Abstract
BackgroundMechanical stress rapidly induces ΔFosB expression in osteoblasts, which binds to interleukin (IL)-11 gene promoter to enhance IL-11 expression, and IL-11 enhances osteoblast differentiation. Because bone morphogenetic proteins (BMPs) also stimulate IL-11 expression in osteoblasts, there is a possibility that BMP-Smad signaling is involved in the enhancement of osteoblast differentiation by mechanical stress. The present study was undertaken to clarify whether mechanical stress affects BMP-Smad signaling, and if so, to elucidate the role of Smad signaling in mechanical stress-induced enhancement of IL-11 gene transcription.Methodology/Principal FindingsMechanical loading by fluid shear stress (FSS) induced phosphorylation of BMP-specific receptor-regulated Smads (BR-Smads), Smad1/5, in murine primary osteoblasts (mPOBs). FSS rapidly phosphorylated Y311 of protein kinase C (PKC)δ, and phosphorylated PKCδ interacted with BR-Smads to phosphorylate BR-Smads. Transfection of PKCδ siRNA or Y311F mutant PKCδ abrogated BR-Smads phosphorylation and suppressed IL-11 gene transcription enhanced by FSS. Activated BR-Smads bound to the Smad-binding element (SBE) of IL-11 gene promoter and formed complex with ΔFosB/JunD heterodimer via binding to the C-terminal region of JunD. Site-directed mutagenesis in the SBE and the AP-1 site revealed that both SBE and AP-1 sites were required for full activation of IL-11 gene promoter by FSS.Conclusions/SignificanceThese results demonstrate that PKCδ-BR-Smads pathway plays an important role in the intracellular signaling in response to mechanical stress, and that a cross-talk between PKCδ-BR-Smads and ΔFosB/JunD pathways synergistically stimulates IL-11 gene transcription in response to mechanical stress.
Highlights
Mechanical stress to bone plays a crucial role in maintaining bone homeostasis
The results demonstrate that mechanical stress by fluid shear stress (FSS) to murine primary osteoblasts in vitro stimulates Smad1/5 phosphorylation, that mechanical stress-induced phosphorylation of Smad1/5 is mediated via phosphorylation and activation of protein kinase C (PKC)d, that activated Smad1/5 forms complex with DFosB and JunD on IL-11 gene promoter, and that IL-11 gene transcription is cooperatively stimulated by DFosB/JunD and Smad1/5 in response to mechanical stress
Because mechanical loading to osteoblasts does not alter the expression levels of bone morphogenetic proteins (BMPs)-2 (Figure S2), these results suggest that mechanical stress to osteoblasts enhances BR-Smads phosphorylation without stimulation by BMP
Summary
Mechanical stress to bone plays a crucial role in maintaining bone homeostasis. Immobilization, long-term bed rest, or microgravity in space causes a marked loss of bone mass and strength, due to reduced bone formation as well as enhanced bone resorption [1,2,3,4]. FSS is shown to be one of the most important signal transduction mechanisms to enhance osteoblastic differentiation and bone formation in response to mechanical loading to bone [6,7]. Transgenic mice overexpressing IL-11 show high bone mass with continued increase of bone mineral density with aging due to an enhanced bone formation without an increase in bone resorption [16]. These observations suggested to us that IL-11 mediates mechanical stress signals to osteoblast differentiation signal. The present study was undertaken to clarify whether mechanical stress affects BMP-Smad signaling, and if so, to elucidate the role of Smad signaling in mechanical stressinduced enhancement of IL-11 gene transcription
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