Beneficial effects of manually assisted chiropractic adjusting instrument in a rabbit model of osteoarthritis.

Osteoarthritis (OA) is a degenerative disease characterized by injury of all joint tissues. Our previous study showed that in experimental osteoporosis, chiropractic manipulation (CM) exerts protective effects on bone. We here assessed whether CM might ameliorate OA by improving subchondral bone sclerosis, cartilage integrity and synovitis. Male New-Zealand rabbits underwent knee surgery to induce OA by anterior cruciate ligament injury. CM was performed using the chiropractic instrument ActivatorV 3 times/week for 8 weeks as follows: force 2 setting was applied to the tibial tubercle of the rabbit right hind limb (TM-OA), whereas the corresponding left hind limb received a false manipulation (FM-OA) consisting of ActivatorV firing in the air and slightly touching the tibial tubercle. After sacrifice, subchondral bone integrity was assessed in the tibiae by microCT and histology. Cartilage damage and synovitis were estimated by Mankin's and Krenn's scores, respectively, and histological techniques. Bone mineral density and content in both cortical and trabecular compartments of subchondral bone decreased in OA rabbits compared to controls, but partially reversed in the TM-OA group. Trabecular bone parameters in the latter group also showed a significant improvement compared to FM-OA group. Moreover RANKL, OPG, ALP and TRAP protein expression in subchondral bone significantly decreased in TM-OA rabbits with respect to FM-OA group. CM was associated with lower Mankin's and Krenn's scores and macrophage infiltrate together with a decreased protein expression of pro-inflammatory, fibrotic and angiogenic factors, in TM-OA rabbits with respect to FM-OA. Our results suggest that CM may mitigate OA progression by improving subchondral bone as well as cartilage and synovial membrane status.

Osteostatin potentiates the bioactivity of mesoporous glass scaffolds containing Zn2+ ions in human mesenchymal stem cells.

There is an urgent need of biosynthetic bone grafts with enhanced osteogenic capacity. In this study, we describe the design of hierarchical meso-macroporous 3D-scaffolds based on mesoporous bioactive glasses (MBGs), enriched with the peptide osteostatin and Zn2+ ions, and their osteogenic effect on human mesenchymal stem cells (hMSCs) as a preclinical strategy in bone regeneration. The MBG compositions investigated were 80%SiO2-15%CaO-5%P2O5 (in mol-%) Blank (BL), and two analogous glasses containing 4% ZnO (4ZN) and 5% ZnO (5ZN). By using additive fabrication techniques, scaffolds exhibiting hierarchical porosity: mesopores (around 4 nm), macropores (1-600 µm) and big channels (∼1000 µm), were prepared. These MBG scaffolds with or without osteostatin were evaluated in hMCSs cultures. Zinc promoted hMSCs colonization (both the surface and inside) of MBG scaffolds. Moreover, Zn2+ ions and osteostatin together, but not independently, in the scaffolds were found to induce the osteoblast differentiation genes runt related transcription factor-2 (RUNX2) and alkaline phosphatase (ALP) in hMSCs after 7 d of culture in the absence of an osteogenic differentiation-promoting medium. These results add credence to the combined use of zinc and osteostatin as an effective strategy for bone regeneration applications. STATEMENT OF SIGNIFICANCE: Mesoporous bioactive glasses (MBGs) are bioceramics whose unique properties make them excellent materials for bone tissue engineering. Physico-chemical characterization of MBGs as scaffolds made by rapid prototyping, doped with zinc (potential osteogenic, angiogenic and bactericidal ion) and loaded with osteostatin (osteogenic peptide) are described. These Zn-MBGs scaffolds showed 3D hierarchical meso-macroporous structure that enables to host and release osteostatin. When decorated with human mesenchymal stem cells (hMSCs), MBGs scaffoldsenriched with both zinc and osteostatin exhibited a synergistic effect to enhance hMSCs growth, and also hMSCs osteogenic differentiationwithout addition of other osteoblastic differentiation factors to the culture medium. This novel strategy has a great potential for use in bone tissue engineering.

Parathyroid hormone-related protein exhibits antioxidant features in osteoblastic cells through its N-terminal and osteostatin domains.

