Resorption controls bone anabolism driven by parathyroid hormone (PTH) receptor signaling in osteocytes.

The contribution of remodeling-based bone formation coupled to osteoclast activity versus modeling-based bone formation that occurs independently of resorption, to the anabolic effect of PTH remains unclear. We addressed this question using transgenic mice with activated PTH receptor signaling in osteocytes that exhibit increased bone mass and remodeling, recognized skeletal effects of PTH elevation. Direct inhibition of bone formation was accomplished genetically by overexpressing the Wnt antagonist Sost/sclerostin; and resorption-dependent bone formation was inhibited pharmacologically with the bisphosphonate alendronate. We found that bone formation induced by osteocytic PTH receptor signaling on the periosteal surface depends on Wnt signaling but not on resorption. In contrast, bone formation on the endocortical surface results from a combination of Wnt-driven increased osteoblast number and resorption-dependent osteoblast activity. Moreover, elevated osteoclasts and intracortical/calvarial porosity is exacerbated by overexpressing Sost and reversed by blocking resorption. Furthermore, increased cancellous bone is abolished by Wnt inhibition but further increased by blocking resorption. Thus, resorption induced by PTH receptor signaling in osteocytes is critical for full anabolism in cortical bone, but tempers bone gain in cancellous bone. Dissecting underlying mechanisms of PTH receptor signaling would allow targeting actions in different bone compartments, enhancing the therapeutic potential of the pathway.

Role of PTHrP in human intestinal Caco-2 cell response to oxidative stress.

We have previously demonstrated that parathyroid hormone (PTH) induces apoptosis in human colon adenocarcinoma Caco-2 cells but the effects of its tumoral analog PTH-related peptide (PTHrP) in this cell line are still unknown. In the present work we investigated whether PTHrP, as PTH, is able to induce Caco-2 cell apoptosis or if it exerts protective effects under apoptotic conditions. Using Caco-2 cells cultured under serum deprivation in the presence or absence of PTHrP we demonstrated that, differently to PTH, its analog employed at the same concentration (10(-8)M) is not a pro-apoptotic hormone. Cells were exposed to an oxidative insult in the form of hydrogen peroxide to induce apoptosis, which leads to a 50% loss of cell viability determined by MTS assay, morphological changes observed under fluorescence microscopy and Western blot analysis. Herein we demonstrate, for the first time, that pre-treatment with PTHrP prior to H2O2 incubation, prevents cell death induced by the apoptotic inductor; and using specific inhibitors we evidenced that protein kinase B (AKT), extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2) and p38 mitogen-activated protein kinase (MAPK) mediate this anti-apoptotic effect. Also, we found that PTHrP decreases the pro-apoptotic protein BAX levels and increases the protein expression of the anti-apoptotic HSP27. Immunoblot analysis revealed that H2O2 increases the phosphorylation levels of AKT and MAPKs, exhibiting a cellular defense response; and consequently increases phospho-BAD levels. The H2O2-induced activation of protein kinases is reverted when cells are pre-treated with PTHrP. Altogether these results evidence a protective effect of PTHrP under apoptotic conditions in intestinal cells, which may be mediated by AKT and MAPKs.

In vitro and in vivo evidence of the antineoplastic activity of quercetin against endothelial cells transformed by Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor.

Quercetin (QUE) is a natural flavonoid with well-known anticancer capabilities, although its effect on viral-induced cancers is less studied. Kaposi's sarcoma (KS) is a viral cancer caused by the human herpesvirus-8, which, during its lytic phase, expresses a constitutively activated viral G protein-coupled receptor (vGPCR) able to induce oncogenic modifications that lead to tumor development. The aim of this work was to investigate the potential effect of QUE on in vitro and in vivo models of Kaposi's sarcoma, developed by transforming endothelial cells with the vGPCR of Kaposi's sarcoma-associated herpesvirus. Initially, the antiproliferative effect of QUE was determined in endothelial cells stably expressing the vGPCR (vGPCR cells), with an IC50 of 30 µM. Additionally, QUE provoked a decrease in vGPCR cell viability, interfered with the cell cycle progression, and induced apoptosis, as revealed by annexin V/PI analysis and caspase-3 activity. The presence of apoptotic bodies and disorganized actin filaments was observed by SEM and phalloidin staining. Furthermore, tumors from vGPCR cells were induced in nude mice, which were treated with QUE (50 or 100 mg/kg/d) resulting in retarded tumor progression and reduced tumor weight. Notably, neither kidney nor liver damage was observed, as indicated by biochemical parameters in serum. In conclusion, this study suggests for the first time that QUE exhibits antineoplastic activity in both in vitro and in vivo models of KS, marking a starting point for further investigations and protocols for therapeutic purpose.

