Functional dissection of osteoprotegerin and its interaction with receptor activator of NF-kappaB ligand

The receptor activator of NF-kappaB (RANK) belongs to the neuregulin/tumor necrosis factor (TNF) receptor superfamily and is activated by RANK ligand (RANK-L), a homotrimeric, TNF-like cytokine. RANK is present on the surface of osteoclast cell precursors, where its interaction with RANK-L induces their terminal differentiation into osteoclasts, thus increasing bone breakdown. The secreted, soluble receptor osteoprotegerin (OPG) interrupts this activation by binding directly to RANK-L. Therefore, osteoclast maturation (and bone homeostasis) is regulated in vivo by OPG levels of expression. We have studied the assembly state and affinity of OPG for RANK-L by sedimentation analyses and surface plasmon resonance (Biacore). Full-length, homodimeric OPG binds to RANK-L with a KD of 10 nM. OPG is also a member of the TNF receptor superfamily and contains four disulfide-rich ligand-binding domains, yet lacks a transmembrane region separating the ligand-binding region from the two death domains, as observed for other receptor family members. We showed that dimerization of OPG results from noncovalent interactions mediated by the death domains and to a lesser extent by a C-terminal heparin-binding region. In contrast, a C-terminal intermolecular disulfide bond does not contribute to the formation or stability of OPG dimers. A truncate of osteoprotegerin, containing the ligand-binding domains but lacking the dimerization domains, bound RANK-L with a KD of approximately 3 microM, indicating that monomer oligomerization for the OPG provides an increase of 3 orders of magnitude in the affinity for RANK-L. Therefore, OPG dimer formation is required for the mechanism of inhibition of the RANK-L/RANK receptor interaction.     

Epidermal receptor activator of NF-kappaB ligand controls Langerhans cells numbers and proliferation

Langerhans cells (LC) are the dendritic APC population of the epidermis, where they reside for long periods and are self-replicating. The molecular signals underlying these characteristics are unknown. The TNF superfamily member receptor activator of NF-kappaB ligand (RANKL, TNFSF11) has been shown to sustain viability of blood dendritic cells in addition to its role in promoting proliferation and differentiation of several cell types, notably osteoclasts. In this study, we have studied expression of the RANKL system in skin and have defined a key role for this molecule in LC homeostasis. In vitro and in vivo, human KC expressed RANKL and epidermal LC expressed cell surface RANK. In vitro, RANKL sustained CD34(+) progenitor-derived LC viability following 72-h cultures in cytokine-free medium (79.5 +/- 1% vs 55.2 +/- 5.7% live cells, respectively; n = 4; p < 0.05). In vivo, RANKL-deficient mice displayed a marked reduction in epidermal LC density (507.1 +/- 77.2 vs 873.6 +/- 41.6 LC per mm(2); n = 9; p < 0.05) and their proliferation was impaired without a detectable effect on apoptosis. These data indicate a key role for the RANKL system in the regulation of LC survival within the skin and suggest a regulatory role for KC in the maintenance of epidermal LC homeostasis.     

Lysophosphatidylcholine induces inflammatory activation of human coronary artery smooth muscle cells

Lysophosphatidylcholine (LPC) is the major bioactive lipid component of oxidized LDL, thought to be responsible for many of the inflammatory effects of oxidized LDL described in both inflammatory and endothelial cells. Inflammation-induced transformation of vascular smooth muscle cells from a contractile phenotype to a proliferative/secretory phenotype is a hallmark of the vascular remodeling that is characteristic of atherogenesis; however, the role of LPC in this process has not been fully described. The present study tested the hypothesis that LPC is an inflammatory stimulus in coronary artery smooth muscle cells (CASMCs). In cultured human CASMCs, LPC stimulated time- and concentration-dependent release of arachidonic acid that was sensitive to phospholipase A₂ and C inhibition. LPC stimulated the release of arachidonic acid metabolites leukotriene-B₄ and 6-keto-prostaglandin F_1α, within the same time course. LPC was also found to stimulate basic fibroblast growth factor release as well as stimulating the release of the cytokines GM-CSF, IL-6, and IL-8. Optimal stimulation of these signals was obtained via palmitic acid-substituted LPC species. Stimulation of arachidonic acid, inflammatory cytokines and growth factor release, implies that LPC might play a multifactorial role in the progression of atherosclerosis, by affecting inflammatory processes.     