OBJECTIVES: Oxidative stress plays a major role in the onset and progression of involutional osteoporosis. However, classical antioxidants fail to restore osteoblast function. Interestingly, the bone anabolism of parathyroid hormone (PTH) has been shown to be associated with its ability to counteract oxidative stress in osteoblasts. The PTH counterpart in bone, which is the PTH-related protein (PTHrP), displays osteogenic actions through both its N-terminal PTH-like region and the C-terminal domain. METHODS: We examined and compared the antioxidant capacity of PTHrP (1-37) with the C-terminal PTHrP domain comprising the 107-111 epitope (osteostatin) in both murine osteoblastic MC3T3-E1 cells and primary human osteoblastic cells. RESULTS: We showed that both N- and C-terminal PTHrP peptides at 100 nM decreased reactive oxygen species production and forkhead box protein O activation following hydrogen peroxide (H2O2)-induced oxidation, which was related to decreased lipid oxidative damage and caspase-3 activation in these cells. This was associated with their ability to restore the deleterious effects of H2O2 on cell growth and alkaline phosphatase activity, as well as on the expression of various osteoblast differentiation genes. The addition of Rp-cyclic 3',5'-hydrogen phosphorothioate adenosine triethylammonium salt (a cyclic 3',5'-adenosine monophosphate antagonist) and calphostin C (a protein kinase C inhibitor), or a PTH type 1 receptor antagonist, abrogated the effects of N-terminal PTHrP, whereas protein phosphatase 1 (an Src kinase activity inhibitor), SU1498 (a vascular endothelial growth factor receptor 2 inhibitor), or an anti osteostatin antiserum, inhibited the effects of C-terminal PTHrP. CONCLUSION: These findings indicate that the antioxidant properties of PTHrP act through its N- and C-terminal domains and provide novel insights into the osteogenic action of PTHrP.Cite this article: S. Portal-Núñez, J. A. Ardura, D. Lozano, I. Martínez de Toda, M. De la Fuente, G. Herrero-Beaumont, R. Largo, P. Esbrit. Parathyroid hormone-related protein exhibits antioxidant features in osteoblastic cells through its N-terminal and osteostatin domains. Bone Joint Res 2018;7:58-68. DOI: 10.1302/2046-3758.71.BJR-2016-0242.R2.

Impact of Chiropractic Manipulation on Bone and Skeletal Muscle of Ovariectomized Rats.

Evidence suggests that chiropractic manipulation might exert positive effects in osteoporotic patients. The aim of this study was to evaluate the effects of chiropractic manipulation on bone structure and skeletal muscle in rats with bone loss caused by ovariectomy (OVX). The 6-month old Sprague-Dawley rats at 10 weeks following OVX or sham operation (Sh) did not suffer chiropractic manipulation (NM group) or were submitted to true chiropractic manipulation using the chiropractic adjusting instrument Activator V® three times/week for 6 weeks as follows: Force 1 setting was applied onto the tibial tubercle of the rat right hind limb (TM group), whereas the corresponding left hind limb received a false manipulation (FM group) consisting of ActivatorV® firing in the air and slightly touching the tibial tubercle. Bone mineral density (BMD) and bone mineral content (BMC) were determined in long bones and L3-L4 vertebrae in all rats. Femora and tibia were analyzed by µCT. Mechano growth factor (MGF) was detected in long bones and soleus, quadriceps and tibial muscles by immunohistochemistry and Western blot. The decrease of BMD and BMC as well as trabecular bone impairment in the long bones of OVX rats vs Sh controls was partially reversed in the TM group versus FM or NM rats. This bone improvement by chiropractic manipulation was associated with an increased MGF expression in the quadriceps and the anterior tibial muscle in OVX rats. These findings support the notion that chiropractic manipulation can ameliorate osteoporotic bone at least partly by targeting skeletal muscle.

Evaluation of bacterial adherence of clinical isolates of Staphylococcus sp. using a competitive model: An in vitro approach to the "race for the surface" theory.