In Vitro Studies of Pegylated Magnetite Nanoparticles in a Cellular Model of Viral Oncogenesis: Initial Studies to Evaluate Their Potential as a Future Theranostic Tool.

Magnetic nanosystems represent promising alternatives to the traditional diagnostic and treatment procedures available for different pathologies. In this work, a series of biological tests are proposed, aiming to validate a magnetic nanoplatform for Kaposi's sarcoma treatment. The selected nanosystems were polyethylene glycol-coated iron oxide nanoparticles (MAG.PEG), which were prepared by the hydrothermal method. Physicochemical characterization was performed to verify their suitable physicochemical properties to be administered in vivo. Exhaustive biological assays were conducted, aiming to validate this platform in a specific biomedical field related to viral oncogenesis diseases. As a first step, the MAG.PEG cytotoxicity was evaluated in a cellular model of Kaposi's sarcoma. By phase contrast microscopy, it was found that cell morphology remained unchanged regardless of the nanoparticles' concentration (1-150 µg mL-1). The results, arising from the crystal violet technique, revealed that the proliferation was also unaffected. In addition, cell viability analysis by MTS and neutral red assays revealed a significant increase in metabolic and lysosomal activity at high concentrations of MAG.PEG (100-150 µg mL-1). Moreover, an increase in ROS levels was observed at the highest concentration of MAG.PEG. Second, the iron quantification assays performed by Prussian blue staining showed that MAG.PEG cellular accumulation is dose dependent. Furthermore, the presence of vesicles containing MAG.PEG inside the cells was confirmed by TEM. Finally, the MAG.PEG steering was achieved using a static magnetic field generated by a moderate power magnet. In conclusion, MAG.PEG at a moderate concentration would be a suitable drug carrier for Kaposi's sarcoma treatment, avoiding adverse effects on normal tissues. The data included in this contribution appear as the first stage in proposing this platform as a suitable future theranostic to improve Kaposi's sarcoma therapy.

Molecular and cellular outcomes of quercetin actions on healthy and tumor osteoblasts.

There is a global trend in the use of natural bioactive compounds to complement conventional therapies in bone diseases. In this work, we studied the effects of the phytoestrogen quercetin (QUE) in healthy and tumor osteoblasts. We found that QUE (1 µM, 48 h) significantly increased the cell number and the viability of healthy human osteoblasts (hFOB cells) determined by a trypan blue and a MTS assay, respectively, among other concentrations tested. In addition, wound healing and cellular adhesion assays also demonstrated that 1 µM of QUE significantly stimulated both parameters in osteoblasts. Moreover, osteoblast differentiation was also triggered by QUE in an osteogenic medium by measuring alkaline phosphatase activity, calcium deposition, and collagen levels. Herein, a concentration of 0.01 µM of QUE showed an increment in these differentiation markers and an activation of AKT/GSK3ß/ß-catenin pathway, determined by a Western blot analysis. In addition, immunocytochemistry and subcellular fraction studies indicated an increase of ß-catenin localization in the plasma membrane after QUE treatment. Otherwise, QUE (20-100 µM) decreased the cell number and the viability in tumor osteoblasts (ROS 17/2.8 cells) after 48 h. Furthermore, QUE (100 µM) decreased AKT(Ser473) and the pro-apoptotic protein BAD(Ser136) phosphorylation. In addition, the ERK1/2 phosphorylation increased leading to osteosarcoma cell death since pre-treatment with the MEK inhibitor PD98059 had reverted QUE effect. Altogether, these results indicate that low concentrations of QUE stimulate osteoblastogenesis but have no effect on the growth of tumor osteoblast cells, for which only high concentrations are efficient.

Connexin43 interacts with ßarrestin: a pre-requisite for osteoblast survival induced by parathyroid hormone.

