cGMP-dependent Protein Kinase Function in Osteoblast Mechanotransduction

描述（由申请人提供）：骨的机械载荷诱导流体剪切应力（FSS），刺激Src和丝裂原活化蛋白激酶Erk1/2，导致成骨细胞/细胞增殖和存活增加。我们发现FSS激活一氧化氮(NO)/cGMP/cGMP依赖性蛋白激酶（PKG）通路，PKG的激活是剪切诱导的Src和Erk激活所必需的。NO供体和cGMP类似物模拟FSS对成骨细胞/细胞Src/Erk的影响，而siRNA敲除膜结合的PKG II则消除了它。FSS或cGMP通过Src Tyr529（抑制位点）的去磷酸化发生Src激活，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共定位于局灶黏附复合物中，PKG II磷酸化了Shp-1和-2，但不磷酸化Src。我们假设PKG II通过激活或募集Shp-1/2（使Src Tyr529去磷酸化）和/或通过抑制或置换C端Src激酶（CSK）（使Tyr529磷酸化）来激活Src。与NO合酶缺陷小鼠的骨骼表型一致，基于fss诱导的PKG II缺失成骨细胞信号传导缺陷，PKG II缺乏可能导致生长和/或对骨骼负荷的反应中骨形成减少。具体目的是：(i)确定NO/cGMP/PKGII在fss刺激的成骨细胞/细胞中活化Src的机制；（ii）表征FSS和NO/cGMP/PKG ii激活的含src的信号复合物；（iii）确定PKG II在小鼠骨（再）造模中的作用。我们将通过PKG II绘制Shp-1/2磷酸化图谱，并测试PKG II对PTP和CSK活性和亚细胞定位的影响。我们将评估PKG II， SHP-1/2和$3整合素在剪切诱导Src激活中的功能，使用siRNA方法重组野生型和突变蛋白。我们将使用共免疫沉淀、免疫荧光染色和双分子荧光互补来表征PKG调控的Src信号复合物，并使用蛋白质组学方法鉴定成骨细胞膜中新的PKG II底物和相互作用伙伴。我们将使用显微ct、组织形态测定和基因表达分析来研究成骨细胞/细胞特异性PKG II敲除小鼠在骨骼生长和衰老过程中的骨骼表型，以及在卸载和重新加载的条件下。我们将分析FSS对原代PKG-/-成骨细胞的反应和分化。这些研究将为NO/cGMP/PKG在骨中的作用提供新的见解，并可能导致改善骨质疏松症的治疗方法。

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.