OBJECTIVES: Implant-related infection is one of the most devastating complications in orthopaedic surgery. Many surface and/or material modifications have been developed in order to minimise this problem; however, most of the in vitro studies did not evaluate bacterial adhesion in the presence of eukaryotic cells, as stated by the 'race for the surface' theory. Moreover, the adherence of numerous clinical strains with different initial concentrations has not been studied. METHODS: We describe a method for the study of bacterial adherence in the presence of preosteoblastic cells. For this purpose we mixed different concentrations of bacterial cells from collection and clinical strains of staphylococci isolated from implant-related infections with preosteoblastic cells, and analysed the minimal concentration of bacteria able to colonise the surface of the material with image analysis. RESULTS: Our results show that clinical strains adhere to the material surface at lower concentrations than collection strains. A destructive effect of bacteria on preosteoblastic cells was also detected, especially with higher concentrations of bacteria. CONCLUSIONS: The method described herein can be used to evaluate the effect of surface modifications on bacterial adherence more accurately than conventional monoculture studies. Clinical strains behave differently than collection strains with respect to bacterial adherence.Cite this article: M. Martinez-Perez, C. Perez-Jorge, D. Lozano, S. Portal-Nuñez, R. Perez-Tanoira, A. Conde, M. A. Arenas, J. M. Hernandez-Lopez, J. J. de Damborenea, E. Gomez-Barrena, P. Esbrit, J. Esteban. Evaluation of bacterial adherence of clinical isolates of Staphylococcus sp. using a competitive model: An in vitro approach to the "race for the surface" theory. Bone Joint Res 2017;6:315-322. DOI: 10.1302/2046-3758.65.BJR-2016-0226.R2.

El estrés oxidativo como posible diana terapéutica en la osteoporosis asociada al envejecimiento / Oxidative stress as a possible therapeutic target for osteoporosis associated with aging

La osteoporosis senil o involutiva es un problema de primera magnitud en el mundo desarrollado. Estudios recientes apuntan al aumento del estrés oxidativo asociado al envejecimiento -cronológico o biológico- como un factor importante en su desarrollo. En esta revisión nos centraremos en las alteraciones del tejido óseo con la edad, en el origen del estrés oxidativo y su influencia negativa en el tejido óseo. Finalmente, abordaremos las posibles terapias antiestrés oxidativo que actualmente se encuentran en desarrollo en esta patología (AU)

Senile or involutional osteoporosis is a major problem in the developed world. Recent studies point to increased oxidative stress associated with aging, whether biological or chronological, as an important factor in its development. In this review paper, we focus on bone tissue disorders related to aging, the source of oxidative stress and negative influence on bone tissue. Finally, we consider the potential oxidative stress therapies currently being developed for this disease (AU)

El receptor 2 de VEGF (VEGFR2) y el receptor 1 de la PTH (PTH1R) actúan como mediadores de la respuesta antiapoptótica al estímulo mecánico en las células osteocíticas MLO-Y4 / The VEGF (VEGFR2) 2 receptor and PTH (PTH1R) 1 receptor act as mediators in the anti-apoptotic response to mechanical stimulus in MLO-Y4 osteocyte-like cell

La estimulación mecánica juega un papel fundamental en el mantenimiento de la masa ósea. Dicha estimulación previene la apoptosis de los osteocitos por un mecanismo que implica la acumulación de β-catenina y la translocación nuclear de quinasas reguladas por señales extracelulares (ERK). El factor de crecimiento del endotelio vascular (VEGF) y la proteína relacionada con la parathormona (PTHrP) modulan la formación ósea, aunque su interacción con los osteocitos es desconocida. En el presente estudio hemos evaluado el posible papel del receptor 2 del VEGF (VEGFR2) y del receptor tipo 1 de PTH (PTH1R) en la respuesta antiapoptótica a la estimulación mecánica en células osteocíticas MLO-Y4. Las células se sometieron a estrés mecánico por flujo laminar de fluido (10 min, 10 dinas/cm2) o choque hipotónico (240 mOsm, 1h), o estimuladas con VEGF165 o PTHrP (1-36). Además, comparamos los efectos de sobre-expresar VEGFR2 y el estímulo mecánico en estas células. La estimulación mecánica, el VEGF165 o la PTHrP (1-36), de manera similar, estimularon la viabilidad celular y la estabilización de β-catenina, relacionada con su localización en la membrana. Además, la estimulación mecánica aumentó la presencia del PTH1R en la membrana. La inhibición del VEGFR2 así como el antagonista PTHrP (7-34) disminuyeron estos efectos. Por otro lado, la sobre-expresión del VEGFR2 en las células MLO-Y4 mimetizó el efecto del estímulo mecánico sobre la β-catenina y la viabilidad celular. Estos hallazgos apoyan un papel funcional de ambos sistemas, VEGF/VEGFR2 y PTHrP/PTH1R, en la respuesta temprana a la estimulación mecánica para promover la viabilidad osteocítica (AU)

