Role of Cholesterol in Age-related Decline in Steroidogenesis

描述（由申请人提供）： 人类和实验动物的年龄增长与类固醇激素合成和分泌的深刻变化有关。这个问题的原因似乎是衰老大鼠模型的肾上腺皮质细胞或Leydig细胞不能有效地将胆固醇转运到细胞的线粒体内膜，在那里发生类固醇激素产生的最初步骤。虽然，控制这种衰老缺陷的各种细胞和分子机制尚未明确确定，但有相当多的证据表明， 实验室指出，过度的自由基形成和氧化损伤（特别是来自终身持续加工胆固醇以产生类固醇）对调节关键蛋白质星星、PBR/TSPO和参与胆固醇转运到线粒体内膜的潜在其他StAR相关（StarD）蛋白质的功能表达的细胞机制的影响。本提案中概述的研究将进一步探讨这些问题，并将具体测试 假设增加的ROS形成和随后的氧化损伤导致衰老动物的类固醇生成组织模型和增加的氧化应激的遗传小鼠模型中的固醇转移蛋白、星星和PBR/TSPO蛋白的表达变化，并且在下游，这导致较少的胆固醇转移到胆固醇侧链裂解发生的线粒体内膜位点，并且第一类固醇，已形成双烯醇酮。我们还测试了第二个假设，即使用小分子量合成抗氧化剂，降低氧化损伤程度的药物干预，将防止，逆转，或减弱与年龄相关的和氧化应激依赖的类固醇生成反应的损失。为了解决这些假设，提出了三个具体目标。目的1：建立一种新的甾体激素合成障碍的衰老模型SAMP 8小鼠。使用肾上腺素（或肾上腺皮质细胞）和睾丸间质细胞（和对照SAMR 1小鼠），我们将测量：a）类固醇生成的激素调节、从细胞内储存的胆固醇的利用、胆固醇诱导的胆固醇向线粒体的转运以及星星和PBR/TSPO的表达; B）细胞抗氧化剂水平、氧化损伤和抗氧化剂状态;和c）ASK 1- p38 MAPK信号级联的改变。目的2是确定氧化应激在年龄相关的肾上腺和睾丸类固醇激素产生下降中的因果作用。进行的第一组研究将包括监测体外类固醇生成的变化，星星、PBR/TSPO和StarD蛋白的mRNA表达，氧化剂和氧化损伤标志物的水平，以及胆固醇诱导的胆固醇转运到肾上腺线粒体的动力学。（肾上腺皮质细胞）和Leydig细胞分离自氧化应激倾向的A/T-Mn-SOD-/-（或Mn-SOD+/-）、GPX 1-/-和Cu，Zn-SOD-/-小鼠。第二组研究将集中于使用从抗氧化应激的Cu、Zn-SODTg、Mn-SODTg和GPX 1 Tg小鼠中分离的肾上腺细胞和Leydig细胞测量这些各种参数的变化。最后一组研究将检查两种抗氧化酶同时过表达对氧化应激诱导的类固醇生成抑制和胆固醇转运至线粒体内膜进行侧链裂解受损的影响。目的3是采用药理学策略来逆转或预防衰老诱导的和氧化应激相关的类固醇合成损失。本节中提出的研究将评估SAMR-1、SAMP-8、A/T-Mn-SOD-/-（或Mn-SOD+/-）、GPX-1-/-和Cu，Zn-SOD-/-，以及对照小鼠与各种低分子量抗氧化剂（NAC、CTMIO、tempol、MnTBAP、EUK-189、依布硒啉）或p38 MAPK抑制剂（SC-409，SD-169; p38 MAPK与氧化应激介导的类固醇生成抑制有关）：a）类固醇生成; B）星星、PBR/TSPO和StarD蛋白的mRNA表达; c）胆固醇诱导的胆固醇递送至线粒体和与线粒体一起递送; d）ASK 1-p38 MAPK信号传导和e）关键氧化应激标志物的水平，使用肾上腺（肾上腺皮质细胞）和睾丸Leydig细胞。

DESCRIPTION (provided by applicant): Advancing age in human and experimental animals is associated with a profound change in the synthesis and secretion of steroid hormones. It appears that the cause of this problem is the inability of adrenocortical cells or Leydig cells of the aging rat model to effectively transport cholesterol to the cell's inner mitochondrial membrrane where the initial steps in steroid hormone production take place. Although, the various cellular and molecular mechanisms controlling this aging defect have not been definitely identified, considerable evidence from this laboratory points to excessive free radical formation and oxidative damage (especially from life-long continued processing of cholesterol for steroid production) to the cell machinery regulating the functional expression of crucial proteins, StAR, PBR/TSPO and potentially other StAR-related (StarD) proteins involved in cholesterol transport to inner mitochondrial membrane. The studies outlined in this proposal will explore these issues further and will specifically test the hypothesis that increased ROS formation and ensuing oxidative damage leads to changes in expression of sterol transfer proteins, StAR, and PBR/TSPO proteins in steroidogenic tissues model of aging animals and genetic mouse models of increased oxidative stress, and downstream, this leads to the transfer of less cholesterol to the inner mitochondrial