Calmodulin and Calmodulin-dependent Kinase IIα Regulate Osteoblast Differentiation by Controlling c-fos Expression

Keertik Fulzele,Majd Zayzafoon,Jay M. McDonald

doi:10.1074/jbc.m412680200

Abstract

Ca(2+)/calmodulin-dependent protein kinase IIalpha (alpha-CaMKII) was once thought to be exclusively expressed in neuronal tissue, but it is becoming increasingly evident that CaMKII is also expressed in various extraneural cells. CaMKII plays a critical role in regulating various signaling pathways leading to modulation of several aspects of cellular functions, including proliferation, differentiation, cytoskeletal structure, and gene expression. The purpose of this study was to examine the expression of CaMKII in osteoblast-like cells (MC4) and to elucidate its role in osteoblast differentiation. We demonstrated that CaMKII, specifically the alpha isoform, is expressed in osteoblasts both in vitro and in vivo. Inhibition of CaMKII by the calmodulin antagonist trifluoperazine or the CaMKII antagonist KN93 reduces alkaline phosphatase activity and mineralization, as well as causes 85 and 56% decreases in alkaline phosphatase and osteocalcin gene expression, respectively. CaM and CaMKII antagonists, using the newborn mouse calvaria in vivo model, cause a 50% decrease in osteoblast number (N.Ob-BS) and a 32% decrease in mineralization (BV/TV). Pharmacologic and genetic inhibition of alpha-CaMKII by using trifluoperazine, KN93, and alpha-CaMKII small interfering RNA decreases the phosphorylation of ERK and of cAMP-response element-binding protein, leading to a significant decrease in the transactivation of serum response element and cAMP-response element. Inhibition of alpha-CaMKII decreases the expression of c-fos, AP-1 transactivation, and AP-1 DNA binding activity. Our findings demonstrated that alpha-CaMKII is expressed in osteoblasts and is involved in c-fos expression via regulation of serum response element and cAMP-response element. Inhibition of alpha-CaMKII results in a decrease in c-fos expression and AP-1 activation, leading to inhibition of osteoblast differentiation.

Highlights

From the ‡Department of Pathology, University of Alabama at Birmingham and the §Veterans Administration Medical Center, Birmingham, Alabama 35233
Our findings demonstrated that ␣-calmodulin-dependent protein kinase II (CaMKII) is expressed in osteoblasts and is involved in c-fos expression via regulation of serum response element and cAMP-response element
To elucidate the mechanisms by which CaM/CaMKII inhibit osteoblast differentiation, we examined the effects of TFP and KN93 on two signaling pathways (ERK/mitogen-activated protein kinase and CREB/ATF) that are known to be regulated by CaMKII and are important for osteoblast differentiation [8, 36]

Summary

Introduction

From the ‡Department of Pathology, University of Alabama at Birmingham and the §Veterans Administration Medical Center, Birmingham, Alabama 35233. CaMKII plays a critical role in regulating various signaling pathways leading to modulation of several aspects of cellular functions, including proliferation, differentiation, cytoskeletal structure, and gene expression. Calmodulin (CaM) is known to bind to and activate numerous targets, including calcium/calmodulin-dependent protein kinase II (CaMKII). It has been shown from transgenic mice models that the elevation of CaM expression leads to an increase in the autonomous activity of CaMKII [3]. The activation of CaMKII and its downstream signaling cascade is involved in regulating a wide variety of cellular events, including proliferation, differentiation, and apoptosis [4].

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