Mechanical stimulation plays a crucial role in bone mineral maintenance. This stimulation prevents osteocyte apoptosis by a mechanism that involves β-catenin accumulation and nuclear translocation of extracellular-signal-regulated kinases (ERKs). The vascular endothelial growth factor (VEGF) and parathyroid hormone-related protein (PTHrP) modulate bone formation, although their interaction with osteocytes is unknown. In this paper we have considered the possible role of VEGF (VEGFR2) 2 receptor and PTH (PTH1R) type 1 receptor in the anti-apoptotic response to mechanical stimulation of MLO-Y4 osteocyte-like cells. The cells were subjected to mechanical stress by laminar fluid flow (10 min, 10 dinas/cm2 ) or hypotonic shock (240 mOsm, 1h), or stimulated with VEGF165 or PTHrP (1-36). We also compared the effects of overexpressed VEGFR2 and mechanical stimulation of these cells. Mechanical stimulation, VEGF165 or PTHrP (1-36) stimulated cellular viability and β-catenin stabilization in a similar manner, associated with its localization in the membrane. Mechanical stimulation increased PTH1R presence in the membrane. VEGFR2 inhibition as well as the PTHrP (7-34) antagonist reduced these effects. On the other hand, VEGFR2 overexpression in MLO-Y4 cells mimicked the mechanical stimulation effect on β-catenin and cellular viability. Our findings support a functional role for both systems, VEGF/VEGFR2 and PTHrP/PTH1R, in the early response to mechanical stimulation in promoting osteocyte-like viability (AU)

La elevada glucosa altera la respuesta anti-apoptótica del estímulo mecánico en osteocitos de ratón MLO-Y4 / High glucose alters the antiapoptotic response to mechanical stimulation in MLO-Y4 osteocytic cells

El estrés oxidativo es clave en el envejecimiento y en los estados diabéticos. La carga mecánica es decisiva para mantener la masa ósea. La respuesta del hueso a los estímulos mecánicos parece reducirse con el envejecimiento y probablemente en la enfermedad ósea provocada por la diabetes. Entender los mecanismos mediante los que el estrés oxidativo afecta a la función de las células óseas, y en concreto a la mecanotransducción en el osteocito, podría proporcionar nuevas dianas moleculares para mejorar los tratamientos actuales y el diseño de otros nuevos para prevenir la pérdida de masa ósea. Nuestros resultados indican que un medio de alta glucosa («diabético») ejerce un efecto negativo sobre la capacidad de los osteocitos para responder a estímulos mecánicos, a través de la interacción con la β-catenina. Además, estos hallazgos sugieren que el estímulo mecánico promueve la viabilidad osteocítica, al menos en parte, a través de la producción de la proteína relacionada con la parathormona (PTHrP) (AU)

Oxidative stress is a key factor in aging and diabetes. Mechanical loading is critical to maintain bone mass. The response of bone to mechanical stimuli appears to be reduced with aging and probably in bone disease caused by diabetes. Understanding the mechanisms by which oxidative stress affects function of bone cells, and specifically to osteocyte mechanotransduction, may provide new molecular targets to improve current treatments and design new treatments to prevent bone loss. Our results indicate that high glucose medium («diabetic ») has a negative effect on the ability of osteocytes to respond to mechanical stimuli affecting b-catenin and apoptosis. Moreover, these findings suggest that the mechanical stimulus promotes viability osteocytic, at least in part, through production of parathyroid hormone related protein (PTHrP) (AU)

Characterization of Hybrid Bioactive Glass-polyvinyl Alcohol Scaffolds Containing a PTHrP-derived Pentapeptide as Implants for Tissue Engineering Applications.

Hybrid foam (BG-PVA) with 50 % Bioactive glass (BG) and 50 % polyvinyl alcohol (PVA) was prepared by sol-gel process to produce scaffolds for bone tissue engineering. The pore structure of hydrated foams was evaluated by 3-D confocal microscopy, confirming 70% porosity and interconnected macroporous network. In this study, we assessed the putative advantage of coating with osteostatin pentapeptide into BG-PVA hybrid scaffolds to improve their bioactivity. In vitro cell culture experiments were performed using mouse pre-osteoblastic MC3T3-E1 cell line. The exposure to osteostatin loaded-BG-PVA scaffolds increase cell proliferation in contrast with the unloaded scaffolds. An in vivo study was selected to implant BG-PVA scaffolds, non-coated (Group A) or coated (Group B) with osteostatin into non critical bone defect at rabbit femur. Both groups showed new compact bone formation on implant surface, with lamellae disposed around a haversian canal forming osteons-like structure. We observed signs of inflammation around the implanted unloaded scaffold at one month, but resolved at 3 months. This early inflammation did not occur in Group B; supporting the notion that osteostatin may act as anti-inflammatory inhibitor. On the other hand, Group B showed increased bone formation, as depicted by many new trabeculae partly mineralized in the implant regenerating area, incipient at 1 month and more evident at 3 months after implantation. PVA/BG hybrid scaffolds present a porous structure suitable to support osteoblast proliferation and differentiation. Our in vitro and in vivo findings indicate that osteostatin coating improves the osteogenic features of these scaffolds.

