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.

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).

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.