ALBERT

Description: We report that two widely-used drugs for erectile dysfunction, tadalafil and vardenafil, trigger bone gain in mice through a combination of anabolic and antiresorptive actions on the skeleton. Both drugs were found to enhance osteoblastic bone formation in vivo using a unique gene footprint and to inhibit osteoclast formation. The target enzyme, phosphodiesterase 5A (PDE5A), was found to be expressed in mouse and human bone as well as in specific brain regions, namely the locus coeruleus, raphe pallidus, and paraventricular nucleus of the hypothalamus. Localization of PDE5A in sympathetic neurons was confirmed by coimmunolabeling with dopamine β-hydroxylase, as well as by retrograde bone-brain tracing using a sympathetic nerve-specific pseudorabies virus, PRV152. Both drugs elicited an antianabolic sympathetic imprint in osteoblasts, but with net bone gain. Unlike in humans, in whom vardenafil is more potent than tadalafil, the relative potencies were reversed with respect to their osteoprotective actions in mice. Structural modeling revealed a higher binding energy of tadalafil to mouse PDE5A compared with vardenafil, due to steric clashes of vardenafil with a single methionine residue at position 806 in mouse PDE5A. Collectively, our findings suggest that a balance between peripheral and central actions of PDE5A inhibitors on bone formation together with their antiresorptive actions specify the osteoprotective action of PDE5A blockade.

Description: Abstract 3277 In recent decades, addition to the diet of synthetically hydrogenated vegetable oils has markedly increased human consumption of trans fatty acids. Epidemiological studies have linked this change in diet to current high rates of atherosclerotic cardiovascular disease. Despite recognition of this important connection, the basic mechanisms by which trans fatty acids contribute to the pathogenesis of atherosclerosis are still not well understood. In the present studies we examined the effects of trans fatty acids on macrophage functions and their possible role in the pathogenesis of atherosclerosis. Human macrophages, derived from peripheral blood mononuclear cells, were treated with the trans fat elaidic acid (C18:Δ9–10 trans), the corresponding cis fatty acid oleic acid (C18:Δ9–10 cis), or the saturated fatty acid stearic acid (C18:0). We examined changes in macrophage fat metabolism using GC/MS to measure cell fatty acid content and intermediates, and MS/MS to identify acylcarnitine derivatives, and assayed fatty acid oxidation using fatty acids radiolabeled at the [1–14C] position and the double bond at the [C9-C103H] position. After 44 hours treatment with 100 micromolar elaidic acid, macrophages showed an accumulation of multiple unsaturated fatty acid intermediates, both long-chain and short-chain, by GC/MS analysis, that were not observed in cultures containing either oleic or stearic acid. Using acylcarnitine analysis, we observed an increase in C12 and C18 intermediates in the macrophages exposed to trans fat (either as fatty acids or partially hydrogenated soy oil) compared to controls. These results suggest a block in acyl-CoA removal one group proximate to the trans bond. Beta-oxidation assays using carbon-1 radiolabeled oleic and elaidic acids revealed enhanced entry of the trans-fat into the catabolic cycle compared to the entry of the natural cis-fatty acid. Using carbon 9–10 radiolabeled oleic acid to study oleic acid catabolism, we discovered that in the presence of the trans fat, oxidation of the cis fat was diminished. Thus, in addition to the block in the catabolism of the trans fat itself, the degradation of the cis monounsaturated fatty acids are also impaired in the presence of the trans fat. We then examined the effects of inhibited fatty acid catabolism on macrophage function by examining changes in gene expression. Initial results from Affymetrix gene expression profiling, were confirmed using quantitative real time PCR. These studies revealed that exposure to trans fatty acid, compared to cis fatty acids, markedly upregulated macrophage expression of interleukin 1 beta, an inflammatory cytokine previously implicated in the pathogenesis of atherosclerosis. Also increased was expression of heparin-binding epidermal growth factor, previously implicated as a stimulus for vascular smooth muscle proliferation in atherosclerosis. The results overall suggest that the deleterious effects of trans fats may be linked to impaired macrophage fatty acid catabolism, contributing to lipid accumulation in the atheroma, and also to increased macrophage production of inflammatory mediators. Disclosures: No relevant conflicts of interest to declare.