Functional roles of the nuclear localization signal of parathyroid hormone-related protein (PTHrP) in osteoblastic cells.

PTHrP is an important regulator of bone remodelling, apparently by acting through several sequence domains. We here aimed to further delineate the functional roles of the nuclear localization signal (NLS) comprising the 88-107 amino acid sequence of PTHrP in osteoblasts. PTHrP mutants from a human PTHrP (-36/+139) cDNA (wild type) cloned into pcDNA3.1 plasmid with deletion (Δ) of the signal peptide (SP), NLS, T(107), or T107A replacing T(107) by A(107) were generated and stably transfected into osteoblastic MC3T3-E1 cells. In these cells, intracellular trafficking, cell proliferation and viability, as well as cell differentiation were evaluated. In these transfected cells, PTHrP was detected in the cytoplasm and also in the nucleus, except in the NLS mutant. Meanwhile, the PTH type 1 receptor (PTH1R) accumulates in the cytoplasm except for the ΔSP mutant in which the receptor remains at the cell membrane. PTHrP-wild type cells showed enhanced growth and viability, as well as an increased matrix mineralization, alkaline phosphatase activity, and osteocalcin gene expression; and these features were inhibited or abolished in ΔNLS or ΔT(107) mutants. Of note, these effects of PTHrP overexpression on cell growth and function were similarly decreased in the ΔSP mutant after PTH1R small interfering RNA transfection or by a PTH1R antagonist. The present in vitro findings suggest a mixed model for PTHrP actions on osteoblastic growth and function whereby this protein needs to be secreted and internalized via the PTH1R (autocrine/paracrine pathway) before NLS-dependent shuttling to the nucleus (intracrine pathway).

Amylin exerts osteogenic actions with different efficacy depending on the diabetic status.

Amylin displays osteogenic features, but its role in diabetic osteopenia is unclear. We examined the possible osteogenic action of amylin infusion for 3days into fructose-induced insulin-resistant (IR) and streptozotocin-induced type 2 diabetic (T2D) and normal (N) rats. Amylin failed to affect glycaemia or parathyroid hormone levels in any group, but reduced hyperinsulinemia in IR rats. In N rats, amylin increased bone formation rate and reduced osteoclast surface and erosive surface in the femoral metaphysis, and increased osteoprotegerin (OPG)/receptor activator of NFκB ligand (RANKL) mRNA ratio in the tibia. In T2D rats, amylin normalized trabecular structure parameters and increased osteoblast number and osteocalcin (OC) expression in long bones. In contrast, in IR rats, no apparent osteogenic effect of amylin in the femur was observed, although both OC and OPG/RANKL ratio were increased in the tibia. Our findings demonstrate a different osteogenic efficacy of amylin in two diabetic settings.

The C-terminal fragment of parathyroid hormone-related peptide promotes bone formation in diabetic mice with low-turnover osteopaenia.

BACKGROUND AND PURPOSE: Current data suggest that parathyroid hormone (PTH)-related peptide (PTHrP) domains other than the N-terminal PTH-like domain contribute to its role as an endogenous bone anabolic factor. PTHrP-107-139 inhibits bone resorption, a fact which has precluded an unequivocal demonstration of its possible anabolic action in vivo. We thus sought to characterize the osteogenic effects of this peptide using a mouse model of diabetic low-turnover osteopaenia. EXPERIMENTAL APPROACH: PTHrP-107-139 was administered to streptozotocin-induced diabetic mice, with or without bone marrow ablation, for 13 days. Osteopaenia was confirmed by dual-energy X-ray absorptiometry and microcomputed tomography analysis. Histological analysis was performed on paraffin-embedded bone tissue sections by haematoxylin/eosin and Masson's staining, and tartrate-resistent acid phosphatase immunohistochemistry. Mouse bone marrow stromal cells and osteoblastic MC3T3-E1 cells were cultured in normal and/or high glucose (HG) medium. Osteogenic and adipogenic markers were assessed by real-time PCR, and PTHrP and the PTH(1) receptor protein expression by Western blot analysis. KEY RESULTS: PTHrP-107-139 reversed the alterations in bone structure and osteoblast function, and also promoted bone healing after marrow ablation without affecting the number of osteoclast-like cells in diabetic mice. This peptide also reversed the high-glucose-induced changes in osteogenic differentiation in both bone marrow stromal cells and the more differentiated MC3T3-E1 cells. CONCLUSIONS AND IMPLICATIONS: These findings demonstrate that PTHrP-107-139 promotes bone formation in diabetic mice. This mouse model and in vitro cell cultures allowed us to identify various anabolic effects of this peptide in this scenario.

