Histochemical localization of heme oxygenase-2 protein and mRNA expression in rat brain
Histochemical localization of heme oxygenase-2 protein and mRNA expression in rat brain
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DOI：
10.1016/s1385-299x(96)00027-x
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发表时间：
1997-05-01
期刊：
影响因子：
--
通讯作者：
Maines, MD
中科院分区：
文献类型：
--
作者：
Ewing, JF;Maines, MD
血红素加氧酶(HO)蛋白是HSP30家族的成员，由两种同工酶组成，分别命名为HO-1和HO-2[1，2]。不同的基因编码同工酶[3]和蛋白质产物，它们在免疫化学上是不同的，在氨基酸序列水平上的相似性不到50%。然而，每一种形式在物种之间都显示出超过90%的相似性，包括人和老鼠(参见参考文献)。[4]))。此外，这些同工酶发挥着明确的作用，与NADPH-细胞色素P450还原酶协同执行血红素分子(Fe-原卟啉IX)的氧化。血红素的氧化是异构体特有的，并导致胆色素、一氧化碳和铁的形成。血红素分子由血红蛋白、肌红蛋白、过氧化氢酶、可溶性鸟苷环化酶、细胞色素b(5)、细胞色素P450和一氧化氮合酶组成。HO-1也称为热休克蛋白(HSP)32，由一种对应激反应敏感的基因编码，在体内[4-7]和体外[8，9]，一系列介导氧化应激的刺激物都能诱导蛋白质的表达。HO-2形式显示出与HO-1形式不同的独特的调控模式。HO-2是一种构成蛋白，其表达不受迄今为止测试的HO-1诱导剂的影响[10]；相反，唯一已知的HO-2调节因子是肾上腺糖皮质激素[11]。这两种同工酶在组织分布上表现出巨大的差异，在正常情况下，HO-1存在于整个大脑中，达到免疫检测的极限[6，12]，并谨慎地定位于选定的神经元群体[13，14]。然而，HO-1蛋白(类似于32 kDa)及其类似于1.8kb的转录本增加[6，13-16]，主要是在非神经细胞群体中对应激刺激的反应。在前一种能力中，它下调了细胞内的血红素和血红素蛋白水平。因此，它会使形成自由基的最有效催化剂--血红素分子失活[17，18]。如上所述，在其合成代谢作用中，血红素加氧酶产生胆色素、一氧化碳和铁，所有这些都具有生物活性：胆色素起抗氧化剂的作用[19]；HO活性产生的一氧化碳与cGMP的产生相关[7，20-24]；铁调节各种基因的表达，包括HO-1本身[25]，以及转铁蛋白受体、铁蛋白和一氧化氮合酶[26-28]。我们使用兔抗大鼠HO-2多克隆抗体和HO-2 cDNA分别定位HO-2免疫活性蛋白和1.3(-)和1.9kb的同源转录本，通过组织化学染色方法显示。这些协议首次详细描述了成功用于在成年大鼠大脑[14，29]和糖皮质激素治疗的新生大鼠[30]的转录和翻译水平上定义HO-2表达模式的方法。这里描述的程序具有非放射性的优点，并且适用于全身器官，如心血管系统[7，23]和男性生殖器官[31-33]。细胞HO-2表达的可视化有助于评估神经系统内一氧化碳、铁和胆红素的潜在产生部位。
Heme oxygenase (HO) proteins are members of the HSP30 family and consist of 2 isozymes identified to date, termed HO-1 and HO-2 [1,2]. Separate genes encode the isozymes [3] and protein products which are immunochemically distinct, share less than 50% similarity at the amino acid sequence level. Each form, however, shows greater than 90% similarity among species, including human and the rat (reviewed in ref. [4]). Furthermore, these isozymes function in a well-defined role to carry out oxidation of the heme molecule (Fe-protoporphyrin IX) in concert with NADPH-cytochrome P450 reductase. The oxidation of heme is isomer specific and results in the formation of bile pigments, carbon monoxide, and iron. The heme molecule constitutes the prosthetic moiety of hemoproteins, such as hemoglobin, myoglobin, catalase, soluble guanylate cyclase, cytochrome b(5), cytochromes P450 and NO synthase.HO-1 also known as heat shock protein (HSP) 32 is encoded by a gene which is exquisitely stress-responsive and a host of stimuli that mediate oxidative stress cause induction of the protein both in vivo [4-7] and in vitro [8,9]. The HO-2 form shows a unique pattern of regulation from that of HO-1. HO-2 is a constitutive protein and its expression is not affected by the inducers of HO-1 tested to date [10]; rather, the only known regulator of HO-2 yet identified is adrenal glucocorticoids [11]. The two isozymes display vast differences in tissue distribution and under normal conditions HO-1 is present in the whole brain at the limit of immunodetection [6,12] and is discreetly localized in select neuronal populations [13,14]. HO-1 protein (similar to 32 kDa) and its similar to 1.8 kb transcript are increased [6,13-16], however, in response to stressful stimuli primarily in non-neuronal cell populations.The heme oxygenase system serves in both a catabolic and anabolic capacity in the cell. In the former capacity, it down-regulates cellular heme and hemoprotein levels. And, as such it inactivates the most effective catalyst for formation of free radicals, the heme molecule [17,18]. In its anabolic role, as noted above, heme oxygenase produces bile pigments, carbon monoxide, and iron, all of which are biologically active: bile pigments function as antioxidants [19]; the carbon monoxide generated by HO activity has been correlated with the generation of cGMP [7,20-24]; and iron regulates expression of various genes, including that of HO-1 itself [25], as well as transferrin receptors, ferritin, and NO synthase [26-28].We used rabbit anti-rat HO-2 polyclonal antibody and HO-2 cDNA to localize HO-2 immunoreactive protein and the 1.3(-) and 1.9 kb homologous transcripts, respectively, in rodent brain as visualized by histochemical staining procedures. These protocols provide the first detailed description of methodologies successfully used to define the pattern of HO-2 expression at the transcriptional and translational levels in the adult rat brain [14,29] and glucocorticoid-treated newborn rats [30]. The procedures described herein have the virtue of being non-radioactive, as well as applicability to the systemic organs, such as the cardiovascular system [7,23] and the male reproductive organs [31-33]. Visualization of cellular HO-2 expression aids in assessment of potential sites of carbon monoxide, iron, and bilirubin production within the nervous system.