Functional characterization of cysteinyl leukotriene CysLT(2) receptor on human coronary artery smooth muscle cells

Cysteinyl leukotrienes (LTC(4), LTD(4), and LTE(4)) are a class of biologically active lipids that exert potent effects on the heart. To assess their roles, we investigated the distribution of their receptors, CysLT(1) and CysLT(2), in the cardiovascular system. CysLT(2) mRNA was detected at high levels in the human atrium and ventricle and at intermediate levels in the coronary artery, whereas CysLT(1) mRNA was barely detected. Further analysis by in situ hybridization revealed that CysLT(2) mRNA was expressed in myocytes, fibroblasts, and vascular smooth muscle cells, but not in endothelial cells. When human coronary smooth muscle cells were stimulated with LTC(4), the intracellular calcium concentration increased in a dose-dependent manner, and this action was partially inhibited by nicardipine. Additionally, these cells showed chemotactic responses to LTC(4). This is the first report on the physiological role of CysLT(2), and the findings suggest that CysLT(2) has biological significance in the cardiovascular system.     

Effects of peroxynitrite on pig coronary artery smooth muscle

We examined the effects of peroxynitrite pretreatment of pig coronary arteries on their sarcoplasmic reticulum (SR) Ca(2+) pump function. Pretreating rings from de-endothelialized arteries with peroxynitrite, followed by a wash to remove this agent, led to a decrease in the force of contraction produced in response to the SR Ca(2+) pump inhibitor cyclopiazonic acid (CPA, IC(50) = 87 +/- 6 microM). Inclusion of catalase and superoxide dismutase with the peroxynitrite did not alter its effect indicating that the inhibition was produced by peroxynitrite. Contractions produced by 30 mM KCl were not affected by up to 250 microM peroxynitrite. Smooth muscle cells cultured from this artery gave a transient increase in cytosolic Ca(2+) in response to CPA. Treating the cells with peroxynitrite inhibited this increase. Treating the SR-enriched isolated subcellular membrane fraction with peroxynitrite produced an inhibition of the ATP-dependent azide-insensitive oxalate-stimulated Ca(2+) uptake. Thus, peroxynitrite damages the SR Ca(2+)pump in the coronary artery, and this inhibition appears to lead to an inability of the arteries to respond to CPA. Thus, peroxynitrite produced from superoxide and NO in the arteries may compromise regulation of coronary tone which requires mobilization of Ca(2+) from the SR.     

Hypoxia divergently regulates production of reactive oxygen species in human pulmonary and coronary artery smooth muscle cells

Acute hypoxia causes pulmonary vasoconstriction and coronary vasodilation. The divergent effects of hypoxia on pulmonary and coronary vascular smooth muscle cells suggest that the mechanisms involved in oxygen sensing and downstream effectors are different in these two types of cells. Since production of reactive oxygen species (ROS) is regulated by oxygen tension, ROS have been hypothesized to be a signaling mechanism in hypoxia-induced pulmonary vasoconstriction and vascular remodeling. Furthermore, an increased ROS production is also implicated in arteriosclerosis. In this study, we determined and compared the effects of hypoxia on ROS levels in human pulmonary arterial smooth muscle cells (PASMC) and coronary arterial smooth muscle cells (CASMC). Our results indicated that acute exposure to hypoxia (Po(2) = 25-30 mmHg for 5-10 min) significantly and rapidly decreased ROS levels in both PASMC and CASMC. However, chronic exposure to hypoxia (Po(2) = 30 mmHg for 48 h) markedly increased ROS levels in PASMC, but decreased ROS production in CASMC. Furthermore, chronic treatment with endothelin-1, a potent vasoconstrictor and mitogen, caused a significant increase in ROS production in both PASMC and CASMC. The inhibitory effect of acute hypoxia on ROS production in PASMC was also accelerated in cells chronically treated with endothelin-1. While the decreased ROS in PASMC and CASMC after acute exposure to hypoxia may reflect the lower level of oxygen substrate available for ROS production, the increased ROS production in PASMC during chronic hypoxia may reflect a pathophysiological response unique to the pulmonary vasculature that contributes to the development of pulmonary vascular remodeling in patients with hypoxia-associated pulmonary hypertension.     

