PTH1R translocation to primary cilia in mechanically-stimulated ostecytes prevents osteoclast formation via regulation of CXCL5 and IL-6 secretion.

Osteocytes respond to mechanical forces controlling osteoblast and osteoclast function. Mechanical stimulation decreases osteocyte apoptosis and promotes bone formation. Primary cilia have been described as potential mechanosensors in bone cells. Certain osteogenic responses induced by fluid flow (FF) in vitro are decreased by primary cilia inhibition in MLO-Y4 osteocytes. The parathyroid hormone (PTH) receptor type 1 (PTH1R) modulates osteoblast, osteoclast, and osteocyte effects upon activation by PTH or PTH-related protein (PTHrP) in osteoblastic cells. Moreover, some actions of PTH1R seem to be triggered directly by mechanical stimulation. We hypothesize that PTH1R forms a signaling complex in the primary cilium that is essential for mechanotransduction in osteocytes and affects osteocyte-osteoclast communication. MLO-Y4 osteocytes were stimulated by FF or PTHrP (1-37). PTH1R and primary cilia signaling were abrogated using PTH1R or primary cilia specific siRNAs or inhibitors, respectively. Conditioned media obtained from mechanically- or PTHrP-stimulated MLO-Y4 cells inhibited the migration of preosteoclastic cells and osteoclast differentiation. Redistribution of PTH1R along the entire cilium was observed in mechanically stimulated MLO-Y4 osteocytic cells. Preincubation of MLO-Y4 cells with the Gli-1 antagonist, the adenylate cyclase inhibitor (SQ22536), or with the phospholipase C inhibitor (U73122), affected the migration of osteoclast precursors and osteoclastogenesis. Proteomic analysis and neutralizing experiments showed that FF and PTH1R activation control osteoclast function through the modulation of C-X-C Motif Chemokine Ligand 5 (CXCL5) and interleukin-6 (IL-6) secretion in osteocytes. These novel findings indicate that both primary cilium and PTH1R are necessary in osteocytes for proper communication with osteoclasts and show that mechanical stimulation inhibits osteoclast recruitment and differentiation through CXCL5, while PTH1R activation regulate these processes via IL-6.

Primary cilia mediate parathyroid hormone receptor type 1 osteogenic actions in osteocytes and osteoblasts via Gli activation.

Mechanical stimulation of primary cilia in osteocytes and osteoblasts has been proposed as a mechanism that participates in bone cell survival and skeletal remodeling. Among different signaling pathways stimulated by primary cilia, the hedgehog signaling pathway has been associated with the regulation of bone development. Parathyroid hormone (PTH)-related protein (PTHrP) signaling through PTH 1 receptor (PTH1R) also regulates bone cell survival and remodeling and has been associated with the hedgehog pathway during skeletal development. We hypothesize that primary cilia and PTH1R concomitantly regulate bone remodeling and cell survival and aim to describe the mechanisms that mediate these effects in osteocytes and osteoblasts. Colocalization of PTH1R with primary cilia was observed in control and PTHrP-stimulated MLO-Y4 osteocytic and MC3T3-E1 osteoblastic cells. Activation of PTH1R by PTHrP increased cell survival, osteoblast gene expression (osteocalcin, runt-related transcription factor 2, and bone alkaline phosphatase) and the expression of the hedgehog transcription factor Gli-1 in osteocytes and osteoblasts. These effects were abrogated by small interfering RNAs for the primary cilia protein IFT88 or by a primary cilia specific inhibitor (chloral hydrate). Preincubation of MLO-Y4 osteocytic and MC3T3-E1 osteoblastic cells with the Gli-1 antagonist GANT61 inhibited PTHrP prosurvival actions but did not affect PTHrP-induced overexpression of osteogenic genes. Mechanical stimulation by fluid flow increased the number of primary cilia-presenting cells in osteocytes and osteoblasts. We propose that PTH1R activation induces prosurvival actions via primary cilia- and Gli-1-dependent mechanism and modulates osteogenic responses via a primary cilia-dependent and Gli-1-independent pathway in osteocytes and osteoblasts.

