Signaling Mechanisms Regulation Muscle Protein Turnover

肌肉蛋白的损失已被证明是由于泛素-蛋白酶体蛋白水解系统的激活，在患者和实验大鼠的分解代谢条件，如肾衰竭，败血症，糖尿病，在每种情况下，糖皮质激素的产量增加。我们的小组是第一个证明糖皮质激素是增加蛋白质降解和编码该系统成分的mrna水平所必需的。我们使用核连续试验表明，这种途径mrna的增加是由于糖尿病或肾功能不全大鼠肌肉中转录增加所致。定义转录反应的机制是很重要的，因为一些报告表明，特定途径蛋白（例如，蛋白酶体C2亚基，泛素）的水平与蛋白质降解率密切相关或决定蛋白质降解率。我们已经确定了糖皮质激素的作用、两种新的细胞信号通路的激活以及调节肌肉中C3蛋白酶体亚基和泛素基因表达的转录因子之间的联系：1)糖皮质激素通过拮抗NF-kB的机制上调蛋白酶体C3亚基的表达（我们之前发现NF-kB抑制肌肉细胞中C3亚基的转录）。我们的初步结果表明，这种糖皮质激素依赖性拮抗剂是通过抑制激酶（IkB激酶或IKK）来实现的，该激酶会启动IkB （NF-kB易位到细胞核的抑制剂）的破坏。这种反应导致细胞质中NF-kB的隔离，缓解C3亚基转录的抑制。也有初步结果表明血清和糖皮质激素调节激酶（SGK）参与调节NF-kB的活化。我们的假设是激活的SGK刺激IKK活性导致IkB的降解，糖皮质激素通过阻止SGK的激活来抑制IKK。2)其他初步结果提示糖皮质激素诱导的泛素转录增加涉及Sp1和MAP激酶途径。我们的假设是糖皮质激素激活ERK1/2，然后使Sp1磷酸化，增加Sp1与泛素启动子的结合。我们检验这些假设的策略将是确定糖皮质激素是否：1)抑制SGK活性；2)激活ERK MAPK并增加Sp1磷酸化。接下来，我们将确定调节SGK或MEK1 （ERK1/2的上游激活因子）是否会改变L6肌肉细胞中蛋白质降解和泛素-蛋白酶体途径基因的表达。最后，我们将对肾上腺切除的大鼠进行研究，以确定糖皮质激素是否与骨骼肌中SGK活性的抑制或MEK/ERK MAPK通路的激活有关。

Loss of muscle protein has been shown to be due to activation of the ubiquitin-proteasome proteolytic system in both patients and experimental rats with catabolic conditions like kidney failure, sepsis, diabetes and in each case, glucocorticoid production is increased. Our group was the first to demonstrate that glucocorticoids are necessary for the increase in protein degradation and levels of mRNAs encoding components of this system. We used nuclear run-on assays to show that this increase in pathway mRNAs results from increased transcription in muscle of rats with diabetes or renal insufficiency. It is important to define the mechanisms for the transcriptional responses because several reports indicate that the level of specific pathway proteins (e.g., proteasome C2 subunit, ubiquitin) is closely linked to or determines the rate of protein degradation. We have identified links among glucocorticoid action, activation of two novel cellular signaling pathways, and transcription factors that regulate the expression of genes for the C3 proteasome subunit and ubiquitin in muscle: 1) glucocorticoids upregulate the expression of the proteasome C3 subunit by a mechanism that antagonizes NF-kB (we showed earlier that NF-kB suppresses transcription of the C3 subunit in muscle cells). Our Preliminary Results indicate that this glucocorticoid-dependent antagonism is achieved through inhibition of the kinase (IkB kinase or IKK) that initiates the destruction of IkB (the inhibitor of NF-kB translocation to the nucleus). This response results in sequestration of NF-kB in the cytosol, relieving suppression of C3 subunit transcription. There also are Preliminary Results that the serum- and glucocorticoid-regulated kinase (SGK) is involved in regulating NF-kB activation. Our hypothesis is that activated SGK stimulates IKK activity leading to degradation of IkB and that glucocorticoids inhibit IKK by preventing the activation of SGK. 2) Other Preliminary Results indicate the increase in ubiquitin transcription induced by glucocorticoids involves Sp1 and the MAP kinase pathway. Our hypothesis is that glucocorticoids activate ERK1/2 which then phosphorylates Sp1, increasing Sp1 binding to the ubiquitin promoter. Our strategy for examining these hypothesis will be to determine if glucocorticoids: 1) inhibit SGK activity; and 2) activate the ERK MAPK and increase Sp1 phosphorylation. Next, we will determine if regulating SGK or MEK1 (the upstream activator of ERK1/2) will change protein degradation and expression of ubiquitin-proteasome pathway genes in L6 muscle cells. Lastly, we will study adrenalectomized rats to determine if glucocorticoids can be linked to inhibition of SGK activity or activation of the MEK/ERK MAPK pathway in skeletal muscle.