Effects of alpha/beta-androstenediol immune regulating hormones on bone remodeling and apoptosis in osteoblasts.

A large body of evidence suggests that the immune system directly impacts bone physiology. We tested whether immune regulating hormones (IRH), 17beta-androstenediol (beta-AED), 7beta,17beta-androstenetriol (beta-AET) or the 17alpha-androstenediol (alpha-AED), and 7alpha,17beta-androstenetriol (alpha-AET) metabolites could directly influence bone remodeling in vitro using human fetal osteoblasts (FOB-9). The impact on bone remodeling was examined by comparing the ratio of RANKL/OPG gene expression in response to AED and AET compounds. The alpha-AED was found to significantly increase in the ratio of RANKL/OPG gene expression and altering the morphology of RANKL stained FOB-9 cells. Cell viability was assessed using a Live/Dead assay. Again alpha-AED was unique in its ability to reduce the proportion of viable cells, and to induce mild apoptosis of FOB-9 cells. Treatment of FOB-9 cells with WY14643, an activator of PPAR-alpha and -gamma, also significantly elevated the percentage of dead cells. This increase was abolished by co-treatment with GW9962, a specific inhibitor of PPAR-gamma. Analysis of PPAR-gamma mRNA by Quantitative RT-PCR and its activation by DNA binding demonstrated that alpha-AED increased PPAR-gamma activation by 19%, while beta-AED conferred a 37% decrease in PPAR-gamma activation. In conclusion, alpha-AED opposed beta-AED by elevating a bone resorption scenario in osteoblast cells. The increase in RANKL/OPG is modulated by an activation of PPAR-gamma that in turn caused mild apoptosis of FOB-9 cells.

Expression of RANKL in osteolytic membranes: association with fibroblastic cell markers.

BACKGROUND: Aseptic loosening is often mentioned as the primary reason for costly revision of total joint arthroplasties. Receptor activator of nuclear factor-kappaB ligand (RANKL) appears to be a major factor in the bone resorption observed in periprosthetic osteolysis. RANKL plays an essential role in the recruitment, differentiation, and survival of the osteoclasts implicated in periprosthetic osteolysis. This study was performed in an effort to identify the cell type in the periprosthetic membrane responsible for expression of RANKL. METHODS: Tissues harvested from osteolytic lesions in nine patients undergoing total joint revision were serially sectioned for immunohistochemical analysis. Intercellular adhesion molecule-1 (ICAM-1) and prolyl 4-hydroxylase (5B5) antibodies were used to detect fibroblasts, and anti-CD-163 (Ber-MAC3) was used to detect macrophages. In addition, antibodies to osteoprotegerin (OPG), RANKL, and receptor activator of nuclear factor-kappaB (RANK) were utilized. The binding pattern of these antibodies was then viewed with confocal microscopy with the use of only secondary antibodies as method controls. RESULTS: Histological analysis was confined to areas of the membrane where cells were detected with use of Hoechst 34580 nuclear stain. In the membrane specimens from all nine patients, diffuse RANKL staining was localized to areas lacking cells and more intense staining was seen in areas containing nucleated cells. There was strong colocalization between RANKL and OPG, and there was weak but specific colocalization between RANKL and both 5B5 and ICAM-1. In contrast, there was complete separation of antibody staining of Ber-MAC3 and RANKL, indicating only generalized overlap of the myeloid markers with the RANKL. CONCLUSIONS: RANKL expression was localized to cells that stained positively for fibroblast markers. The data also indicated that there is an intact RANKL/RANK/OPG system in the periprosthetic membrane that could regulate focalized bone resorption in osteolysis. CLINICAL RELEVANCE: Identifying the cell types responsible for RANKL production is critical to the development of a strategy to prevent periprosthetic osteolysis.

Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus.

KCl has long been used as a convenient stimulus to bypass G protein-coupled receptors (GPCR) and activate smooth muscle by a highly reproducible and relatively "simple" mechanism involving activation of voltage-operated Ca2+ channels that leads to increases in cytosolic free Ca2+ ([Ca2+]i), Ca2+-calmodulin-dependent myosin light chain (MLC) kinase activation, MLC phosphorylation and contraction. This KCl-induced stimulus-response coupling mechanism is a standard tool-set used in comparative studies to explore more complex mechanisms generated by activation of GPCRs. One area where this approach has been especially productive is in studies designed to understand Ca2+ sensitization, the relationship between [Ca2+]i and force produced by GPCR agonists. Studies done in the late 1980s demonstrated that a unique relationship between stimulus-induced [Ca2+]i and force does not exist: for a given increase in [Ca2+]i, GPCR activation can produce greater force than KCl, and relaxant agents can produce the opposite effect to cause Ca2+ desensitization. Such changes in Ca2+ sensitivity are now known to involve multiple cell signaling strategies, including translocation of proteins from cytosol to plasma membrane, and activation of enzymes, including RhoA kinase and protein kinase C. However, recent studies show that KCl can also cause Ca2+ sensitization involving translocation and activation of RhoA kinase. Rather than complicating the Ca2+ sensitivity story, this surprising finding is already providing novel insights into mechanisms regulating Ca2+ sensitivity of smooth muscle contraction. KCl as a "simple" stimulus promises to remain a standard tool for smooth muscle cell physiologists, whose focus is to understand mechanisms regulating Ca2+ sensitivity.

K+ depolarization induces RhoA kinase translocation to caveolae and Ca2+ sensitization of arterial muscle.

KCl causes smooth muscle contraction by elevating intracellular free Ca2+, whereas receptor stimulation activates an additional mechanism, termed Ca2+ sensitization, that can involve activation of RhoA-associated kinase (ROK) and PKC. However, recent studies support the hypothesis that KCl may also increase Ca2+ sensitivity. Our data showed that the PKC inhibitor GF-109203X did not, whereas the ROK inhibitor Y-27632 did, inhibit KCl-induced tonic (5 min) force and myosin light chain (MLC) phosphorylation in rabbit artery. Y-27632 also inhibited BAY K 8644- and ionomycin-induced MLC phosphorylation and force but did not inhibit KCl-induced Ca2+ entry or peak ( approximately 15 s) force. Moreover, KCl and BAY K 8644 nearly doubled the amount of ROK colocalized to caveolae at 30 s, a time that preceded inhibition of force by Y-27632. Colocalization was not inhibited by Y-27632 but was abolished by nifedipine and the calmodulin blocker trifluoperazine. These data support the hypothesis that KCl caused Ca2+ sensitization via ROK activation. We discuss a novel model for ROK activation involving translocation to caveolae that is dependent on Ca2+ entry and involves Ca2+-calmodulin activation.