cGMP-dependent Protein Kinase Function in Osteoblast Mechanotransduction

描述(申请人提供)：骨的机械负荷导致流体剪应力(FSS)，从而刺激Src和丝裂原激活的蛋白激酶ERK1/2，从而增加成骨细胞/细胞的增殖和存活。我们发现，FSS激活了一氧化氮(NO)/cGMP/cGMP依赖的蛋白激酶(PKG)通路，而PKG激活是剪切诱导的Src和Erk激活所必需的。没有供体和cGMP类似物模拟FSS对成骨细胞/细胞中Src/Erk的影响，而膜结合的PKG II的siRNA敲除了它。FSS或cGMP通过去磷酸化Src Tyr529(抑制位点)激活SRC，这需要蛋白酪氨酸磷酸酶(PTP)SHP-1和-2，并通过$3整合素附着细胞。在FSS刺激的成骨细胞中，PKG II、Src和SHP-2与3美元共同定位于局部黏附复合体，PKG II可磷酸化SHP-1和SHP-2，但不能磷酸化Src。我们假设PKG II通过激活或招募SHP-1/2和/或通过抑制或置换C-末端使Tyr529磷酸化的Src来激活Src，SHP-1/2使Tyr529去磷酸化。与一氧化氮合酶缺陷小鼠的骨骼表型一致，并基于PKG II缺失的成骨细胞中FSS诱导的信号缺陷，PKG II缺陷可能导致生长过程中和/或骨骼负荷反应中的骨形成减少。本研究的具体目的是：(1)研究FSS刺激的成骨细胞/细胞中NO/cGMP/PKGⅡ激活Src的机制(S)；(2)研究FSS和NO/cGMP/PKGⅡ激活的含Src的信号复合体的特征；(3)明确PKG II在小鼠骨(Re)模型中的作用。我们将用PKG II定位SHP-1/2的磷酸化，并检测PKG II对PTP和CSK活性及亚细胞定位的影响。我们将使用siRNA方法结合野生型和突变蛋白的重组来评估PKG II、SHP-1/2和$3整合素在剪切诱导的Src激活中的功能。我们将使用免疫共沉淀、免疫荧光染色和双分子荧光互补来表征PKG调节的Src信号复合体，并使用蛋白质组学方法来鉴定新的PKG II底物和成骨细胞膜中的相互作用伙伴。我们将利用显微CT、组织形态计量学和基因表达分析，检测成骨细胞/细胞特异性PKG II基因敲除小鼠在骨骼生长和衰老过程中以及在卸载和再加载条件下的骨骼表型。我们将分析原代PKG-/-成骨细胞的FSS反应和分化。这些研究将为NO/cGMP/PKG在骨骼中的作用提供新的见解，并可能导致改进骨质疏松的治疗方法。 与公共健康相关：机械刺激是骨细胞(成骨细胞)生长和分化、提高骨骼强度和预防骨质疏松症的有效刺激；然而，成骨细胞将机械刺激转化为生化变化的分子机制(称为机械转导过程)仍然知之甚少。我们最近定义了一氧化氮/cGMP/cGMP依赖的蛋白激酶(PKG)信号转导通路在成骨细胞力学转导中的新功能，现在我们提议研究PKG在机械刺激的成骨细胞中控制重要下游信号蛋白的机制，并确定PKG II在转基因小鼠骨重建中的作用。这些研究将为骨质疏松症的新的和改进的治疗策略提供基础。

DESCRIPTION (provided by applicant): Mechanical loading of bone induces fluid shear stress (FSS), which stimulates Src and the mitogen- activated protein kinases Erk1/2 leading to increased osteoblast/cyte proliferation and survival. We found that FSS activates the nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) pathway, and that PKG activation is necessary for shear-induced Src and Erk activation. NO donors and cGMP analogs mimicked the effect of FSS on Src/Erk in osteoblasts/cytes, while siRNA knock-down of membrane-bound PKG II abolished it. Src activation by FSS or cGMP occurred through de-phosphorylation of Src Tyr529 (an inhibitory site), which required the protein tyrosine phosphatases (PTP) Shp-1 and -2, and cell attachment through $3 integrins. PKG II, Src, and Shp-2 co-localized with $3 in focal adhesion complexes in FSS-stimulated osteoblasts, and PKG II phosphorylated Shp-1 and -2, but not Src. We hypothesize that PKG II activates Src through activation or recruitment of Shp-1/2, which de-phosphorylate Src Tyr529, and/or through inhibition or displacement of C- terminal Src kinase (CSK), which phosphorylates Tyr529. Consistent with the skeletal phenotype of NO synthase-deficient mice, and based on defective FSS-induced signaling in PKG II-null osteoblasts, PKG II deficiency may lead to decreased bone formation during growth and/or in response to skeletal loading. The Specific Aims are: (i) to determine the mechanism(s) of Src activation by NO/cGMP/PKGII in FSS-stimulated osteoblasts/cytes; (ii) to characterize the Src-containing signaling complex activated by FSS and NO/cGMP/PKG II; and (iii) to define the role of PKG II in bone (re)modeling in mice. We will map Shp-1/2 phosphorylation by PKG II, and test the effects of PKG II on PTP and CSK activity and subcellular localization. We will assess PKG II, SHP-1/2, and $3 integrin functions in shear-induced Src activation using siRNA approaches with reconstitution of wild type and mutant proteins. We will use co-immunoprecipitation, immuno- fluorescence staining, and bimolecular fluorescence complementation to characterize the PKG-regulated Src signaling complex, and use a proteomics approach to identify novel PKG II substrates and interacting partners in osteoblast membranes. We will examine the skeletal phenotype of osteoblast/cyte-specific PKG II knockout mice during skeletal growth and aging, and under conditions of unloading and reloading, using micro-CT, histo- morphometry, and gene expression analysis. We will analyze FSS responses and differentiation of primary PKG-/- osteoblasts. These studies will provide new insights into NO/cGMP/PKG actions in bone, and could lead to improved therapies for osteoporosis. PUBLIC HEALTH RELEVANCE: Mechanical stimulation is a potent stimulus for bone cell (osteoblast) growth and differentiation, improving bone strength and preventing osteoporosis; however, the molecular mechanisms by which osteoblasts convert mechanical stimuli into biochemical changes (a process known as mechanotransduction) remain poorly understood. We recently defined a novel function of the nitric oxide/cGMP/cGMP-dependent protein kinase (PKG) signal transduction pathway in osteoblast mechanotransduction, and we now propose to study the mechanisms whereby PKG controls important down-stream signaling proteins in mechanically-stimulated osteoblasts and determine the role of PKG II in bone remodeling in genetically-modified mice. These studies will provide the foundation for novel and improved treatment strategies in osteoporosis.