Targeting PHLPP1 to Inhibit Progression of Intervertebral Disc Degeneration

项目摘要 疼痛性椎间盘（IVD）退变（IDD）的快速增加使得迫切需要 为预防碘缺乏病和促进其再生提供解决方案。IDD与IVD丢失相关 细胞结构、基质降解和细胞凋亡，并确定预防IDD这些方面的特定靶点 将为新的和特定的治疗方法打开大门。最近，PH结构域富含亮氨酸重复蛋白 磷酸酶1（PHLPP 1）被鉴定为在胰岛素抵抗、肥胖和骨关节炎中起关键作用。 重要的是，PHLPP1在健康和疾病中的作用是高度环境特异性的。胰岛素抵抗 PHLPP 1通过激活糖原合成酶激酶3 β（GSK 3 β）诱导糖原合成 其在NP中以高水平表达，并且与细胞凋亡和基质降解相关， 提示IVD中存在组织特异性PHLPP1机制。PHLPP 1尚未在IVD中进行研究， 我们首次发现PHLPP 1在退化的人IVD和受损的小鼠IVD中有更高的表达。 并证明PHLPP1敲除增加了小鼠中的NP细胞性。我们的目标是确定 PHLPP 1在IDD发病中的特异性作用，并确定PHLPP 1作为防治IDD新靶点的可行性 进展由于IDD的高影响力和翻译的可行性，本研究具有高影响力， 存在一些风险，因为对PHLPP 1在IDD中的作用一无所知。 拟议的研究将测试PHLPP1是诱导IDD的关键因素的总体假设。我们 假设PHLPP 1存在将导致细胞凋亡和基质降解，而其缺失将 诱导细胞增殖和基质产生。我们认为PHLPP1通过调节IDD的表达， 因此，通过抑制PTEN/PI3K/AKT和PKC通路，靶向PHLPP 1，可以阻止IDD的进展。 目的1将使用体内PHLPP1-ko小鼠测定PHLPP1缺失对IDD进展的作用， 健康和损伤的IVD，测量IVD形态、结构和细胞构成。目标2将 通过测定PHLPP 1，确定PHLPP 1表达与人IDD之间的关系， 在来自尸检的人IVD中的表达，并将其与退行性分级相关联。目标3将决定 特异性阻断剂抑制PHLPP 1活性和诱导细胞增殖的功效， 使用取自尸检标本的人IVD细胞的基质生产。区组研究将评价是否 PHLPP 1通过评估AKT和PKC信号通路的几个下游分子发挥作用。 这个项目是非常重要的，因为IDD在美国的主要负担。调查 PHLPP1及其在IDD中的作用是非常创新的，因为PHLPP1从未在IVD研究中被探索过。 这项提议的结果是非常有影响力的，因为如果PHLPP 1可以被确定为治疗药物， 目标;可以进行进一步的研究，将这些知识转化为微创治疗 为美国和全世界数百万IDD患者制定战略。

PROJECT SUMMARY The rapid increase in painful intervertebral disc (IVD) degeneration (IDD) makes it an urgent need to provide solutions for preventing IDD and promoting its regeneration. IDD is associated with loss of IVD cellularity, matrix degradation and apoptosis, and identifying specific targets to prevent these aspects of IDD would open the door for novel and specific treatments. Recently, the PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) was identified as key player in insulin resistance, obesity and osteoarthritis. Importantly, effects of PHLPP1 in healthy and disease are highly context-specific. In insulin resistant patients, PHLPP1 induces glycogen synthesis via activation of glycogen synthase kinase 3β, (GSK3β) which is expressed at high levels in NPs, and has been correlated to apoptosis and matrix degradation, suggesting a tissue-specific PHLPP1 mechanism in IVDs. PHLPP1 has not been investigated in IVDs and we, for the first time, show higher PHLPP1 expression in degenerated human IVDs and injured mice IVDs. And demonstrate that PHLPP1 knockout increased NP cellularity in mice. Our goal is to determine the specific contributions of PHLPP1 to IDD and to identify the feasibility of PHLPP1 as novel target to stop IDD progression. This study has high impact because of the high impact of IDD and feasibility of translation, yet there are some risks because nothing is known about the role of PHLPP1 in IDD. The proposed studies will test the overall hypothesis that PHLPP1 is a key player in inducing IDD. We hypothesize that PHLPP1 presence will cause apoptosis and matrix degradation while its absence will induce cell proliferation and matrix production. We believe that PHLPP1 regulates IDD via the PTEN/PI3K/AKT and PKC pathways and that targeting PHLPP1 will prevent the progression of IDD. Aim 1 will determine the role of PHLPP1 depletion on IDD progression using in vivo PHLPP1-ko mice on healthy and injured IVDs with measurements of IVD morphology, structure, and cellularity. Aim 2 will determine the relationship between PHLPP1 expression and human IDD by determining PHLPP1 expression in human IVDs from autopsy and correlating this with degenerative grade. Aim 3 will determine the efficacy of specific blocking agents for inhibiting PHLPP1 activity and inducing cell proliferation and matrix production using human IVD cells taken from autopsy specimens. Blocking studies will evaluate if PHLPP1 act via AKT and PKC signaling by assessing several downstream molecules of these pathways. This project is highly significant because the major burden of IDD in the United States. Investigating PHLPP1 and its role in IDD is very innovative because PHLPP1 has never been explored in IVD research. The outcome of this proposal is highly impactful because if PHLPP1 could be identified as a therapeutic target; further studies could be performed to translate this knowledge into minimal invasive treatment strategies for millions of people suffering from IDD in the United States and Worldwide.