Alterations of the Wnt/beta-catenin pathway and its target genes for the N- and C-terminal domains of parathyroid hormone-related protein in bone from diabetic mice.

Type 1 diabetes mellitus (T1D) is associated with bone loss. Given that the Wnt/beta-catenin pathway is a major regulator of bone accrual, we assessed this pathway in mice with streptozotozin-induced T1D. In diabetic mouse long bones, we found alterations favouring the suppression of this pathway by using PCR arrays and beta-catenin immunostaining. Downregulation of sclerostin, an inhibitor of this pathway, also occurred, and related to increased osteocyte apoptosis. Our data show that both N- and C-terminal parathyroid hormone-related peptide fragments might exert osteogenic effects in this setting by targeting several genes of this pathway and increasing beta-catenin in osteoblastic cells.

Phytoestrogen modulation of bone-related cytokines and its impact on cell viability in human prostate cancer cells.

AIMS: Prostate cancer (PCa) has a high propensity to metastasize to the bone. PCa cells produce several bone-related factors, namely parathyroid hormone related protein (PTHrP), its PTH type 1 receptor (PTH1R), osteoprotegerin (OPG), and receptor activator of NF-kappa B ligand (RANKL). The effects of these factors might explain, at least in part, the ability of PCa cells to grow in and interact with bone. MAIN METHODS: We first analyzed the expression of the aforementioned factors (by western blot and flow cytometry), and their modulation by the phytoestrogens genistein and daidzein (as potential anti-tumoral agents), in human PCa cells in vitro. We also assessed the impact of these osteomimetic factors on PCa cell viability (by propidium iodide staining and flow cytometry, and trypan blue staining). KEY FINDINGS: Genistein and daidzein, at nM range, increased both the PTHrP/PTH1R system and the OPG/RANKL protein ratio, while genistein and, to a lesser extent, daidzein, at >microM doses, inhibited cell viability in PCa cells. Both N- and C-terminal domains of PTHrP inhibited genistein-induced cell death by modulating transcription factor Runx-2 and the Bcl-2/Bax protein ratio in PCa cells. SIGNIFICANCE: Our findings indicate that high doses of genistein and daidzein cause PCa cell death. On the other hand, low doses of these phytoestrogens induce some osteomimetic features in PCa cells with putative impact on PCa development.

Parathyroid hormone-related protein promotes inflammation in the kidney with an obstructed ureter.

Parathyroid hormone-related protein (PTHrP) promotes fibrogenesis in the acutely damaged kidney. Considering the relation between fibrosis and inflammation, we studied transgenic mice that overexpress PTHrP in the proximal tubule. When unilateral ureteric obstruction was induced in these transgenic mice, we found that they had more renal tubulointerstitial damage, leukocyte influx, and expression of proinflammatory factors than their control littermates. Reversal of PTHrP constitutive overexpression in these transgenic mice or treatment of control mice with the PTHrP antagonist (7-34) decreased this inflammatory response. Losartan, which abolished obstruction-induced endogenous PTHrP upregulation, also decreased the latter response but less effectively in transgenic mice. The PTHrP fragment (1-36) induced nuclear factor-kappaB (NF-kappaB) activation and proinflammatory cytokine overexpression in mouse cortical tubule cells in culture as well as migration of the macrophage cell line Raw 264.7. All these effects were decreased by PTHrP (7-34) and NF-kappaB or extracellular signal-regulated kinase (ERK) activation inhibitors. Our findings suggest a critical role of PTHrP in the renal inflammatory process that results from ureteral obstruction and indicate that ERK-mediated NF-kappaB activation seems to be an important mechanism whereby PTHrP triggers renal inflammation.