Parathyroid hormone (PTH) promotes osteoblast survival through a mechanism that depends on cAMP-mediated signaling downstream of the G protein-coupled receptor PTHR1. We present evidence herein that PTH-induced survival signaling is impaired in cells lacking connexin43 (Cx43). Thus, expression of functional Cx43 dominant negative proteins or Cx43 knock-down abolished the expression of cAMP-target genes and anti-apoptosis induced by PTH in osteoblastic cells. In contrast, cells lacking Cx43 were still responsive to the stable cAMP analog dibutyril-cAMP. PTH survival signaling was rescued by transfecting wild type Cx43 or a truncated dominant negative mutant of ßarrestin, a PTHR1-interacting molecule that limits cAMP signaling. On the other hand, Cx43 mutants lacking the cytoplasmic domain (Cx43(Δ245)) or unable to be phosphorylated at serine 368 (Cx43(S368A)), a residue crucial for Cx43 trafficking and function, failed to restore the anti-apoptotic effect of PTH in Cx43-deficient cells. In addition, overexpression of wild type ßarrestin abrogated PTH survival signaling in Cx43-expressing cells. Moreover, ßarrestin physically associated in vivo to wild type Cx43 and to a lesser extent to Cx43(S368A) ; and this association and the phosphorylation of Cx43 in serine 368 were reduced by PTH. Furthermore, induction of Cx43(S368) phosphorylation or overexpression of wild type Cx43, but not Cx43(Δ245) or Cx43(S368A) , reduced the interaction between ßarrestin and the PTHR1. These studies demonstrate that ßarrestin is a novel Cx43-interacting protein and suggest that, by sequestering ßarrestin, Cx43 facilitates cAMP signaling, thereby exerting a permissive role on osteoblast survival induced by PTH.

Protein phosphatases: possible bisphosphonate binding sites mediating stimulation of osteoblast proliferation.

We investigated the existence of a bisphosphonate (BP) target site in osteoblasts. Binding assays using [³H]-olpadronate ([³H]OPD) in whole cells showed the presence of specific, saturable and high affinity binding for OPD (K(d)=1.39 ± 0.33 µM) in osteoblasts. [³H]OPD was displaced from its binding site by micromolar concentrations of lidadronate, alendronate and etidronate (K(d)=1.42 ± 0.15 µM, 2.00 ± 0.2 µM and 2.4 ± 0.4 µM, respectively), and by millimolar concentrations of the non-permeant protein phosphatase (PP) substrates p-nitrophenylphosphate and α-naphtylphosphate. PP inhibitors orthovanadate, NaF or vpb(bipy) did not displace [³H]OPD. As expected, specific OPD binding was detected in the plasma membrane of ROS 17/2.8 cells, although significant BP binding was also found intracellularly. Moreover, OPD increased DNA synthesis in these cells with a temporal profile similar to the protein tyrosine phosphatase (PTP) inhibitors, Na3VO4 and vpb(bipy); but different from a general PP inhibitor (NaF). The stimulatory effect of OPD and PTP inhibitors on osteoblast proliferation was inhibited by the protein tyrosine kinase inhibitors genistein and geldanamycin. These results provide new evidence on the existence of a BP target in osteoblastic cells, presumably a PTP, which may be involved in the stimulatory action of BPs on osteoblast proliferation.

Parathyroid hormone receptor signaling in osteocytes increases the expression of fibroblast growth factor-23 in vitro and in vivo.

Mice with constitutive activation of parathyroid hormone (PTH) receptor signaling in osteocytes (DMP1-caPTHR1 transgenic mice) exhibit increased bone mass and remodeling, two of the recognized skeletal actions of PTH. Moreover, similar to PTH administration, DMP1-caPTHR1 mice exhibit decreased expression of the osteocyte-derived Wnt antagonist Sost/sclerostin. We now report that PTH receptor activation also regulates in vivo and in vitro the expression of fibroblast growth factor 23 (FGF23), an osteocyte product involved in inorganic phosphate (Pi) homeostasis and bone mineralization. Whole bones and osteocytes, but not osteoblasts, from DMP1-caPTHR1 mice exhibit elevated FGF23 expression, which is corrected in double transgenic mice overexpressing Sost in osteocytes. PTH, PTH related protein (PTHrP), or a cAMP stable analog, increase FGF23 transcripts in a time- and dose-dependent manner in osteocyte-containing calvarial cell cultures. Circulating FGF23 is also elevated in DMP1-caPTHR1 mice; however, plasma Pi or renal Pi reabsorption is not altered. Furthermore, the FGF23 receptor complex comprising FGFR1 and KLOTHO is expressed in osteoblastic cells; and FGFR1, GALNT3, as well as downstream targets of FGF23 signaling, are increased in osteocytes but not in osteoblasts from DMP1-caPTHR1 mice. Thus, PTH receptor signaling has the potential to modulate the endocrine and auto/paracrine functions of osteocytes by regulating FGF23 through cAMP- and Wnt-dependent mechanisms.

