Abstract
Parathyroid hormone (PTH) and its related peptide (PTH-related peptide 1-34) are two of the Food and Drug Administration-approved bone-promoting drugs for age-related osteoporosis. Treatment with PTH stimulates bone formation. However, the molecular mechanisms of PTH-mediated osteoblast differentiation and cell proliferation are still not completely understood. In this study, we showed that PTH induced endoplasmic reticulum (ER) stress in osteoblasts through the PKR-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2α (EIF2α)-activating transcription factor 4 (ATF4)-signaling pathway. After separately blocking PERK-EIF2α-ATF4 signaling with two different inhibitors [AMG'44 and integrated stress response inhibitor (ISRIB)] or specific small interfering RNA for PERK and ATF4, the following targets were all downregulated: expression of osteoblast differentiation markers [runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), type I collagen (Col1a1), and osteocalcin (Ocn)], cell proliferation markers (CyclinE, CyclinD, and CDC2), amino acid import (Glyt1), and metabolism-related genes (Asns). Additionally, Alp-positive staining cells, Alp activity, matrix mineralization, Ocn secretion, and cell proliferation indexes were inhibited. Interestingly, we found that salubrinal enhanced PTH-induced osteoblast differentiation and proliferation by maintenance of phosphorylation of EIF2α. Furthermore, we observed that PTH increased the association between heat shock protein 90 (HSP90) and PERK and maintained PERK protein stabilization in the early stages of PTH-induced ER stress. Treatment of MC3T3-E1 cells with geldanamycin, an HSP90 inhibitor, decreased PERK protein expression and inhibited osteoblast differentiation and cell proliferation upon PTH treatment. Taken together, our data demonstrate that PTH regulates osteoblast differentiation and cell proliferation, partly by activating the HSP90-dependent PERK-EIF2α-ATF4 signaling pathway.
Highlights
The imbalance between bone formation and bone resorption processes leads to osteoporosis and secondary fractures, which seriously harm the patient’s quality of life, especially in elderly people
We found that PKR-like endoplasmic reticulum kinase (PERK) protein levels were significantly upregulated approximately two- to threefold at 6 h Parathyroid hormone (PTH) treatment (Fig. 2, A and B)
We found that PTH elicited an early endoplasmic reticulum (ER) stress response by the activation of the PERK-EIF2␣-activating transcription factor 4 (ATF4) signaling branch in osteoblasts (Fig. 9)
Summary
The imbalance between bone formation and bone resorption processes leads to osteoporosis and secondary fractures, which seriously harm the patient’s quality of life, especially in elderly people. Drugs with antibone absorption properties are conventional clinical therapies for osteoporosis. These drugs do not improve the integrity of bone structure and are problematic due to side effects [31]. The use of osteogenic drugs for the purpose of improving osteoblast activity, or the use of antibone absorption drugs in combination with bone-promoting drugs, is becoming a popular therapeutic approach [19]. PTH binding to parathyroid hormone type-1 receptor (PTH1R) leads to regulation of multiple signaling pathways, including G␣s-cAMP-PKA signaling, PLC-PKC signaling [5], and non-PLC-PKC signaling [43]. PTH can promote bone mesenchymal stem cells to differentiate into osteoblasts through Wnt--catenin signaling [27]. The detailed molecular mechanisms of PTH modulation on osteoblast differentiation and proliferation are not completely understood [35]
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