Diabetes Mellitus y pérdida de masa ósea / Diabetes Mellitus and bone mass loss

La diabetes mellitus es una enfermedad que afecta a unos 200 millones de personas en el mundo. Estudios epidemiológicos y experimentales demuestran la existencia de una pérdida de masa ósea asociada a la diabetes de tipo 1. Esta pérdida parece estar mediada por diversos factores endocrinos y/o locales, entre los que se incluyen: la insulina, el factor similar a la insulina tipo 1 (IGF-1), péptidos pancreáticos (amilina, leptina y preptina), hormonas intestinales (GLP-1 y GLP-2) y la vitamina D. Sin embargo, en la diabetes tipo 2 no existen datos concluyentes que la relacionen con una disminución de la masa ósea

Diabetes mellitus is a disease affecting about 200 million people worldwide. Both epidemiological and experimental studies have demonstrated the existence of a diabetes-related bone loss in type 1 diabetic patients. This relationship seems to be mediated by different endocrine and local factors, namely insulin, insulin-like growth factor 1 (IGF-1), pancreatic peptides (amylin, leptin, and preptin), intestinal hormones (GLP-1 y GLP-2), and vitamin D. However, no such relationship has been demonstrated in type 2 diabetes

Transient exposure to PTHrP (107-139) exerts anabolic effects through vascular endothelial growth factor receptor 2 in human osteoblastic cells in vitro.

Intermittent administration of the N-terminal fragment of parathyroid hormone (PTH) and PTH-related protein (PTHrP) induces bone anabolic effects. However, the effects of the C-terminal domain of PTHrP on bone turnover remain controversial. We examined the putative mechanisms whereby this PTHrP domain can affect osteoblastic differentiation, using human osteosarcoma MG-63 cells and osteoblastic cells from human trabecular bone. Intermittent exposure to PTHrP (107-139), within 10-100 nM, for only

Diferentes dosis de los fitoestrógenos genisteína y daidzeína afectan de distinto modo a la viabilidad celular y a la expresión de factores implicados en el desarrollo de cáncer de próstata / Different doses of genistein and daidzein phytoestrogens affect cell viability and expression of factors involved in the development of prostrate carcinoma differently

Introducción. Estudios epidemiológicos sugieren que los fitoestrógenos de la soja podrían ser responsables de la baja incidencia del carcinoma prostático (CaP) en poblaciones asiáticas. Las células de CaP expresan factores reguladores de la proliferación/viabilidad celular, así como factores capaces de interaccionar con las células en el microambiente óseo. En el presente estudio hemos evaluado los efectos de los fitoestrógenos de la soja, genisteína y daidzeína, sobre la viabilidad celular/apoptosis y sobre la expresión de la proteína relacionada con la parathormona (PTHrP) y su receptor (PTH1R), la osteoprotegerina (OPG) y el ligando del receptor activador del NF-kappa B (RANKL) en las células de CaP humano PC-3 y LNCaP. Pacientes y métodos. Las células, sembradas a 20.000 células/cm2 en RPMI con suero fetal bovino al 10%, se incubaron con distintas concentraciones de cada fitoestrógeno o el vehículo salino (control basal) durante 1-4 días. La viabilidad celular se evaluó por exclusión con azul de tripán y la apoptosis se determinó por citometría de flujo. La expresión génica de PTHrP se analizó por RT-PCR semicuantitativa con cebadores específicos. La expresión proteica de PTHrP, PTH1R, OPG y RANKL se determinó por transferencia western en extractos de proteína celular total o de membrana (RANKL). Resultados. Hemos encontrado que tanto la genisteína como la daidzeína en el rango µM disminuyen la viabilidad celular e incrementan la apoptosis; siendo la genisteína más potente y eficaz que la daidzeína en ambos tipos celulares. Además, estas isoflavonas a dosis ¾ nM aumentan la expresión de PTHrP y del PTH1R, así como la relación OPG/RANKL en estas células. Conclusiones. Estos hallazgos demuestran que diferentes dosis de genisteína y daidzeína inducen efectos diversos sobre las células de CaP que podrían afectar al desarrollo de este tumor(AU)