Osteocytes and Diabetes: Altered Function of Diabetic Osteocytes.

PURPOSE OF REVIEW: Diabetes mellitus is a prevalent chronic disease affecting millions of people in the world. Bone fragility is a complication found in diabetic patients. Although osteoblasts and osteoclasts are directly affected by diabetes, herein we focus on how the diabetic state-based on hyperglycemia and accumulation of advanced glycation end products among other features-impairs osteocyte functions exerting deleterious effects on bone. RECENT FINDINGS: In the last years, several studies described that diabetic conditions cause morphological modifications on lacunar-canalicular system, alterations on osteocyte mechanoreceptors and intracellular pathways and on osteocyte communication with other cells through the secretion of proteins such as sclerostin or RANKL. This article gives an overview of events occurring in diabetic osteocytes. In particular, mechanical responses seem to be seriously affected in these conditions, suggesting that mechanical sensibility could be a target for future research in the field.

Gene expression, protein profiling, and chemotactic activity of infrapatellar fat pad mesenchymal stem cells in pathologies of the knee joint.

The infrapatellar fat pad (IPFP) is a periarticular adipose knee tissue. This tissue contains a large number of mesenchymal stem cells (MSCs). In the present work, we wanted to study the IPFP MSCs and their relationship and differences in two groups, anterior cruciate ligament (ACL) ruptures knees and ostheoarthrosis (OA). The IPFP of 42 patients with OA or ACL rupture were analyzed. Isolation, primary culture, and a genetic and proteomic study of MSCs from IPFP were performed. Gene expression of IL-6, tumor necrosis factor (TNF), IL-8, HSPA1A (Hsp70), CXCL10, RANTES, MMP1, MMP3, TIMP1, and BMP7 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). We analyzed MSCs from from 12 diferents patients in two cellular pools (6 from AO disease and 6 from ALC rupture to form two cell pool), for the iTRAQ Proteomic Assay. The conditional media were used in quantitative analysis of MSC soluble factors by Luminex and for de migration assay. A higher gene expression of IL-6, TNF, CXCL10, RANTES, and MMP1 and OPG in MSCs from OA versus ACL (p < 0.05) was observed. Conversely HSPA1A, TIMP1, and RANKL showed a significant lower expression in OA-MSCs (p < 0.05). In the secretome analysis, adipsin and visfantin levels in the supernatants from OA-MSCs were lower (p < 0.05) respect to ACL-MSCs. Also, the monocytic cells migrated two-folds in the presence of conditioned media from OA-MSCs patients versus patients with ACL-MSC. The infrapatellar pad should be considered as an adipose tissue capable of producing and excreting inflammatory mediators directly in the knee joint, influencing the development and progression of knee joint pathologies.

Opposite Effects of Mechanical Action of Fluid Flow on Proangiogenic Factor Secretion From Human Adipose-Derived Stem Cells With and Without Oxidative Stress.

Mechanical forces, hypoxia, and oxidative stress contribute to skin renewal, perfusion, and wound healing, but how are they regulating subcutaneous adipose-derived stem cells (ASCs) in the inflammatory microenvironment associated to skin repair and disorders is unknown. In this study, ASCs were isolated from lipoaspirate samples from plastic surgery patients, primary cultured and their differentiation and secretion of a panel of cytokines with pronounced effects on skin repair and angiogenesis were studied under mechanical stimulation by intermittent fluid flow, 1% hypoxia and oxidative stress by glutathione (GSH) depletion with buthionine sulfoximine (BSO) treatment. Mechanical action of fluid flow did not alter mesenchymal phenotype of CD90+ /CD29+ /CD44+ /CD34- /CD106- /CD45- ASCs; however, it remarkably induced ASC secretion of human umbilical vein endothelial cell (HUVEC) migration-stimulating factors. Multiplex Luminex assay further confirmed an increased secretion of VEGF, G-CSF, HGF, Leptin, IL-8, PDGF-BB, Angiopoietin-2, and Follistatin from mechanically-stimulated ASCs via cyclooxygenase-2. Consistent with this mechanism, GSH depletion and hypoxia also increased ASC secretion of VEGF, IL-8, leptin, Angiopoitein-2, and PDGF-BB. However, mechanical action of fluid flow abrogated VEGF and HUVEC migration-stimulating activity from GSH-depleted and hypoxic ASCs. Conversely, GSH depletion and hypoxia abrogated VEGF and HUVEC migration-stimulating activity from mechano-stimulated ASCs. Although mechanical action of fluid flow, hypoxia, and GSH-depletion had independent proangiogenic-stimulating activity on ASCs, mechanical stimulation had opposite effects on proangiogenic factor secretion from ASCs with and without oxidative stress. These data uncover the role of hypoxia and endogenous redox balance during the proangiogenic response of ASCs and other mesenchymal-derived cell types to mechanical action of interstitial fluid flow. J. Cell. Physiol. 232: 2158-2167, 2017. © 2016 Wiley Periodicals, Inc.

