Type II cGMP-dependent Protein Kinase Mediates Osteoblast Mechanotransduction

Hema Rangaswami,Nisha Marathe,Shunhui Zhuang,Yongchang Chen,Jiunn-Chern Yeh,John A Frangos,Gerry R Boss,Renate B Pilz

doi:10.1074/jbc.m806486200

Abstract

Continuous bone remodeling in response to mechanical loading is critical for skeletal integrity, and interstitial fluid flow is an important stimulus for osteoblast/osteocyte growth and differentiation. However, the biochemical signals mediating osteoblast anabolic responses to mechanical stimulation are incompletely understood. In primary human osteoblasts and murine MC3T3-E1 cells, we found that fluid shear stress induced rapid expression of c-fos, fra-1, fra-2, and fosB/DeltafosB mRNAs; these genes encode transcriptional regulators that maintain skeletal integrity. Fluid shear stress increased osteoblast nitric oxide (NO) synthesis, leading to activation of cGMP-dependent protein kinase (PKG). Pharmacological inhibition of the NO/cGMP/PKG signaling pathway blocked shear-induced expression of all four fos family genes. Induction of these genes required signaling through MEK/Erk, and Erk activation was NO/cGMP/PKG-dependent. Treating cells with a membrane-permeable cGMP analog partly mimicked the effects of fluid shear stress on Erk activity and fos family gene expression. In cells transfected with small interfering RNAs (siRNA) specific for membrane-bound PKG II, shear- and cGMP-induced Erk activation and fos family gene expression was nearly abolished and could be restored by transducing cells with a virus encoding an siRNA-resistant form of PKG II; in contrast, siRNA-mediated repression of the more abundant cytosolic PKG I isoform was without effect. Thus, we report a novel function for PKG II in osteoblast mechanotransduction, and we propose a model whereby NO/cGMP/PKG II-mediated Erk activation and induction of c-fos, fra-1, fra-2, and fosB/DeltafosB play a key role in the osteoblast anabolic response to mechanical stimulation.

Highlights

Mechanical stress is a primary determinant of bone growth and remodeling; the strength of bone increases with weight bearing and muscular activity and decreases with unloading and disuse [1, 2]
We found that c-fos, fra-1, fra-2, and fosB/⌬fosB mRNAs were strongly induced 30 min after the onset of flow in both MC3T3 cells and Human primary osteoblasts (hPOBs) and remained elevated for at least 2 h (Fig. 1A shows hPOBs, results for MC3T3 cells are shown in supplemental Fig. 1A)
We found that fluid shear stress induced a rapid and sustained increase in c-fos, fra-1, fra-2, and fosB/⌬fosB mRNA in human primary osteoblasts, MC3T3, and UMR106 cells

Summary

Introduction

Mechanical stress is a primary determinant of bone growth and remodeling; the strength of bone increases with weight bearing and muscular activity and decreases with unloading and disuse [1, 2]. In primary human osteoblasts and murine MC3T3-E1 cells, we found that fluid shear stress induced rapid expression of c-fos, fra-1, fra-2, and fosB/⌬fosB mRNAs; these genes encode transcriptional regulators that maintain skeletal integrity.

Results

Conclusion