PTH receptor signaling in osteocytes governs periosteal bone formation and intracortical remodeling.

The periosteal and endocortical surfaces of cortical bone dictate the geometry and overall mechanical properties of bone. Yet the cellular and molecular mechanisms that regulate activity on these surfaces are far from being understood. Parathyroid hormone (PTH) has profound effects in cortical bone, stimulating periosteal expansion and at the same time accelerating intracortical bone remodeling. We report herein that transgenic mice expressing a constitutive active PTH receptor in osteocytes (DMP1-caPTHR1 mice) exhibit increased cortical bone area and an elevated rate of periosteal and endocortical bone formation. In addition, DMP1-caPTHR1 mice display a marked increase in intracortical remodeling and cortical porosity. Crossing DMP1-caPTHR1 mice with mice lacking the Wnt coreceptor, LDL-related receptor 5 (LRP5), or with mice overexpressing the Wnt antagonist Sost in osteocytes (DMP1-Sost mice) reduced or abolished, respectively, the increased cortical bone area, periosteal bone formation rate, and expression of osteoblast markers and Wnt target genes exhibited by the DMP1-caPTHR1 mice. In addition, DMP1-caPTHR1 lacking LRP5 or double transgenic DMP1-caPTHR1;DMP1-Sost mice exhibit exacerbated intracortical remodeling and increased osteoclast numbers, and markedly decreased expression of the RANK decoy receptor osteoprotegerin. Thus, whereas Sost downregulation and the consequent Wnt activation is required for the stimulatory effect of PTH receptor signaling on periosteal bone formation, the Wnt-independent increase in osteoclastogenesis induced by PTH receptor activation in osteocytes overrides the effect on Sost. These findings demonstrate that PTH receptor signaling influences cortical bone through actions on osteocytes and defines the role of Wnt signaling in PTH receptor action.

Connexin 43 is required for the anti-apoptotic effect of bisphosphonates on osteocytes and osteoblasts in vivo.

Connexin (Cx)43 is required for inhibition of osteocyte and osteoblast apoptosis by bisphosphonates in vitro. Herein, we evaluated its requirement for the in vivo actions of bisphosphonates using mice in which Cx43 was deleted specifically from osteocytes and osteoblasts (Cx43(DeltaOb-Ot/-) mice). Effective removal of Cx43 was confirmed by the presence of the deleted form of the gene and by reduced mRNA and protein expression in osteoblastic cells and bones obtained from Cx43(DeltaOb-Ot/-) mice. The amino-bisphosphonate alendronate (2.3 micromol/kg/d) was injected daily into 5-mo-old female mice (n = 6-11) for 31 days, starting 3 days before implantation of pellets releasing the glucocorticoid prednisolone (2.1 mg/kg/d). Cx43(DeltaOb-Ot/-) mice and their littermates (Cx43(fl/-), Cx43(DeltaOb-Ot/+), and Cx43(fl/+)) gained bone with similar kinetics and exhibited identical bone mass from 2 to 4.5 mo of age, indicating that Cx43 deletion from osteocytes and mature osteoblasts does not impair bone acquisition. In addition, prednisolone induced a similar increase in osteocyte and osteoblast apoptosis in Cx43(DeltaOb-Ot/-) or in control Cx43(fl/-) littermates. However, whereas alendronate prevented prednisolone-induced apoptosis in control Cx43(fl/-) mice, it was ineffective in Cx43(DeltaOb-Ot/-) mice. In contrast, alendronate inhibited glucocorticoid-induced bone loss in both type of animals, suggesting that inhibition of resorption is the predominant effect of alendronate against the early phase of glucocorticoid-induced bone loss. Taken together with earlier in vitro evidence, these findings show that Cx43 is required for the anti-apoptotic effect of bisphosphonates on osteocytes and osteoblasts.