membrane sites where cholesterol side chain cleavage takes place, and the first steroid, pregnenolone is formed. We also test a second hypothesis that pharmacological intervention with the use of small molecular weight synthetic antioxidants that reduce the degree of oxidative damage will prevent, reverse, or attenuate the age-related and oxidative stress dependent loss of steroidogenic response. To address these hypotheses, three Specific Aims are proposed. Aim 1 is to establish Senescence-Accelerated-Mouse-Prone 8 (SAMP8) mice as a new aging model of impaired steroidogenesis. Using adrenal (or adrenocortical cells) and testicular Leydig cells from aging SAMP8 mice (and control SAMR1 mice) we will measure: a) hormonal regulation of steroidogensis, utilization of cholesterol from intracellular stores, hormone-induced cholesterol transport to mitochondria and expression of StAR and PBR/TSPO; b) cellular antioxidant levels, oxidative damage and antioxidant status; and c) alteration in the ASK1- p38 MAPK signaling cascade. Aim 2 is to establish the causal role of oxidative stress in age-related decline in adrenal and testicular steroid hormone production. The first set of studies to be conducted will include monitoring changes in in vitro steroidogenesis, mRNA expression of StAR, PBR/TSPO and StarD proteins, levels of oxidants and markers of oxidative damage and the kinetics of hormone-induced cholesterol transport to mitochondria in adrenal (adrenocortical cells) and Leydig cells isolated from oxidative stress-prone A/T-Mn-SOD-/- (or Mn-SOD+/-), GPX1-/- and Cu,Zn-SOD-/- mice. The second of set studies will focus on measuring changes in these various parameters using adrenal cells and Leydig cells isolated from oxidative stress resistant Cu,Zn-SODTg, Mn-SODTg, and GPX1Tg mice. The final set of studies will examine the impact of simultaneous over-expression of the two antioxidant enzymes on oxidative stress-induced inhibition of steroidogenesis and impaired cholesterol transport to the inner mitochondrial membrane for side-chain cleavage. Aim 3 is to employ pharmacological strategies to reverse or prevent aging-induced and oxidative stress-associated loss of steroidogenesis. Studies proposed in this section will evaluate the effect of treatment of SAMR-1, SAMP-8, A/T-Mn-SOD-/- (or Mn-SOD+/-), GPX-1-/-, and Cu,Zn-SOD-/-, and control mice, with various low molecular weight antioxidants (NAC, CTMIO, tempol, MnTBAP, EUK-189, ebselen) or p38 MAPK inhibitors (SC-409, SD-169; p38 MAPK is implicated in oxidative stress-mediated inhibition of steroidogenesis) on: a) steroidogenesis; b) mRNA expression of StAR, PBR/TSPO and StarD proteins; c) hormone-induced cholesterol delivery to and with the mitochondria; d) ASK1-p38 MAPK signaling and e) levels of key oxidative stress markers, using adrenal (adrenocortical cells) and testicular Leydig cells.