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Osteoporosis is a common disease that increases individual's fragility fracture risk. PTH is the only therapeutic agent for severe osteoporosis that requires anabolic action of bone formation. Although a part of the PTH actions is explained by increased proliferation of osteoblastic precursor cells, the mechanisms involved in the proliferation of osteoblastic cells by PTH have not been clarified yet. Therefore, in this study, we investigated the effects of PTH on gene expression in the cultured osteoblastic MC3T3-E1 cells, and found that the ubiquitin-specific peptidase 2 (Usp2) may be one of the direct target genes of PTHR signaling. Usp2 is a deubiquitination enzyme targeting various factors including CyclinD1 in cancer cells and PTH receptor 1 in osteoblasts. We confirmed that consistent induction of Usp2 expression peaked at 1 h by PTH1-34 (teriparatide) in MC3T3-E1 cells and primary calvarial osteoblasts. Among the three known splicing variants of the Usp2, we found the isoforms 1 and 2 are predominantly expressed in osteoblasts. Live-imaging analysis of the Fucci-transgenic mouse-derived primary osteoblasts indeed demonstrated that Usp2 is required for the PTH1-34-induced osteoblast proliferation. Western blotting analysis of the CyclinD1 indicated that Usp2 knock-down influences the paradoxical changes of CyclinD1 protein levels in this condition. Our data indicate that Usp2 is required for the PTH1-34-induced proliferation of osteoblasts.

Osteoporosis affects over 20 million patients in the United States. Among those, disuse osteoporosis is serious as it is induced by bed-ridden conditions in patients suffering from aging-associated diseases including cardiovascular, neurological, and malignant neoplastic diseases. Although the phenomenon that loss of mechanical stress such as bed-ridden condition reduces bone mass is clear, molecular bases for the disuse osteoporosis are still incompletely understood. In disuse osteoporosis model, bone loss is interfered by inhibitors of sympathetic tone and adrenergic receptors that suppress bone formation. However, how beta adrenergic stimulation affects osteoblastic migration and associated proliferation is not known. Here we introduced a live imaging system, fluorescent ubiquitination-based cell cycle indicator (FUCCI), in osteoblast biology and examined isoproterenol regulation of cell cycle transition and cell migration in osteoblasts. Isoproterenol treatment suppresses the levels of first entry peak of quiescent osteoblastic cells into cell cycle phase by shifting from G1 /G0 to S/G2 /M and also suppresses the levels of second major peak population that enters into S/G2 /M. The isoproterenol regulation of osteoblastic cell cycle transition is associated with isoproterenol suppression on the velocity of migration. This isoproterenol regulation of migration velocity is cell cycle phase specific as it suppresses migration velocity of osteoblasts in G1 phase but not in G1 /S nor in G2 /M phase. Finally, these observations on isoproterenol regulation of osteoblastic migration and cell cycle transition are opposite to the PTH actions in osteoblasts. In summary, we discovered that sympathetic tone regulates osteoblastic migration in association with cell cycle transition by using FUCCI system.

β adrenergic stimulation suppresses bone formation in vivo while its actions in osteoblastic differentiation are still incompletely understood. We therefore examined the effects of β2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells focusing on BMP-induced alkaline phosphatase expression. Morphologically, isoproterenol treatment suppresses BMP-induced increase in the numbers of alkaline phosphatase-positive small foci in the cultures of MC3T3-E1 cells. Biochemically, isoproterenol treatment suppresses BMP-induced enzymatic activity of alkaline phosphatase in a dose-dependent manner. Isoproterenol suppression of alkaline phosphatase activity is observed even when the cells are treated with high concentrations of BMP. With respect to cell density, isoproterenol treatment tends to suppress BMP-induced increase in alkaline phosphatase expression more in osteoblasts cultured at higher cell density. In terms of treatment protocol, continuous isoproterenol treatment is compared to cyclic treatment. Continuous isoproterenol treatment is more suppressive against BMP-induced increase in alkaline phosphatase expression than cyclic regimen. At molecular level, isoproterenol treatment suppresses BMP-induced enhancement of alkaline phosphatase mRNA expression. Regarding the mode of isoproterenol action, isoproterenol suppresses BMP-induced BRE-luciferase activity. These data indicate that isoproterenol regulates BMP-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

As the aged population is soaring, prevalence of osteoporosis is increasing. However, the molecular basis underlying the regulation of bone mass is still incompletely understood. Sympathetic tone acts via beta2 adrenergic receptors in bone and regulates the mass of bone which is the target organ of parathyroid hormone (PTH). However, whether beta2 adrenergic receptor is regulated by PTH in bone cells is not known. We therefore investigated the effects of PTH on beta2 adrenergic receptor gene expression in osteoblast-like MC3T3-E1 cells. PTH treatment immediately suppressed the expression levels of beta2 adrenergic receptor mRNA. This PTH effect was dose-dependent starting as low as 1 nM. PTH action on beta2 adrenergic receptor gene expression was inhibited by a transcriptional inhibitor, DRB, but not by a protein synthesis inhibitor, cycloheximide suggesting direct transcription control. Knockdown of beta2 adrenergic receptor promoted PTH-induced expression of c-fos, an immediate early response gene. With respect to molecular basis for this phenomenon, knockdown of beta2 adrenergic receptor enhanced PTH-induced transcriptional activity of cyclic AMP response element-luciferase construct in osteoblasts. Knockdown of beta2 adrenergic receptors also enhanced forskolin-induced luciferase expression, revealing that adenylate cyclase activity is influenced by beta2 adrenergic receptor. As for phosphorylation of transcription factor, knockdown of beta2 adrenergic receptor enhanced PTH-induced phosphorylation of cyclic AMP response element binding protein (CREB). These data reveal that beta2 adrenergic receptor is one of the targets of PTH and acts as a suppressor of PTH action in osteoblasts.

Bone metabolism is maintained via balanced repetition of bone resorption by osteoclasts and bone formation by osteoblasts. Osteoblastic cells are capable of conducting self-renewal and differentiation that are basically associated with cell-cycle transition to enable cell specification and bone formation. Osteoblasts are also migrating to fill the resorption cavity curved by osteoclasts during bone remodeling to maintain homeostasis of bone mass whose imbalance leads to osteoporosis. However, technical difficulties have hampered the research on the dynamic relationship between cell cycle and migration in osteoblasts. In this report, we overcome these problems by introducing fluorescent ubiquitination-based cell cycle indicator (FUCCI) reporter system in calvarial osteoblastic cells and reveal that the cells in G1 as well as S/G2 /M phase are migrating. Furthermore, the osteoblastic cells in S/G2 /M phase migrate faster than those in G1 phase. Interestingly, parathyroid hormone (PTH) as an anabolic agent enhances migration velocity of the cells. Mechanical stress, another anabolic signal, also enhances migration velocity. In contrast, in the presence of both PTH and mechanical stress, the migration velocity returns to the base line levels revealing the interaction between the two anabolic stimuli in the regulation of cell migration. Importantly, PTH and mechanical stress also interact when they regulate the transition of cell cycle. These data demonstrate that osteoblastic migration is linked to cell cycle and it is under the control of mechanical and chemical stimuli that coordinate to regulate bone mass.