Intermittent parathyroid hormone (1-34) application regulates cAMP-response element binding protein activity to promote the proliferation and osteogenic differentiation of bone mesenchymal stromal cells, via the cAMP/PKA signaling pathway

Bailing Chen; Tao Lin; Xiaoxi Yang; Yiqiang Li; Denghui Xie; Haowen Cui

doi:10.3892/etm.2016.3177

Intermittent parathyroid hormone (1-34) application regulates cAMP-response element binding protein activity to promote the proliferation and osteogenic differentiation of bone mesenchymal stromal cells, via the cAMP/PKA signaling pathway

Exp Ther Med. 2016 Jun;11(6):2399-2406. doi: 10.3892/etm.2016.3177. Epub 2016 Mar 22.

Authors

Bailing Chen¹, Tao Lin¹, Xiaoxi Yang², Yiqiang Li³, Denghui Xie⁴, Haowen Cui¹

Affiliations

¹ Department of Spine Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong 510080, P.R. China.
² Department of Spine Surgery, Chinese PLA General Hospital (301 Hospital), Beijing 100853, P.R. China.
³ Department of Orthopedics, Guangzhou Women and Children's Medical Center, Guangzhou, Guangdong 510623, P.R. China.
⁴ Department of Spine Surgery, The Third Affiliated Hospital of Southern Medical University, Guangzhou, Guangdong 510630, P.R. China.

Abstract

The potential effects of intermittent parathyroid hormone (1-34) [PTH (1-34)] administration on bone formation have previously been investigated. A number of studies have suggested that the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway is associated with PTH-induced osteogenic differentiation. However, the precise signaling pathways and molecular mechanism by which PTH (1-34) induces the osteogenic differentiation of bone mesenchymal stromal cells (BMSCs) remain elusive. The purpose of the present study was to investigate the mechanism underlying the effect of intermittent PTH (1-34) application on the proliferation and osteogenic differentiation of BMSCs. BMSCs were randomly divided into four groups, as follows: Osteogenic medium (control group); osteogenic medium and intermittent PTH (1-34); osteogenic medium and intermittent PTH (1-34) plus the adenylyl cyclase activator forskolin; and osteogenic medium and intermittent PTH (1-34) plus the PKA inhibitor H-89. A cell proliferation assay revealed that PTH (1-34) stimulates BMSC proliferation via the cAMP/PKA pathway. Furthermore, reverse transcription-quantitative polymerase chain reaction, alkaline phosphatase activity testing and cell examination using Alizarin Red S staining demonstrated that PTH (1-34) administration promotes osteogenic differentiation and mineralization, mediated by the cAMP/PKA pathway. Crucially, the results of western blot analyses suggested that PTH (1-34) treatment and, to a greater degree, PTH (1-34) plus forskolin treatment caused an increase in phosphorylated cAMP response element binding protein (p-CREB) expression, but the effect of PTH on p-CREB expression was blocked by H-89. In conclusion, the current study demonstrated that intermittent PTH (1-34) administration regulates downstream proteins, particularly p-CREB, in the cAMP/PKA signaling pathway, to enhance the proliferation, osteogenic differentiation and mineralization of BMSCs.

Keywords: bone mesenchymal stromal cells; cyclic adenosine monophosphate/protein kinase A pathway; osteogenic differentiation; parathyroid hormone (1-34).