Effects of C-reactive protein on atherogenic mediators and adrenomedullin in human coronary artery endothelial and smooth muscle cells

We examined the effects of recombinant human C-reactive protein (rhCRP) on atherosclerosis-related factors in cultured human coronary artery endothelial and smooth muscle cells (HCAECs and HCASMCs). After removing endotoxin from commercial rhCRP preparations using the appropriate column, the purified (P)-rhCRP retained the ability to Ca(2+)-dependently bind to phosphorylcholine, but did not augment the secretion of interleukin-6 and MCP-1 from HCAECs, as non-purified (NP)-rhCRP did. By contrast, P-rhCRP elicited 2- to 3-fold increases in the secretion of both hormones from HCASMCs, though the effect was smaller than that obtained with NP-rhCRP. Production of PAI-1 and endothelin-1 was little affected by either rhCRP preparation in either cell type. In addition, P-rhCRP dose-dependently diminished adrenomedullin release from both cell types, but did not affect adrenomedullin receptor expression or function. Our findings highlight the importance of removing endotoxin from commercial rCRP preparations and show that hCRP elicits atherogenic responses from HCASMCs, but not HCAECs.     

Effect of YM471, a nonpeptide AVP receptor antagonist,on human coronary artery smooth muscle cells

The antagonistic properties of YM471, a potent nonpeptide vasopressin (AVP) V(1A) and V(2) receptor antagonist, were characterized using human coronary artery smooth muscle cells (CASMC). YM471 potently inhibited specific binding of 3H-AVP to V(1A) receptors on human CASMC, exhibiting a K(i) value of 0.49 nM. Furthermore, YM471 inhibited the AVP-induced increase in intracellular free Ca(2+) concentration with an IC(50) value of 1.42 nM, but exerted no agonistic activity on CASMC. Additionally, while AVP concentration-dependently induced hyperplasia and hypertrophy in CASMC, YM471 prevented these AVP-induced growth effects, exhibiting IC(50) values of 0.93 and 2.64 nM, respectively. These results indicate that YM471 has high affinity for V(1A) receptors on, and high potency in inhibiting AVP-induced physiologic responses of, human CASMC.     

Regulation of endotoxin-induced proinflammatory activation in human coronary artery cells: expression of functional membrane-bound CD14 by human coronary artery smooth muscle cells

Low-level endotoxemia has been identified as a powerful risk factor for atherosclerosis. However, little is known about the mechanisms that regulate endotoxin responsiveness in vascular cells. We conducted experiments to compare the relative responses of human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC) to very low levels of endotoxin, and to elucidate the mechanisms that regulate endotoxin responsiveness in vascular cells. Endotoxin (10-fold higher in magnitude at >10-fold lower threshold concentrations (10-30 pg/ml) compared with HCAEC. This remarkable sensitivity of HCASMC to very low endotoxin concentrations, comparable to that found in circulating monocytes, was not due to differential expression of TLR4, which was detected in HCAEC, HCASMC, and intact coronary arteries. Surprisingly, membrane-bound CD14 was detected in seven different lines of HCASMC, conferring responsiveness to endotoxin and to lipoteichoic acid, a product of Gram-positive bacteria, in these cells. These results suggest that the low levels of endotoxin associated with increased risk for atherosclerosis are sufficient to produce inflammatory responses in coronary artery cells. Because CD14 recognizes a diverse array of inflammatory mediators and functions as a pattern recognition molecule in inflammatory cells, expression of membrane-bound CD14 in HCASMC implies a potentially broader role for these cells in transducing innate immune responses in the vasculature.     

Activation of peroxisome proliferator-activated receptor gamma inhibits osteoprotegerin gene expression in human aortic smooth muscle cells

Increasing evidence indicates an important role of PPAR gamma activation in modulating the development and progression of atherosclerosis, however, the mechanisms involved in these effects are not well understood since the PPAR gamma-regulated genes in vascular smooth muscle cells (VSMC) are poorly defined. Here we reported that PPAR gamma ligands, GW7845, ciglitazone and troglitazone had the effect of inhibiting osteoprotegerin (OPG) expression in human aortic smooth muscle cells (HASMC). The effect of GW7845 and ciglitazone on OPG expression was completely abolished by GW9662, a PPAR gamma antagonist. Overexpression of PPAR gamma in HASMC by the infection of a PPAR gamma adenovirus dramatically decreased OPG expression. In addition, PPAR gamma activation inhibited OPG promoter activity. Taken together, our data suggest that OPG expression is a novel PPAR gamma target gene in VSMC and downregulation of OPG expression by PPAR gamma activation provides a new insight into the understanding of the role of PPAR gamma in atheroscelrosis and hypertension.     

Tumor necrosis factor-alpha downregulates adrenomedullin receptors in human coronary artery smooth muscle cells

We examined the effects of tumor necrosis factor (TNF)-alpha on the expression and functionality of adrenomedullin (AM) receptors in cultured human coronary artery smooth muscle cells. Analysis of real-time quantitative polymerase chain reactions showed that these cells abundantly express two AM receptors comprised of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein 1 (RAMP1) or RAMP2. TNF-alpha induced time- and dose-dependent decreases in the expression of CRLR and RAMP1/2 mRNAs, thereby diminishing AM-evoked cAMP production. The suppression of these three mRNAs was unaffected by inhibiting NOS, protein kinase G, protein kinase A, superoxide formation or NF-kappaB activation.     