High glucose alters the secretome of mechanically stimulated osteocyte-like cells affecting osteoclast precursor recruitment and differentiation.

Diabetes mellitus (DM) induces bone deterioration, while mechanical stimulation promotes osteocyte-driven bone formation. We aimed to evaluate the interaction of acute exposure (24 h) to high glucose (HG) with both the pro-survival effect conferred to osteocytic MLO-Y4 cells and osteoblastic MC3T3-E1 cells by mechanical stimulation and the interaction of these cells with osteoclast precursor RAW264.7 cells. We found that 24 h of HG (25 mM) pre-exposure prevented both cell survival and ERK and ß-catenin nuclear translocation upon mechanical stimulation by fluid flow (FF) (10 min) in both MLO-Y4 and MC3T3-E1 cells. However, migration of RAW 264.7 cells was inhibited by MLO-Y4 cell-conditioned medium (CM), but not by MC3T3-E1 cell-CM, with HG or FF. This inhibitory effect was associated with consistent changes in VEGF, RANTES, MIP-1α, MIP-1ß MCP-1, and GM-CSF in MLO-Y4 cell-CM. RAW264.7 proliferation was inhibited by MLO-Y4 CM under static or HG conditions, but it increased by FF-CM with or without HG. In addition, both FF and HG abrogated the capacity of RAW 264.7 cells to differentiate into osteoclasts, but in a different manner. Thus, HG-CM in static condition allowed formation of osteoclast-like cells, which were unable to resorb hydroxyapatite. In contrast, FF-CM prevented osteoclastogenesis even in HG condition. Moreover, HG did not affect basal RANKL or IL-6 secretion or their inhibition induced by FF in MLO-Y4 cells. In conclusion, this in vitro study demonstrates that HG exerts disparate effects on osteocyte mechanotransduction, and provides a novel mechanism by which DM disturbs skeletal metabolism through altered osteocyte-osteoclast communication.

Distinct Osteomimetic Response of Androgen-Dependent and Independent Human Prostate Cancer Cells to Mechanical Action of Fluid Flow: Prometastatic Implications.