Description: Increased osteoclastogenesis and angiogenesis occur in physiologic and pathologic conditions. However, it is unclear if or how these processes are linked. To test the hypothesis that osteoclasts stimulate angiogenesis, we modulated osteoclast formation in fetal mouse metatarsal explants or in adult mice and determined the effect on angiogenesis. Suppression of osteoclast formation with osteoprotegerin dose-dependently inhibited angiogenesis and osteoclastogenesis in metatarsal explants. Conversely, treatment with parathyroid hormone related protein (PTHrP) increased explant angiogenesis, which was completely blocked by osteoprotegerin. Further, treatment of mice with receptor activator of nuclear factor-κB ligand (RANKL) or PTHrP in vivo increased calvarial vessel density and osteoclast number. We next determined whether matrix metalloproteinase-9 (MMP-9), an angiogenic factor predominantly produced by osteoclasts in bone, was important for osteoclast-stimulated angiogenesis. The pro-angiogenic effects of PTHrP or RANKL were absent in metatarsal explants or calvaria in vivo, respectively, from Mmp9−/− mice, demonstrating the importance of MMP-9 for osteoclast-stimulated angiogenesis. Lack of MMP-9 decreased osteoclast numbers and abrogated angiogenesis in response to PTHrP or RANKL in explants and in vivo but did not decrease osteoclast differentiation in vitro. Thus, MMP-9 modulates osteoclast-stimulated angiogenesis primarily by affecting osteoclasts, most probably by previously reported migratory effects on osteoclasts. These results clearly demonstrate that osteoclasts stimulate angiogenesis in vivo through MMP-9.

Description: Atherosclerosis, through its sequelae of heart disease, stroke and peripheral vascular disease, underlies substantial human morbidity and mortality. The incidence of atherosclerosis increased markedly over the last century paralleling the increase in fat, particularly saturated fat, in the modern diet. Furthermore, artificial hydrogenation of saturated fats, originally introduced to provide a ‘healthier’ dietary substitute, resulted in consumption of non-physiological trans fatty acids that are themselves now strongly linked to the development of atherosclerosis. A major cellular target for the negative effects of fatty acids in the vasculature appears to be the macrophage, which through actions including the production of inflammatory cytokines, plays a central role of the pathophysiology of atherosclerosis. Despite the importance of these effects, our understanding of the fundamental molecular changes that underlie the negative effects of fatty acids, and especially trans fatty acids, remains fragmentary. In previous work we showed that prolonged exposure of human macrophages to the 18 carbon trans-monoenoic fatty acid elaidic acid impaired lipid metabolism, partially blocking beta-oxidation and thereby altering cellular lipid composition. To determine the functional consequences of this alteration in cellular lipids, we next examined changes in macrophage gene expression. Primary human macrophages derived from peripheral blood monocytes were treated with the cis fatty acid oleate or the trans fat elaidate for 44 hours, and gene expression was evaluated using the affymetrix gene array, with significant differences confirmed by real time quantitative PCR. Expression of genes linked directly to lipid metabolism differed, as expected, but the most striking differences between cis and trans fatty acid treated macrophages were in their expression of regulators of zinc homeostasis. In particular, there was a marked divergence in expression levels for metallothioneins, the zinc binding proteins which regulate intracellular zinc levels: metallothionein expression was consistently lower in the elaidate-treated macrophages. In contrast, the zinc transporter SLC39A10 was significantly upregulated in macrophages by exposure to the trans but not the cis fatty acid. SLC39A10 mediates uptake of extracellular zinc by cells and its release from organellar stores; its effects are opposed by SLC30, which export zinc from the cytosol but were not induced by exposure to elaidate. These results suggested that trans fats would increase zinc activity in macrophages. To test this we used the fluorescent zinc indicator FluoZin3 (N-(carboxymethyl)-N-[2-[2-[2(carboxymethyl)amino]-5-(2,7,-difluoro-6-hydroxy-3-oxo-3H-xanthen-9-yl)phenoxy]ethoxy]-4-methoxyphenyl]glycine). These studies confirmed that macrophages exposed to elaidate showed increased intracellular Zn2+, compared to both untreated or oleate-treated macrophages. Furthermore, the increase in zinc activity was associated with activation of the NFkB signaling pathway: macrophages treated with elaidic acid showed prominent nuclear translocation of NFkB p65, and the effect was abrogated by cotreatment with the zinc chelator TPEN. We next compared the effects of the trans fat with those of saturated fatty acids. Macrophages exposed to stearate or palmitate showed a milder and delayed increase in metallothionein expression, compared to those treated with oleate, but, like oleate, stearate and palmitate had no effect on the expression of zinc transporters. Studies using FluoZin3 showed a mild early increase in cytosolic Zn2+ following stearate or palmitate treatment, but at the later time points, when metallothionein expression was increased by the saturated fatty acids as well as by oleate, zinc activity returned to baseline. Only elaidic acid, which upregulated the zinc transporter, produced a sustained increase in intracellular macrophage Zn2+. Stearate and palmitate also stimulated early translocation of NFkB to the nucleus. These results suggest that trans fatty acids, while mimicking some activation pathways stimulated by the saturated fatty acids, exert distinct effects through sustained alterations in zinc regulatory proteins that promote elevated intracellular Zn2+. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 928 Calcium signals are major regulators of human osteoclast formation and function, and the molecular mechanisms underlying calcium effects are of interest as possible targets for pharmacologic regulation of bone resorption. IP3-receptor regulated release of calcium stores is linked to NFATc1 activation, which stimulates expression of key osteoclast genes in precursors, but the roles of other calcium channels in osteoclastogenesis are not clear. In particular, the identity of the channel(s) mediating extracellular calcium influx triggered by release of calcium stores remains uncertain. In lymphoid cells, a major mediator of this extracellular calcium influx is the Calcium-Release Activated Calcium (CRAC) channel consisting of Orai1, a plasma-membrane calcium channel, and the calcium-sensitive regulatory protein, STIM1. Calcium released from intracellular stores binds to a low affinity EF-hand in STIM1 causing a conformational change in STIM1 that permits binding to Orai1, aggregation in microscopically distinct puncta at the cell membrane, and opening of the Orai1 channel with consequent influx of extracellular calcium. Targeted deletion of Orai1 or Stim1 in mice results in severe immunodeficiency and early death; this has limited the assessment of Orai1 effects in other tissues. To evaluate the specific role of Orai1 in human osteoclasts, we used peripheral blood monocytes which form multinucleated osteoclasts capable of bone resorption when treated with CSF1 and RANKL. We confirmed Orai1 expression in human monocytes using Western blot and quantitative PCR assays, and found that the protein was down-regulated in mature osteoclasts. We used fura-2 to measure store-dependent and -independent changes in intracellular calcium during osteoclastic differentiation of monocytes over 10–14 days in RANKL and CSF1. RANKL-associated calcium oscillations were detected throughout differentiation, but calcium-release activated influx of extracellular calcium was markedly lower in the mature osteoclasts compared to precursors, paralleling their expression of Orai1. Human monocytes, transfected with Orai1-specific siRNA producing an 80% reduction in Orai1 protein compared to control siRNA treated cells, showed inhibition of store-regulated calcium influx during osteoclastogenesis. Furthermore, monocytes deficient in Orai1 showed impaired osteoclast formation; in particular, multinucleation resulting from osteoclast precursor cell fusion was markedly reduced, impairing bone resorption. Orai1 deficiency in T-cells inhibits activation of NFATc1, but this did not appear prominent in our cells: we found no significant inhibition of NFATc1 regulated gene expression in Orai1 siRNA-transfected cells compared to control siRNA-transfected cells, despite the marked difference in Orai1 protein. Other calcium channels may mediate calcium dependent NFATc1 activation in osteoclast precursors; alternatively, the low level of Orai1 protein remaining in Orai1 siRNA treated monocytes may be sufficient for NFATc1 activation. To define the effects of complete Orai1 deficiency, we examined osteoclast formation and in vivo skeletal development in mice with targeted deletion of the Orai1 gene (Gwack et al. Mol Cell Biol 28 (2008) 5209-22). Consistent with our in vitro results, multinucleated osteoclasts were nearly absent from Orai1-/- mice, but mononuclear cells expressing osteoclast markers such as TRAcP, were seen. Surprisingly, the knock-out mice did not show the osteopetrotic phenotype typical of osteoclast deficiency. Retention of fetal cartilage was seen, indicating defective osteoclastic function in Orai1-/- mice, but marked inhibition of bone formation was also present. Using micro-computed tomography we found significant reductions in both cortical ossification and trabecular bone formation in Orai1-/- mice. This raised the possibility of a previously unrecognized role for Orai1 in osteoblasts, or the osteoblast defect might simply reflect abnormalities of Orai1-/- osteoclasts and/or lymphocytes, since both cell types have regulatory effects on osteoblast formation and function. In summary, our studies identify a requirement for CRAC channel mediated calcium influx, and specifically the Orai1 channel, for normal formation and activity of human osteoclasts; these results are confirmed in an Orai1 knock-out mouse which also shows defective bone formation. Disclosures: No relevant conflicts of interest to declare.