Negative regulation of osteoclastogenesis by ectodomain shedding of receptor activator of NF-kappaB ligand

Receptor activator of NF-kappaB ligand (RANKL) is a transmembrane glycoprotein that has an essential role in the development of osteoclasts. The extracellular portion of RANKL is cleaved proteolytically to produce soluble RANKL, but definite RANKL sheddase(s) and the physiologic function of RANKL shedding have not yet been determined. In the present study, we found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 have strong RANKL shedding activity. In Western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in a reduction of both the lower and higher molecular weight products. Suppression of MMP14 in primary osteoblasts increased membrane-bound RANKL and promoted osteoclastogenesis in cocultures with macrophages. Soluble RANKL produced by osteoblasts from MMP14-deficient mice was markedly reduced, and their osteoclastogenic activity was promoted, consistent with the findings of increased osteoclastogenesis in vivo. RANKL shedding is an important process that down-regulates local osteoclastogenesis.     

Role of NADPH oxidase in cytomegalovirus-induced proliferation of human coronary artery smooth muscle cells

A number of infectious agents have been implicated in the development of vascular diseases such as atherosclerosis and posttransplantation arterial restenosis. Cytomegalovirus (CMV) has been reported to cause obliteration of coronary arteries by a progressive vasculopathy that involves proliferation of medial smooth muscle cells (SMC). In this study, we report that CMV enhances the serum-induced proliferation of human coronary SMC through activation of a superoxide-generating NADPH oxidase. Exposure of SMC to CMV for 2 h was associated with an 80% increase in NADPH oxidase. This increase in oxidase activity was associated with a two-fold increase in serum-induced DNA synthesis (5-bromo-2'-deoxyuridine incorporation) and significant interleukin-8 (IL-8) production by SMC. Diphenylene iodonium, an inhibitor of NADPH oxidase, significantly inhibited CMV-induced IL-8 production and promotion of serum-induced DNA synthesis. Similar effects were seen following pretreatment of SMC with N-acetyl cysteine, a potent antioxidant, suggesting that oxidative stress following CMV exposure might be responsible for triggering the proliferation of SMC. From this study, we conclude that CMV-mediated promotion of SMC growth is redox sensitive and may be mediated by NADPH oxidase.     

Growth and phenotypic characterization of porcine coronary artery smooth muscle cells

Summary Vascular smooth muscle cell (VSMC) proliferation significantly contributes to atherosclerotic plaque formation and limits the success rate of percutaneous transluminal coronary angioplasty. We derived a population of porcine coronary artery SMCs to characterize VSMC proliferation and phenotype in preparation to study the molecular actions of VSMC mitogens and antiproliferative agents. Growth assays were designed to minimize the estrogen content in the culture medium, since this steroid hormone significantly influences VSMC growth and the expression of VSMC mitogens and their receptors. Culture conditions were identified such that this criterion was achieved while maintaining a significant VSMC growth rate. Cells cultured in serum-free medium, regardless of growth factor supplements, did not remain adherent to a plastic culture substrate, nor did they proliferate. Dextran-coated charcoal (DCC)-treated sera, including fetal bovine, calf, and porcine, supported VSMC adhesion, but not growth. Whole fetal bovine serum (FBS) produced the best proliferative response. A type-I collagen-coated culture surface significantly enhanced VSMC growth, but only in culture medium containing non-DCC-treated FBS. Flow cytometry analyses confirmed the mitogenic effects of this substrate. The VSMCs exhibited a morphological change on type-I collagen, but this was not accompanied by a change in VSMC phenotype. Our data indicate that culture of these porcine coronary artery SMCs in 2.5% FBS plus 10 ng platelet-derived growth factor-BB per ml in phenol red-free medium on type-I collagen may be the optimal conditions for studying the molecular aspects of VSMC mitogens and antiproliferative agents.     