BACKGROUND AND METHODS: Prostate cancer frequently expresses an osteomimetic phenotype, but it is unclear how it is regulated and what biological and clinical implications it confers. Because mechanical forces physiologically regulate bone-remodeling activity in osteocytes, we hypothesized that mechanical action of fluid flow (MAFF) at the cancer microenvironment may similarly foster prostate cancer cell osteomimicry. RESULTS: We showed that in vitro MAFF on androgen-dependent (LNCap) and androgen-independent (PC3) prostate cancer cells remarkably increased OPG, VEGF, RunX2, PTH1R, and PTHrP gene expression in both cell lines irrespective of their androgen dependency. MAFF also altered the cytokine secretion pattern of prostate cancer cells, including Ang2, SCF, and TNFα increase with TRAIL decrease in the supernatant of both cell lines; preferential increase of Leptin and PDGF-BB in LnCap and of VEGF, IL-8, and G-CSF in PC3; and exclusive increase of FGFß, MIF, and PECAM-1 with HGF decrease in LnCap, and of TGBß1, HGF, M-CSF, CXCL1, and CCL7 with NGF decrease in PC3. Murine MLO-Y4 osteocyte-conditioned medium (CM) abrogated M-CSF, G-CSG, IL-8, TNFα, and FGFß secretion-stimulating activity of mechanical stimulation on PC3 cells, and did the opposite effect on LnCap cells. However, MAFF fostered osteomimetic gene expression response of PC3 cells, but not of LnCap cells, to mechanically stimulated osteocyte-CM. Moreover, it abrogated TNFα and IL-8 secretion inhibitory effect of osteocyte-CM on mechanically stimulated PC3 cells and G-CSF, TNFα, and FGFß-stimulating effect on mechanically stimulated LnCap cells. CONCLUSIONS: MAFF activated osteoblast-like phenotype of prostate cancer cells and altered their responses to osteocyte soluble factors. It also induced osteocyte production of osteomimetic gene expression- and cytokine secretion-stimulating factors for prostate cancer cells, particularly, when they were mechanically stimulated. Importantly, MAFF induced a prometastatic response in androgen-independent prostate cancer cells, suggesting the interest of mechanical stimulation-dependent transcription and secretion patterns as diagnostic biomarkers, and as therapeutic targets for the screening of bone-metastasizing phenotype inhibitors upregulated during prostate cancer cell response to MAFF at the cancer microenvironment. Prostate 77:321-333, 2017. © 2016 Wiley Periodicals, Inc.

Inhibition of osteocyte apoptosis prevents the increase in osteocytic receptor activator of nuclear factor κB ligand (RANKL) but does not stop bone resorption or the loss of bone induced by unloading.

Apoptosis of osteocytes and osteoblasts precedes bone resorption and bone loss with reduced mechanical stimulation, and receptor activator of NF-κB ligand (RANKL) expression is increased with unloading in mice. Because osteocytes are major RANKL producers, we hypothesized that apoptotic osteocytes signal to neighboring osteocytes to increase RANKL expression, which, in turn, increases osteoclastogenesis and bone resorption. The traditional bisphosphonate (BP) alendronate (Aln) or IG9402, a BP analog that does not inhibit resorption, prevented the increase in osteocyte apoptosis and osteocytic RANKL expression. The BPs also inhibited osteoblast apoptosis but did not prevent the increase in osteoblastic RANKL. Unloaded mice exhibited high serum levels of the bone resorption marker C-telopeptide fragments of type I collagen (CTX), elevated osteoclastogenesis, and increased osteoclasts in bone. Aln, but not IG9402, prevented all of these effects. In addition, Aln prevented the reduction in spinal and femoral bone mineral density, spinal bone volume/tissue volume, trabecular thickness, mechanical strength, and material strength induced by unloading. Although IG9402 did not prevent the loss of bone mass, it partially prevented the loss of strength, suggesting a contribution of osteocyte viability to strength independent of bone mass. These results demonstrate that osteocyte apoptosis leads to increased osteocytic RANKL. However, blockade of these events is not sufficient to restrain osteoclast formation, inhibit resorption, or stop bone loss induced by skeletal unloading.

VEGF Receptor 2 (VEGFR2) Activation Is Essential for Osteocyte Survival Induced by Mechanotransduction.

Mechanical loading plays a key role in bone formation and maintenance. While unloading induces osteocyte apoptosis and bone loss in vivo, mechanical stimuli prevents osteocyte death through a mechanism involving ß-catenin accumulation and ERK nuclear translocation. Vascular endothelial growth factor (VEGF) has a crucial role in bone formation, but its interaction with osteocytes is not completely understood. Of interest, VEGF receptor 2 (VEGFR2) has recently been shown to mediate the mechanical response of endothelial cells. The present study aimed to evaluate the putative role of the VEGF system in osteocyte mechanosensing. We show that either short (10 min) mechanical stimulus by pulsatile fluid flow (FF) (10 dyn/cm(2), 8 Hz) or exogenous VEGF165 (6 ng/ml) similarly stimulated cell viability, ERK phosphorylation, and ß-catenin membrane translocation. A VEGFR2 antagonist (SU5416) or transfection with specific VEGFR2 siRNAs (siVEGFR2) decreased these events. FF for 10 min increased VEGFR2 phosphorylation at both Tyr-1059 and Tyr-1175; an effect that was mimicked by VEGF165 but was unaffected by a VEGF neutralizing antibody. Subsequently (at 6 h), this mechanical stimulus induced VEGF gene overexpression, which was prevented by siVEGFR2 transfection. Depletion of the structural protein caveolin-1 by using siRNA technology impaired FF-induced VEGFR2 phosphorylation. In conclusion, these in vitro findings point to caveolin-1-dependent VEGFR2 activation as an important mechanism whereby mechanical stimuli promote osteocyte viability.