Description: Estrogen is a major positive regulator of bone mass with important inhibitory effects on bone resorption. However, knowledge of the mechanisms underlying estrogen effects remains incomplete. We investigated whether estrogen altered osteoclastic differentiation of human monocytes in response to Receptor Activator of NF-kB (RANK) Ligand (RANKL), a critical stimulus for osteoclast formation. For these studies, human monocytic cells were isolated from peripheral blood by positive selection for CD14 expression (“CD14 cells”) and maintained in charcoal stripped medium with monocyte colony-stimulating factor. Compared to RANKL treatment alone, co-treatment with 17-β -estradiol reduced osteoclast formation, as shown by decreased development of tartrate resistant acid phosphatase (TRAP) activity, multinucleation, and resorption pit formation on bone. A possible mechanism for the estrogen effects was interference with RANKL signal transduction. We examined estrogen effects on a key event in the RANKL signaling: nuclear translocation of NF-kB following phosphorylation and degradation of IkB. Estrogen cotreatment of RANKL-stimulated CD14 cells inhibited the nuclear translocation of NF-kB observed with RANKL alone. This effect was evident after only 30 minutes of treatment. Cotreatment with estrogen also inhibited RANKL-stimulated IkB phosphorylation; this was evident within 5 minutes after treatment. The time course of the estrogen effects argued strongly for a non-transcriptional mechanism. We therefore investigated the involvement of proteins previously implicated in rapid estrogen effects, including the breast cancer anti-estrogen resistance protein 1(BCAR1, also called p130Cas), a scaffolding/adaptor protein recently reported to interact with estrogen receptors in breast cancer cells. Co-immunoprecipitation from ligand treated CD14 cells revealed an estrogen-dependent association of estrogen receptor-α (ER) with BCAR1 that was enhanced by cotreatment with RANKL. The coordinate regulation of this complex by both RANKL and estrogen suggested that BCAR1 might represent a link between the two signaling systems. In addition, in cells cotreated with estrogen and RANKL, co-immunoprecipitation revealed a further association of ER with Traf6, a critical proximal signaling intermediate for RANKL. These protein interactions were also rapid, evident 5 minutes after ligand addition. To test the functional significance of BCAR1 in estrogen effects, its expression was inhibited by transduction with BCAR1 siRNA. In cells deficient in BCAR1, estrogen no longer inhibited RANKL-stimulated NF-kB nuclear translocation. Unexpectedly, suppression of BCAR1 itself partially blocked RANKL stimulated NF-kB translocation, suggesting a role for BCAR1 even in the absence of estrogen. Furthermore, when BCAR1 deficient CD14 cells were treated with RANKL, osteoclastic differentiation, as assessed by formation of TRAP-positive multinucleated cells, was reduced compared to RANKL-treated cells transduced with control siRNA. The results suggest that BCAR1 is involved in the RANK-NF-kB pathway that mediates osteoclast differentiation signals in monocytes. In addition, BCAR1 appears necessary for estrogen inhibition of RANKL signaling. The estrogen dependent association of ER with BCAR1 may, like BCAR1 deficiency, block BCAR1 actions in the RANKL-NF-kB pathway, and thus inhibit the osteoclastic differentiation of monocytes.