Introduction. Epidemiological studies suggest that soy phytoestrogens might be responsible for the low incidence of prostate carcinoma (PCa) in Asian populations. PCa cells express regulatory factors of cell proliferation and viability, and also several factors that interact with cells in the bone microenvironment. In the present study, we evaluated the effects of soy phytoestrogens genistein and daidzein on cell viability and apoptosis, and also on the expression of parathormone-related protein (PTHrP) and its receptor (PTH1R), osteoprotegerina (OPG) and receptor activator of nuclear factor kappa B ligand (RANK-L) in the human PCa cell lines PC-3 and LNCaP. Patients and methods. Cells were seeded at 20,000 cells/cm2 in RPMI with 10% fetal bovine serum, and then were incubated with different concentrations of each phytoestrogen or saline vehicle (basal control) for 1-4 days. Cell viability was evaluated by Trypan blue exclusion, and apoptosis was determined by flow citometry. Gene expression of PTHrP was analyzed by semiquantitative RT-PCR with specific primers. Protein expression of PTHrP, the PTH1R, OPG, and RANKL was determined by western blot in total cell protein or cell membrane (RANKL) extracts. Results. We found that both genistein and daidzein, at µM range, decrease cell viability and increase apoptosis; but genistein was more potent and efficient than daidzein in both PCa cell lines. In addition, these isoflavones, at ¾ nM, increase the expression of PTHrP and the PTH1R, and also the OPG/RANKL ratio in these cells. Conclusions. These findings demonstrate that different concentrations of genistein and daidzein induce distinct effects on PCa cells that might affect PCa development(AU)

The parathyroid hormone-related protein system and diabetic nephropathy outcome in streptozotocin-induced diabetes.

The pathophysiology of the diabetic kidney (e.g., hypertrophy, increase urinary albumin excretion (UAE) is still ill-defined. Parathyroid hormone-related protein (PTHrP) is overexpressed in several nephropathies, but its role remains unclear. We evaluated the effect of high glucose on PTHrP and the PTH1 receptor (PTH1R) protein (by Western blot and immunohistochemistry) in the kidney of mice ith streptozotocin-induced diabetes, and in several mouse renal cells in vitro. Diabetic mice showed a significantly increased renal expression of PTHrP and PTH1R proteins with 2-8 weeks from the onset of diabetes. These animals exhibited an intense immunostaining for both proteins in the renal tubules and glomeruli. Using transgenic mice overexpressing PTHrP targeted to the renal proximal tubule, we found a significant increase in the renal hypertrophy index and in UAE in these diabetic mice relative to their control littermates. Moreover, logistic regression analysis showed a significant association between both PTHrP and PTH1R protein levels and UAE in all diabetic mice throughout the study. High-glucose (25 mm) medium was found to increase PTHrP and PTH1R in tubuloepithelial cells, mesangial cells and podocytes in vitro. Moreover, this increase in PTHrP (but not that of PTH1R) was inhibited by the AT1 receptor antagonist losartan. Collectively, these results indicate that the renal PTHrP/PTH1R system is upregulated in streptozotozin-induced diabetes in mice, and appears to adversely affect the outcome of diabetic renal disease. Our findings also suggest that angiotensin II might have a role in the PTHrP upregulation in this condition.

Sequential changes of parathyroid hormone related protein (PTHrP) in articular cartilage during progression of inflammatory and degenerative arthritis.

OBJECTIVE: To investigate immunolocalisation of parathyroid hormone related protein (PTHrP) in two sequential models of experimental cartilage damage (inflammatory and degenerative) in order to elucidate differences in chondrocyte response to the disease. METHODS: Immunohistochemistry with a polyclonal rabbit antiserum to the N-terminal domain of PTHrP was used to detect this protein in two different rabbit models sharing progressive cartilage damage: antigen induced arthritis (AIA) and osteoarthritis (OA) secondary to partial medial meniscectomy. Cartilage specimens from early (2 days in AIA; 8 weeks in experimental OA) and late (3 weeks in AIA; 52 weeks in OA) disease were compared. RESULTS: Cell and matrix PTHrP staining in early AIA and OA was similar to that in controls. Late AIA cartilage showed a significant decrease in PTHrP positive cells and in the cartilage matrix. In contrast, at late OA stages, distinct PTHrP positivity was detected in proliferating cell clones, as assessed by proliferating cell nuclear antigen staining around cartilage damaged areas. CONCLUSION: PTHrP staining of hyaline articular cartilage shows a different pattern during progression of each type of arthritis: an overall decrease associated with the inflammatory disease, and an increase in the proliferating chondrocyte clones with degenerative arthritis.