Crosstalk between caveolin-1/extracellular signal-regulated kinase (ERK) and ß-catenin survival pathways in osteocyte mechanotransduction.

Osteocyte viability is a critical determinant of bone strength and is promoted by both mechanical stimulation and activation of the Wnt signaling pathway. Earlier studies demonstrated that both stimuli promote survival of osteocytes by activating the ERKs. Here, we show that there is interaction between the caveolin-1/ERK and Wnt/ß-catenin signaling pathways in the transduction of mechanical cues into osteocyte survival. Thus, ERK nuclear translocation and anti-apoptosis induced by mechanical stimulation are abolished by the Wnt antagonist Dkk1 and the ß-catenin degradation stimulator Axin2. Conversely, GSK3ß phosphorylation and ß-catenin accumulation induced by mechanical stimulation are abolished by either pharmacologic inhibition of ERKs or silencing caveolin-1. In contrast, the canonical Wnt signaling inhibitor dominant-negative T cell factor does not alter ERK nuclear translocation or survival induced by mechanical stimulation. These findings demonstrate that ß-catenin accumulation is an essential component of the mechanotransduction machinery in osteocytes, albeit ß-catenin/T cell factor-mediated transcription is not required. The simultaneous requirement of ß-catenin for ERK activation and of ERK activation for ß-catenin accumulation suggests a bidirectional crosstalk between the caveolin-1/ERK and Wnt/ß-catenin pathways in mechanotransduction leading to osteocyte survival.

Osteostatin, a peptide for the future treatment of musculoskeletal diseases.

Nowadays, the treatment of musculoskeletal diseases represents a major challenge in the developed world. Diseases such as osteoporosis, osteoarthritis and arthritis have a high incidence and prevalence as a consequence of population aging, and they are also associated with a socioeconomic burden. Many efforts have been made to find a treatment for these diseases with various levels of success, but new approaches are still needed to deal with these pathologies. In this context, one peptide derived for the C-terminal extreme of the Parathormone related Peptide (PTHrP) called Osteostatin can be useful to treat musculoskeletal diseases. This pentapeptide (TRSAW) has demonstrated both in different in vitro and in vivo models, its role as a molecule with anti-resorptive, anabolic, anti-inflammatory, and anti-antioxidant properties. Our aim with this work is to review the Osteostatin main features, the knowledge of its mechanisms of action as well as its possible use for the treatment of osteoporosis, bone regeneration and fractures and against arthritis given its anti-inflammatory properties.

Abaloparatide is more potent than teriparatide in restoring bone mass and strength in type 1 diabetic male mice.

