic acid A and was prevented by adding low-density lipoprotein. At 300 microM, N-BPs reduced expression of cyclin-dependent kinase (cdk) 2 and cdk4 and enhanced expression of p21(waf1) and p27(kip1) and their binding to cdks with corollary hypophosphorylation of retinoblastoma. Lovastatin and zaragozic acid A produced similar effects, except that p21(waf1) expression and binding to cdks was not induced. Growth inhibition, but not binucleation, was also caused by the geranylgeranyl transferase I inhibitor, GGTI-298, which also enhanced cdk2 and cdk4 association with p27(kip1). These findings are consistent with suppression of epithelial cell growth by N-BPs via inhibition of the mevalonate pathway and the consequent reduction in cholesterol synthesis, which blocks cytokinesis, and in geranylgeranylation, which interferes with progression through the cell cycle.
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. Bisphosphonates (BPs)1 include potent inhibitors of bone resorption used for the treatment of osteoporosis, Paget's disease, bone metastases, and other bone diseases. It is generally accepted that BPs inhibit bone resorption by acting directly or indirectly on osteoclasts, cells of hematopoietic origin. Until recently the molecular mechanism of action of BPs was not known (1, 2). Recent pharmacological studies suggest that nitrogen-containing BPs (N-BPs), such as alendronate (ALN), risedronate (RIS), ibandronate, and pamidronate (PAM), act on the cholesterol biosynthesis pathway (3,4). N-BPs were recently shown to inhibit either isopentenyl diphosphate synthase or the downstream enzyme, farnesyl diphosphate (FPP) synthase, or both (5), while clodronate (CL2) had little or no effect on these enzymes. Inhibition of either enzyme in this pathway blocks cholesterol biosynthesis, as well as farnesylation and geranylgeranylation. We recently showed that only geranylgeraniol (GGOH) prevented the effects of ALN on bone resorption (pit formation) in vitro (4), suggesting that geranylgeranylation is rate-limiting for osteoclast function. CL2 and EHDP may inhibit osteoclasts by forming toxic ATP analogs or by inhibiting protein-tyrosine phosphatases (6 -9).The final outcome of BP action appears to be osteoclast apoptosis, which was suggested to be the means by which BPs inhibit bone resorption (10, 11). It is not known if the induction of apoptosis is the result of direct action of BPs on the osteoclast, and little is known about the biochemical pathways involved. Coxon et al. (12) have shown the induction of caspases in BP-trea...
Colony-stimulating factor-1 (CSF-1) is synthesized as a secreted or membrane-bound molecule. We investigated whether osteoblastic cells produce these forms of CSF-1. Glutaraldehyde-fixed cell layers supported proliferation of the macrophage cell line BAC1.2F5, suggesting the presence of membrane- or/and matrix-associated CSF-1. Furthermore, CSF-1 activity could be either extracted from the matrix or released from the cell membrane. A neutralizing antiserum against CSF-1 inhibited these activities. After labeling the cellular proteins with [35S] met/cys or [35S] SO4(2-), CSF-1 was immunoprecipitated and analyzed by SDS-PAGE. Under nonreducing conditions, bands with MW more than 200, 200, 100, and 50 kd were detected. These bands shifted to lower MW under reducing conditions. Treatment with chondroitin lyase ABC decreased the MW of the 200 kd monomer, proving the proteoglycan structure. Much smaller quantities of CSF-1 were found in the matrix extract than in the conditioned medium. Transforming growth factor beta (TGF-beta) increased both the synthesis of CSF-1 and its accumulation in the matrix. CSF-1 released with trypsin from the membrane fraction yielded on SDS-PAGE a band with MW of 60 and 30 kd under nonreducing and reducing conditions, respectively. Transcripts encoding both the secreted and the membrane-associated forms of the cytokine were detected in osteoblasts by reverse transcription polymerase chain reaction. These data indicate that osteoblastic cells produce the secreted forms, either remaining in the culture supernatant, or being associated to the matrix, and the membrane associated form of CSF-1.
Colony-stimulating factor-1 (CSF-1), originally described as a growth factor for macrophages, is essential for the proliferation and differentiation of the cells of the osteoclast lineage. The cytokine is synthesized either as a secreted or a membrane-bound protein, which are encoded by four transcripts. The aim of the present study was to investigate the expression of CSF-1 in vivo at the mRNA level. Transcripts encoding CSF-1 were determined in total RNA from fetal murine metatarsals of different ages by a quantitative reverse-transcription polymerase chain reaction assay. Within the investigated period of time, the bone rudiments contain cells of the osteoclastic lineage representing well-defined differentiation stages. We found that only low levels of transcripts encoding CSF-1 could be detected in metatarsals from 15-day-old fetuses. Transcript levels increased slowly during the following days to reach a maximum in the rudiments from 18-day-old fetuses. After birth, in newborn animals, transcript levels were lowered again. While in rudiments from 15-day-old fetuses a considerable portion of the transcripts encoded the membrane-bound molecule, a transcript encoding the secreted form of the cytokine was the predominant species during the following days. These results suggest that the maintenance of proliferating and postmitotic osteoclast precursors requires low levels of CSF-1 only. Highest levels of locally synthesized CSF-1 are required, however, during the initial recruitment and activation of osteoclasts. After birth, levels of CSF-1 transcripts decrease again, suggesting that newly synthesized CSF-1 may be replaced by protein released from the mineralized matrix during resorption. In conclusion, the present data further strengthen the notion that CSF-1 produced locally acts in a paracrine fashion during the formation of osteoclasts. (J Bone Miner Res 1998;13:1267-1274)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.