Angiogenin activates human umbilical artery smooth muscle cells

Angiogenin stimulates proliferation of human umbilical artery smooth muscle cells. This activity of angiogenin depends on the cell density and requires nuclear translocation of the ligand as well as activation of SAPK/JNK MAP kinase. Angiogenin binds to a 170-kDa putative receptor on the cell surface and induces phosphorylation of SAPK/JNK. It also undergoes nuclear translocation in a time and concentration dependent manner. Neomycin inhibits nuclear translocation of angiogenin and abolishes angiogenin-induced cell proliferation but does not inhibit SAPK/JNK phosphorylation. The data demonstrate that smooth muscle cells are targets for angiogenin and that both SAPK/JNK phosphorylation and nuclear translocation of the ligand are required for angiogenin to activate smooth muscle cells.     

Effects of lubiprostone on human uterine smooth muscle cells

Lubiprostone, a bicyclic fatty acid derivative and member of a new class of compounds called prostones, locally activates ClC-2 Cl(-) channels without activation of prostaglandin receptors. The present study was specifically designed to test and compare lubiprostone and prostaglandin effects at the cellular level using human uterine smooth muscle cells. Effects on [Ca(2+)](i), membrane potential and [cAMP](i) in human uterine smooth muscle cells were measured. 10 nM lubiprostone significantly decreased [Ca(2+)](i) from 188 to 27 nM, which was unaffected by 100 nM SC-51322, a prostaglandin EP receptor antagonist. In contrast 10nM PGE(2) and PGE(1) both increased [Ca(2+)](i) 3-5-fold which was blocked by SC-51322. Similarly, lubiprostone and prostaglandins had opposite/different effects on membrane potential and [cAMP](i). Lubiprostone caused SC-51322-insensitive membrane hyperpolarization and no effect on [cAMP](i). PGE(2) and PGE(1) both caused SC-51322-sensitive membrane depolarization and increased [cAMP](i). Lubiprostone has fundamentally different cellular effects from prostaglandins that are not mediated by EP receptors.     

Calcification of human vascular smooth muscle cells: associations with osteoprotegerin expression and acceleration by high-dose insulin

Arterial medial calcifications occur often in diabetic individuals as part of the diabetic macroangiopathy. The pathogenesis is unknown, but the presence of calcifications predicts risk of cardiovascular events. We examined the effects of insulin on calcifying smooth muscle cells in vitro and measured the expression of the bone-related molecule osteoprotegerin (OPG). Human vascular smooth muscle cells (VSMCs) were grown from aorta from kidney donors. Induction of calcification was performed with beta-glycerophosphate. The influence of insulin (200 microU/ml or 1,000 microU/ml) on calcification was judged by measuring calcium content in the cell layer and by von Kossa staining. OPG was measured in the medium by ELISA. Histochemistry was used for determination of alkaline phosphatase (ALP). Bone sialoprotein (BSP) and OPG mRNA expressions were done by RT-PCR. beta-Glycerophosphate was able to induce calcification in human smooth muscle cells from a series of donors after variable time in culture. Decreased OPG amounts were observed from the cells during the accelerated calcification phase. High dose of insulin (1,000 microU/ml) accelerated the calcification, whereas lower concentrations (200 microU/ml) did not. Calcified cells expressed ALP and BSP activity in high levels. In conclusion, high concentration of insulin enhances in vitro-induced calcification in VSMCs. Altered OPG levels during the calcification raise the possibility that OPG may have a potent function in regulating the calcification process or it may represent a consequence of mineralization. Effects of insulin and modulations by OPG on the calcification process in arterial cells may play a role in the development of calcifications as part of the diabetic macroangiopathy.     

mu-Calpain regulates receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis via NF-kappaB activation in RAW 264.7 cells

To clarify the role of calpain in the receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis, RANKL-induced calpain activation was examined by using murine RAW 264.7 cells and bone marrow-derived monocyte/macrophage progenitors. We found that calpain activity increased in response to RANKL in both cell types based on alpha-spectrinolysis and that mu-calpain, rather than m-calpain, was activated during RANKL-supported osteoclastogenesis in RAW 264.7 cells. Overexpression of mu-calpain clearly augmented RANKL-supported osteoclastogenesis in RAW 264.7 cells, thereby implicating its pivotal role in this process. Cell-permeable calpain inhibitors, including calpastatin and calpeptin, were sufficient to suppress RANKL-supported osteoclastogenesis based on decreased expression of the osteoclastogenic marker, matrix metalloproteinase 9, and the generation of tartrate-resistant acid phosphatase-positive multinucleated cells in both cell types. Calpain inhibitors suppressed NF-kappaB activation via inhibition of the cleavage of inhibitor of NF-kappaB(IkappaBalpha)in RAW 264.7 cells. Taken together, our findings suggest that mu-calpain is essential to the regulation of RANKL-supported osteoclastogenesis via NF-kappaB activation.