This study investigated the efficacy of the two FDA-approved bone anabolic ligands of the parathyroid hormone receptor 1 (PTH1R), teriparatide or human parathyroid hormone 1-34 (PTH) and abaloparatide (ABL), to restoring skeletal health using a preclinical murine model of streptozotocin-induced T1-DM. Intermittent daily subcutaneous injections of equal molar doses (12 pmoles/g/day) of PTH (50 ng/g/day), ABL (47.5 ng/g/day), or vehicle, were administered for 28 days to 5-month-old C57Bl/6 J male mice with established T1-DM or control (C) mice. ABL was superior to PTH in increasing or restoring bone mass in control or T1-MD mice, respectively, which was associated with superior stimulation of trabecular and periosteal bone formation, upregulation of osteoclastic/osteoblastic gene expression, and increased circulating bone remodeling markers. Only ABL corrected the reduction in ultimate load, which is a measure of bone strength, induced by T1-DM, and it also increased energy to ultimate load. In addition, bones from T1-DM mice treated with PTH or ABL exhibited increased ultimate stress, a material index, compared to T1-DM mice administered with vehicle. And both PTH and ABL prevented the increased expression of the Wnt antagonist Sost/sclerostin displayed by T1-DM mice. Further, PTH and ABL increased to a similar extent the circulating bone resorption marker CTX and the bone formation marker P1NP in T1-DM after 2 weeks of treatment; however, only ABL sustained these increases after 4 weeks of treatment. We conclude that at equal molar doses, ABL is more effective than PTH in increasing bone mass and restoring the cortical and trabecular bone lost with T1-DM, due to higher and longer-lasting increases in bone remodeling.

IGF-I increases markers of osteoblastic activity and reduces bone resorption via osteoprotegerin and RANK-ligand.

BACKGROUND: Bone is one of the major target tissues for Insulin-like Growth Factor I (IGF-I). Low doses of IGF-I were able to improve liver-associated osteopenia. In the present work, a model of partial IGF-I deficiency was used in order to provide insight into the mechanisms of the beneficial actions of IGF-I replacement therapy in bone. METHODS: Several proteins involved in osteoblastic/osteocyte and osteoclastic differentiation and activity were studied in the three experimental groups: control (CO) group (wild type mice, Igf+/+, n=10), heterozygous Igf+/- group with partial IGF-I deficiency (Hz, n=10), and heterozygous Igf+/- mice treated with IGF-I for 10 days (Hz+IGF-I, n=10). RESULTS: Data in this paper confirm that the simple partial IGF-I deficiency is responsible for osteopenia, determined by densitometry and histopathology. These findings are associated with a reduced gene expression of osteoprotegerin, sclerostin, calcitonin receptor (CTR), insulin-like growth factor binding protein 5 and RUNX2. IGF-I replacement therapy normalized CTR gene expression and reduced markers of osteoclastic activity. CONCLUSIONS: Low doses of IGF-I constituted a real replacement therapy that normalized IGF-I serum levels improving the expression of most of these proteins closely involved in bone-forming, and reducing bone resorption by mechanisms related to osteoprotegerin, RANKL and PTH receptor.

Pleiotrophin-Loaded Mesoporous Silica Nanoparticles as a Possible Treatment for Osteoporosis.

Osteoporosis is the most common type of bone disease. Conventional treatments are based on the use of antiresorptive drugs and/or anabolic agents. However, these treatments have certain limitations, such as a lack of bioavailability or toxicity in non-specific tissues. In this regard, pleiotrophin (PTN) is a protein with potent mitogenic, angiogenic, and chemotactic activity, with implications in tissue repair. On the other hand, mesoporous silica nanoparticles (MSNs) have proven to be an effective inorganic drug-delivery system for biomedical applications. In addition, the surface anchoring of cationic polymers, such as polyethylenimine (PEI), allows for greater cell internalization, increasing treatment efficacy. In order to load and release the PTN to improve its effectiveness, MSNs were successfully internalized in MC3T3-E1 mouse pre-osteoblastic cells and human mesenchymal stem cells. PTN-loaded MSNs significantly increased the viability, mineralization, and gene expression of alkaline phosphatase and Runx2 in comparison with the PTN alone in both cell lines, evidencing its positive effect on osteogenesis and osteoblast differentiation. This proof of concept demonstrates that MSN can take up and release PTN, developing a potent osteogenic and differentiating action in vitro in the absence of an osteogenic differentiation-promoting medium, presenting itself as a possible treatment to improve bone-regeneration and osteoporosis scenarios.

Primary cilia and PTH1R interplay in the regulation of osteogenic actions.

