Role of HIF-1 in Intervertebral Disc Function

描述(申请人提供)：在椎间盘中，髓核(NP)的细胞在一个独特的环境中发挥作用，其特征是非常有限的血管供应，对椎间盘细胞施加新陈代谢限制和富含蛋白多糖的细胞外基质。我们已经证明，NP的氧分压非常低，与所有其他组织不同，NP的细胞显示出HIF-1a的结构性表达。我们提出了新的假设，即HIF-1a的结构性表达有助于NP细胞适应其无血管环境，并且HIF-1活性的变化是其生存和功能所必需的。第一个具体目的是验证低氧椎间盘中NP细胞的生存需要高结构性HIF-1a表达的假说；研究调节HIF-1a结构性表达和活性的机制；使用来自人类退变椎间盘的组织来关联NP HIF-1活性与退变和对细胞凋亡的敏感性。我们将暂时和稳定地抑制HIF-1a，并评估HIF-1a下调对细胞存活和死亡的影响。然后，我们将恢复受抑制细胞中HIF-1a的表达，并评估存活、凋亡和细胞表型。我们还将确定pVHL、FIH和PhDS表达的变化是否调节NP细胞中HIF-1a的表达和转录活性。我们将使用从具有不同程度退变的临床人类椎间盘样本中分离的细胞来确定对凋亡原治疗的敏感性是否与内源性HIF-1a活性和退变程度有关。第二个具体目的是验证结构性HIF-1a表达调节蛋白多糖合成、GAG链形成和硫酸盐化的假设；在人类退变的椎间盘标本中将HIF-1活性与蛋白多糖合成联系起来。我们将确定构成HIF-1a在aggrecan核心蛋白的表达和合成中的作用，aggrecan核心蛋白是一种具有HIF-1响应启动子元件的分子。此外，我们将评估HIF-1a在调节GAG合成所需的酶(葡萄糖醛酸基转移酶I；GlcAT-I)和软骨素硫酸盐化(软骨素-4-O-磺基转移酶2；C4ST-2)中的作用。我们将研究从临床人类椎间盘样本中分离的细胞合成蛋白多糖是否与HIF-1a活性有关。最后一个具体目的是利用慢病毒HIF-1a-siRNA建立一种在NP细胞中抑制HIF-1a的兔模型；了解HIF-1a失活是否会促进细胞密度的丧失、蛋白多糖合成的减少和椎间盘的退变。为了补充体内研究，并调查NP变性的早期细胞事件，我们将使用FLOXED-HIF-1a小鼠进行椎间盘器官培养研究。通过注射表达cre重组酶的腺病毒可以灭活器官培养盘中的HIF-1a基因。这些研究提供的信息将为深入了解NP细胞早期退变的机制提供依据，并为开发预防退行性腰椎间盘疾病的干预策略提供基础。公共卫生相关性腰痛的发生率非常高，美国医疗保健行业每年的成本超过330亿美元。腰痛通常与椎间盘退行性变化有关。目前的治疗方法都不能完全恢复退变的椎间盘的功能，从而防止受损脊柱的进一步恶化。这项拟议的研究将为椎间盘早期退行性改变的机制提供洞察力，并可能允许开发干预策略来预防退行性疾病。

DESCRIPTION (provided by applicant): In the intervertebral disc, the cells of the nucleus pulposus (NP) function in a unique environmental niche, characterized by a very limited vascular supply that imposes metabolic constraints on the disc cells and a proteoglycan-rich extracellular matrix. We have shown that the oxygen tension of the NP is very low and unlike all other tissues, cells of the NP exhibit constitutive expression of HIF-1a. We advance the new hypothesis that constitutive expression of HIF-1a serves to adapt the NP cells to their avascular environment and that changes in HIF-1 activity is required for their survival and function. The first Specific Aims is to test the hypotheses that high constitutive HIF-1a expression is required for survival of the NP cells in the hypoxic disc; to investigate mechanisms regulating constitutive expression and activity of HIF-1a; using tissue derived from human degenerative discs to correlate NP HIF-1 activity with degeneration and susceptibility to apoptosis. We will transiently and stably suppress HIF-1a and assess the impact of HIF-1a downregulation on cell survival and death. We will then restore HIF-1a expression in suppressed cells and evaluate survival, apoptosis and cell phenotype. We will also determine if changes in expression of pVHL, FIH and PHDs regulate HIF-1a expression and transcriptional activity in NP cells. We will use cells isolated form clinical human disc samples, with varying degree of degeneration, to determine if susceptibility to apoptogen treatment is linked to endogenous HIF-1a activity and to the level of degeneration. The second specific aim is to test the hypothesis that constitutive HIF-1a expression regulates proteoglycan synthesis and GAG chain formation and sulfation; to correlate HIF-1 activity to proteoglycan synthesis in human degenerative disc samples. We will determine the role of constitutive HIF-1a in the expression and synthesis of aggrecan core protein, a molecule that has a HIF-1 responsive promoter element. In addition, we will evaluate the role of HIF-1a in regulating expression of enzymes required for GAG synthesis (glucuronosyltransferase I; GlcAT-I) and chondroitin sulfation (chondroitin-4-O-sulfosyl transferase 2; C4ST-2). We will investigate if proteoglycan synthesis by cells isolated form clinical human disc samples is linked to HIF-1a activity. The last Specific Aim is to develop a rabbit model with suppression of HIF-1a in NP cells using lentiviral HIF-1a-SiRNA; to learn if inactivation of HIF-1a promotes a loss of cellularity, a decrease in proteoglycan synthesis and degeneration of the intervertebral disc. To complement the in vivo studies, and to investigate early cellular events in NP degeneration, we will perform disc organ culture studies using floxed-HIF-1a mouse. Inactivation of the HIF-1a gene in organ cultured discs will be achieved by injecting adenovirus expressing cre recombinase. The information provided by proposed studies will provide insights into mechanisms of early degenerative changes in the NP cells and provide foundation for development of interventional strategies to prevent degenerative disc disease. PUBLIC HEALTH RELEVANCE The incidence of low back pain, which is often linked to degenerative changes in the intervertebral disc, is extraordinary high with annual costs to the US health care industry exceeding 33 billion dollars. None of the current therapies can completely restore the function of the degenerative intervertebral disc and thereby prevent further deterioration of the compromised spine. The proposed studies will provide insights into mechanisms of early degenerative changes in the disc and may permit development of interventional strategies to prevent degenerative disease.