Regulation of IGF-1 and Insulin Signaling by O-GlcNAc Glycosylation

描述（由申请人提供）：三分之一的糖尿病患者表现出严重的牙周病，这被认为是由多种因素的相互作用引起的，包括对感染的易感性增加、促炎细胞因子升高、对激素的敏感性改变和高血糖。受损牙周组织的牙槽骨再生延迟，部分反映了对IGF-1和胰岛素合成代谢作用的反应不足，而IGF-1和胰岛素可能被细胞因子和高血糖下调。IGF-1刺激成骨细胞具有抗凋亡作用，有助于细胞增殖、分化、迁移、基质沉积和矿化，并支持破骨细胞的发生。胰岛素受体底物IRS-1和IRS-2介导了IGF-1R激活引发的多效效应的特异性，它们对新骨的形成和维持成骨细胞和破骨细胞之间的偶联至关重要。通过IRS蛋白向PI3K/Akt和/或Grb2/ERK通路传递信号是通过翻译后调控介导和调节的。IRS-1的Tyr/Ser/Thr磷酸化发生在糖尿病和骨质疏松症中。IRS-1和IRS-2也被n -乙酰氨基葡萄糖（O-GlcNAc）在丝氨酸/苏氨酸残基上糖基化。这种独特的糖基化是一种可逆的翻译后修饰，以类似于磷酸化的方式调节蛋白质功能。使用最先进的串联质谱方法，我们已经确定了人类IRS-1和IRS-2上O-GlcNAc修饰的多个位点，并开发了以特定位点的方式测量这种不稳定修饰的相对化学计量学的方法。蛋白o - glcn酰化程度在葡萄糖、激素和细胞应激的反应中迅速变化，并与胰岛素信号的改变和糖尿病相关的葡萄糖诱导并发症有关。在Aim 1中，我们提出表征成骨细胞系中IRS-1和IRS-2的O- GlcNAc修饰位点，并确定调节修饰程度的因素。我们将研究磷酸化和O-GlcNAc修饰的相互作用，因为这些修饰可能相互排斥。到目前为止，IRS-1的O-GlcNAc修饰位点与多个SH2结构域结合基序非常接近，因此在Aim 2中，我们将研究位点特异性的O-GlcNAc修饰对与受体和已知结合伙伴相互作用的影响，并探索这种修饰可能诱导的新的蛋白质相互作用。O-GlcNAc转移酶（OGT）和O-GlcNAc casase （OGA）这两种酶在成骨细胞中调节IGF-1作用中的作用，负责催化蛋白质中GlcNAc的结合和去除，但尚未得到解决。因此，在Aim 3中，我们将确定这些酶的表达调节对IGF-1刺激引起的细胞效应的影响，以及改变的O-GlcNAc修饰IRS-1是否会影响成骨细胞中的IGF-1信号传导。

DESCRIPTION (provided by applicant): One third of all diabetic patients exhibit severe periodontal disease which is postulated to stem from an interplay of factors including increased susceptibility to infection, elevated pro-inflammatory cytokines, altered sensitivity to hormones, and hyperglycemia. The delayed regeneration of alveolar bone in the damaged periodontium reflects, in part, inadequate response to the anabolic actions of IGF-1 and insulin which may be down-regulated by cytokines and hyperglycemia. IGF-1 stimulation of osteoblasts, is anti-apoptotic and contributes to cell proliferation, differentiation, migration, matrix deposition and mineralization, and supports osteoclastogenesis. The specificity of the pleiotropic effects elicited by IGF-1R activation are mediated by the insulin receptor substrates, IRS-1 and IRS-2, which are critical to the formation of new bone and maintaining coupling between osteoblasts and osteoclasts. Signaling through the IRS proteins to PI3K/Akt and/or the Grb2/ERK pathways is mediated and modulated by posttranslational regulation. Perturbations in Tyr/Ser/Thr phosphorylation of IRS-1 occur in diabetes and osteoporosis. IRS-1 and IRS-2 are also glycosylated at Ser/Thr residues by N-acetylglucosamine (O-GlcNAc). This unique glycosylation is a reversible posttranslational modification proposed to regulate protein function in a manner analogous to phosphorylation. Using state-of-the-art tandem mass spectrometric approaches, we have identified multiple sites of O-GlcNAc modification on human IRS-1 and IRS-2 and developed methodology to measure the relative stoichiometry of this labile modification in a site-specific manner. The extent of protein O-GlcNAcylation changes rapidly in response to glucose, hormones, and cellular stress and it is implicated in altered insulin signaling and the glucose-induced complications associated with diabetes. In Aim 1, we propose to characterize the sites of O- GlcNAc modification of IRS-1 and IRS-2 in an osteoblast cell line and define the factors that modulate the extent of modification. We will investigate the interplay of phosphorylation and O-GlcNAc modification as these modifications may mutually exclude one another. The sites of O-GlcNAc modification of IRS-1 identified thus far are in close proximity to multiple SH2 domain binding motifs, thus in Aim 2 we will interrogate the effect of site-specific O-GlcNAc modification on interactions with the receptor and known binding partners and probe for novel protein interactions that may be induced by this modification. The role of the enzymes responsible for catalyzing the incorporation and removal of GlcNAc from proteins, O-GlcNAc transferase (OGT) and O- GlcNAcase (OGA), in modulating IGF-1 action in osteoblasts has not been addressed. Thus, in Aim 3, we will determine the impact of modulation of the expression of these enzymes on the cellular effects elicited by IGF-1 stimulation and whether altered O-GlcNAc modification of IRS-1 influences IGF-1 signaling in osteoblasts. PUBLIC HEALTH RELEVANCE: With the predicted escalation in the number of newly diagnosed cases of diabetes annually in the U.S., understanding the regulation of key molecules critical to the pathogenesis of diabetes and its complications is essential. One-third of diabetic patients exhibit severe periodontal disease, the cause of which is a chronic bacterial infection, but with the underlying changes associated with the diabetic state, the stage is set for an altered response to hormones and cytokines culminating in the degradation of alveolar bone and tooth loss. This research will provide novel information concerning the regulation of two proteins crucial for normal bone formation that are known to be perturbed in diabetes with the ultimate goal of elucidating therapeutic targets to facilitate regeneration of alveolar bone in diabetes-associated periodontal disease.