Primary cilia are subcellular structures specialized in sensing different stimuli in a diversity of cell types. In bone, the primary cilium is involved in mechanical sensing and transduction of signals that regulate the behavior of mesenchymal osteoprogenitors, osteoblasts and osteocytes. To perform its functions, the primary cilium modulates a plethora of molecules including those stimulated by the parathyroid hormone (PTH) receptor type I (PTH1R), a master regulator of osteogenesis. Binding of the agonists PTH or PTH-related protein (PTHrP) to the PTH1R or direct agonist-independent stimulation of the receptor activate PTH1R signaling pathways. In turn, activation of PTH1R leads to regulation of bone formation and remodeling. Herein, we describe the structure, function and molecular partners of primary cilia in the context of bone, playing special attention to those signaling pathways that are mediated directly or indirectly by PTH1R in association with primary cilia during the process of osteogenesis.

Optimization and validation of a chiral CE-LIF method for quantitation of aspartate, glutamate and serine in murine osteocytic and osteoblastic cells.

Asp, Glu, and D-Ser are chiral amino acids and neurotransmitters binding to the N-methyl-D-aspartate receptor (NMDA) and they participate in glutamate signalization. D-amino acids are increasingly being recognized as important signaling molecules and variations in their levels are considered a marker of different pathologies, however, there is still a lack of knowledge about the role of most of D-amino acids in living organisms such as bone cells. A method for determination of concentrations of L/D-Asp, L/D-Glu and L/D-Ser in two types of bone cell lines: murine osteocytes (MLOY4) and osteoblasts (MC3T3-E1) is presented. It is based on capillary electrophoresis coupled to laser-induced fluorescence detection in normal polarity with 4-fluoro-7-nitro-2,1,3-benzoxadiazole as derivatizing agent suitable for an Argon ion laser source. The electrolyte consists of 137.5 mM borate buffer and 12.5 mM ß-cyclodextrins as chiral selectors and the separation lasts 25 min. The method was optimized and validated for specificity, sensitivity, linearity, accuracy, and precision in murine osteocytes and osteoblasts. LLOQ was 0.25 µmol L-1 for the three D-amino acids and linearity was confirmed with r > 0.995 for all D-and L-amino acids. Accuracy ranged between 81.9% and 111.7% and intra-day precision ranged between 1.8% and 10.9%. Concentrations of D- and L- Asp, Glu, and Ser are given and statistical differences between osteocytes and osteoblasts were found. The highest differences corresponded to L- and D-Glu. This method could play a fundamental role in the study of therapeutic targets in the treatment of bone diseases.

Osteoarthritis-related biomarkers profile in chronic anterior cruciate ligament injured knee.

OBJECTIVE: Increasing evidence has shown the involvement of the innate immune system and inflammatory response in osteoarthritis (OA) pathogenesis; however, anterior cruciate ligament (ACL) tears are recognized risk factors for development of post-traumatic OA. We investigated (1) whether inflammatory mediators involved in OA pathogenesis are also present at significant concentrations in the knee joint sometime after ACL complete tear and may be considered as prognostic biomarkers of progression to secondary OA; and (2) whether quantification in serum may surrogate synovial fluid (SF) measurements in both cases. METHODS: Thirty-seven end-stage OA patients and 33 patients with ACL complete tear that were included on the waiting list for knee surgery were consecutively recruited. Serum and SF samples were taken before surgery, and tumor necrosis factor-alpha, (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), matrix metalloproteinase-1 (MMP1), matrix metalloproteinase-3 (MMP3), tissue inhibitor of metalloproteinase-1 (TIMP1), bone morphogenetic protein-7 (BMP7), regulated upon activation normal t-cell expressed and secreted (RANTES), cytokine interferon-γ-induced protein 10 (IP-10) and heat shock protein family A (Hsp70) member 1A (HSPA1A) were quantified by enzyme-linked immunosorbent assay (ELISA.) Normally distributed data were compared using a one-way analysis of variance (ANOVA) test. Data not normally distributed were analyzed using a nonparametric Mann-Whitney rank sum test. Differences in means were compared using a Student's t-test. Correlations were analyzed using Pearson's coefficient of variation. RESULTS: Eighty-seven percent of patients with OA and 86% of those with ACL tear had quantifiable levels of biomarkers in SF. SF levels of IL-6, IL-8, MMP1, MMP3, RANTES, IP-10, BMP7 and HSPA1A were significantly lower in ACL injury knees compared with those with OA, but much higher than those reported in control subjects. Serum levels of IL-6, IP-10, and MMP1 were also lower in patients with ACL tears, who had, however, significantly higher TNF-α, HSPA1A, and TIMP1 levels when compared with OA patients. Levels of biomarkers tested in serum and SF samples were significantly different. CONCLUSIONS: Our data propose that cytokines IL-6 and IL-8 and the chemokines RANTES, IP-10, MMP1, MMP3, and HSPA1A may be involved in the inflammatory process leading to synovitis, the central lesion in OA onset and development; persistent high levels of these substances sometime after ACL injury suggest that they could play an etiopathogenic role in the maintenance of the inflammatory environment leading to post-traumatic OA. Serum biomarker levels do not appear to faithfully reflect what occurs inside the joint. Thus, most biomarkers cannot yet be considered as useful inflammatory biomarkers of knee joint diseases.

