Neuropeptide Y1 Receptor Regulates Glucocorticoid-Induced Inhibition of Osteoblast Differentiation in Murine MC3T3-E1 Cells via ERK Signaling.

Wei Yu,Shengdan Jiang,Chao Zhu,Leisheng Jiang,Wenning Xu

doi:10.3390/ijms17122150

Abstract

High dose glucocorticoid (GC) administration impairs the viability and function of osteoblasts, thus causing osteoporosis and osteonecrosis. Neuropeptide Y1 receptor (Y1 receptor) is expressed in bone tissues and cells, and regulates bone remodeling. However, the role of Y1 receptor in glucocorticoid-induced inhibition of osteoblast differentiation remains unknown. In the present study, osteoblastic cell line MC3T3-E1 cultured in osteogenic differentiation medium was treated with or without of 10−7 M dexamethasone (Dex), Y1 receptor shRNA interference, Y1 receptor agonist [Leu31, Pro34]-NPY, and antagonist BIBP3226. Cell proliferation and apoptosis were assessed by cell counting kit-8 (CCK-8) assay and cleaved caspase expression, respectively. Osteoblast differentiation was evaluated by Alizarin Red S staining and osteogenic marker gene expressions. Protein expression was detected by Western blot analysis. Dex upregulated the expression of Y1 receptor in MC3T3-E1 cells associated with reduced osteogenic gene expressions and mineralization. Blockade of Y1 receptor by shRNA transfection and BIBP3226 significantly attenuated the inhibitory effects of Dex on osteoblastic activity. Y1 receptor signaling modulated the activation of extracellular signal-regulated kinases (ERK) as well as the expressions of osteogenic genes. Y1 receptor agonist inhibited ERK phosphorylation and osteoblast differentiation, while Y1 receptor blockade exhibited the opposite effects. Activation of ERK signaling by constitutive active mutant of MEK1 (caMEK) abolished Y1 receptor-mediated Dex inhibition of osteoblast differentiation in MC3T3-E1 cells. Taken together, Y1 receptor regulates Dex-induced inhibition of osteoblast differentiation in murine MC3T3-E1 cells via ERK signaling. This study provides a novel role of Y1 receptor in the process of GC-induced suppression in osteoblast survival and differentiation.

Highlights

Glucocorticoids (GCs) are extensively used as immunosuppressive and anti-inflammatory drugs for various disorders including autoimmune diseases and inflammatory [1,2]
To examine the role of the Y1 receptor in the glucocorticoid-induced suppression of osteoblast differentiation, we first detected the expression of Y1 receptor in MC3T3-E1 cells with or without dexamethasone (Dex) treatment in osteogenic differentiation media
Western blot demonstrated that cells with 10−7 M dexamethasone treatments for 24 h displayed a high level of Y1 receptor expression (Figure 1E)

Summary

Introduction

Glucocorticoids (GCs) are extensively used as immunosuppressive and anti-inflammatory drugs for various disorders including autoimmune diseases and inflammatory [1,2]. Excessive or long-term administration of glucocorticoids causes several adverse effects on the bones, including osteoporosis and osteonecrosis [3,4,5]. Glucocorticoids inhibit the survival and differentiation capacity of osteoblasts, which is considered a prominent mechanism in the process of GC-induced bone loss [6]. Previous studies have shown that induction of cell apoptosis or autophagy contributes to glucocorticoid-induced loss of bone cell viability [4,7]. The precise mechanisms by which glucocorticoids regulate the proliferation and differentiation pathways in osteoblasts are still unknown

Methods

Results

Discussion

Conclusion