Normoxic Expression of Hypoxia-Inducible Factor 1 in Rat Leydig Cells In Vivo and In Vitro
Normoxic Expression of Hypoxia-Inducible Factor 1 in Rat Leydig Cells In Vivo and In Vitro
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DOI：
10.2164/jandrol.110.011494
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发表时间：
2011-05-01
影响因子：
--
通讯作者：
Hermo, L.
中科院分区：
文献类型：
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作者：
Palladino, M. A.;Pirlamarla, P. R.;Hermo, L.
缺氧诱导因子（Hypoxia-inducible factors，HIF）是一类在细胞和分子对氧债反应中起重要调节作用的转录因子。HIF由缺氧依赖性α亚基（1 α，2 α，3 α）和氧非依赖性β亚基组成。以前，我们证明了HIF-1，缺氧反应的主调节器，在成年大鼠睾丸中表达。我们假设HIF-1参与调节睾丸对氧张力的反应。本研究的目的是确定HIF-2 α和HIF-3 α是否在大鼠睾丸中表达，鉴定表达HIF-1 α的睾丸细胞类型，并检查体内和体外缺血和缺氧条件下睾丸HIF-1 α蛋白表达的模式。逆转录聚合酶链反应显示Hif-1 α、Hif-2 α和Hif-3 α的mRNA在睾丸中表达。HIF-1 α蛋白是睾丸中的主要亚基。HIF-1 α蛋白在含氧量正常的睾丸中含量丰富，并且在手术诱导的睾丸扭转和再灌注造成的缺血后其水平保持不变。免疫印迹和免疫细胞化学实验表明，Leydig细胞是低氧和常氧睾丸中HIF-1 α的主要来源。为了研究睾丸HIF-1稳定化的潜在机制，通过免疫印迹法分析了在5%或21%氧气中培养的Leydig细胞或用H2 O2培养的细胞的核蛋白。与新鲜分离的细胞相比，在5%或21%氧培养物中HIF-1 α a的水平显著降低。用H2 O2作为活性氧的来源处理Leydig细胞不影响HIF-1 α水平。高水平的组成型表达HIF-1 α在常氧Leydig细胞表明潜在的独特作用，HIF-1在Leydig细胞对氧的反应。
Hypoxia-inducible factors (HIF) are transcription factors that serve essential regulatory roles in cellular and molecular responses to oxygen debt. HIFs are composed of hypoxia-dependent alpha subunits (1 alpha, 2 alpha, 3 alpha) and an oxygen-independent beta subunit. Previously we demonstrated that HIF-1, the master regulator of hypoxic responses, is expressed in the adult rat testis. We hypothesized that HIF-1 is involved in regulating responses to oxygen tension in the testis. Goals of this study were to determine if HIF-2 alpha and HIF-3 alpha are expressed in rat testis, identify testis cell types that express HIF-1 alpha, and examine patterns of testicular HIF-1 alpha protein expression under conditions of ischemia and hypoxia in vivo and in vitro. Reverse transcriptase polymerase chain reaction revealed that mRNA for Hif-1 alpha, Hif-2 alpha, and Hif-3 alpha is expressed in the testis. The HIF-1 alpha protein is the predominant subunit in testis. HIF-1 alpha protein was abundant in normoxic testis, and its levels remained unchanged following ischemia created by surgically induced testicular torsion and reperfusion. Immunoblot and immunocytochemical experiments demonstrated that Leydig cells are the major source of HIF-1 alpha in normoxic and hypoxic testes. To examine potential mechanisms of testicular HIF-1 stabilization, nuclear proteins from Leydig cells cultured in 5% or 21% oxygen, or cells cultured with H2O2, were analyzed by immunoblotting. Levels of HIF-1 alpha a were significantly diminished in 5% or 21% oxygen cultures compared with freshly isolated cells. Treating Leydig cells with H2O2 as a source of reactive oxygen species did not affect HIF-1 alpha levels. High levels of constitutively expressed HIF-1 alpha in normoxic Leydig cells suggest potentially unique roles for HIF-1 in Leydig cell responsiveness to oxygen.