MINDIN secretion by prostate tumors induces premetastatic changes in bone via ß-catenin.

Bone metastases are common in advanced prostate cancer patients, but mechanisms by which specific pro-metastatic skeletal niches are formed before tumor cell homing are unclear. We aimed to analyze the effects of proteins secreted by primary prostate tumors on the bone microenvironment before the settlement and propagation of metastases. Here, using an in vivo pre-metastatic prostate cancer model based on the implantation of prostate adenocarcinoma TRAMP-C1 cells in immunocompetent C57BL/6 mice, we identify MINDIN as a prostate tumor secreted protein that induces bone microstructural and bone remodeling gene expression changes before tumor cell homing. Associated with these changes, increased tumor cell adhesion to the endosteum ex vivo and to osteoblasts in vitro was observed. Furthermore, MINDIN promoted osteoblast proliferation and mineralization and monocyte expression of osteoclast markers. ß-catenin signaling pathway revealed to mediate MINDIN actions on osteoblast gene expression but failed to affect MINDIN-induced adhesion to prostate tumor cells or monocyte differentiation to osteoclasts. Our study evidences that MINDIN secretion by primary prostate tumors creates a favorable bone environment for tumor cell homing before metastatic spread.

T cells with dysfunctional mitochondria induce multimorbidity and premature senescence.

The effect of immunometabolism on age-associated diseases remains uncertain. In this work, we show that T cells with dysfunctional mitochondria owing to mitochondrial transcription factor A (TFAM) deficiency act as accelerators of senescence. In mice, these cells instigate multiple aging-related features, including metabolic, cognitive, physical, and cardiovascular alterations, which together result in premature death. T cell metabolic failure induces the accumulation of circulating cytokines, which resembles the chronic inflammation that is characteristic of aging ("inflammaging"). This cytokine storm itself acts as a systemic inducer of senescence. Blocking tumor necrosis factor-α signaling or preventing senescence with nicotinamide adenine dinucleotide precursors partially rescues premature aging in mice with Tfam-deficient T cells. Thus, T cells can regulate organismal fitness and life span, which highlights the importance of tight immunometabolic control in both aging and the onset of age-associated diseases.

Handling Parathormone Receptor Type 1 in Skeletal Diseases: Realities and Expectations of Abaloparatide.

Musculoskeletal disorders represent an elevated socioeconomic burden for developed aging societies. Osteoporosis (OP) has been treated with antiresorptive therapies or with teriparatide that was until recently the only anabolic therapy. However, approval of osteoporosis treatment in postmenopausal women with abaloparatide, which is an analog of parathyroid hormone-related peptide (PTHrP), has created a new alternative for OP management. The success of this new treatment is related to differential mechanisms of activation of PTH receptor type 1 (PTH1R) by abaloparatide and PTH. Here, we address the distinguishing mechanisms of PTH1R activation; the effects of PTH1R stimulation in osteoblast, osteocytes, and chondrocytes; the differences between PTH and abaloparatide actions on PTH1R; potential safety concerns; and future perspectives about abaloparatide use in other musculoskeletal disorders.