Analysis of molecular mechanisms by which heme oxygenase-carbon monoxide pathway regulates cellular functions under physiological and pathophysiological conditions using newly developed gene targeting mice.

血红素加氧酶(HO)是一种能将原血红素IX降解为一氧化碳(CO)、游离二价铁和胆绿素IXα的酶，在许多细胞功能的调节中起着重要作用。然而，HO依赖的生物学功能的详细分子机制仍然不清楚。在本研究中，我们旨在建立新的HO基因打靶小鼠，以解决同工酶的特异性作用，寻找内源性CO的新靶点，并检测CO和其他气体分子如一氧化氮(NO)在体内对相同靶分子的影响。为了避免HO-1基因失活导致的胚胎死亡和不育，我们构建了一个新的靶向载体，该基因的外显子2两侧带有loxP序列，可以在体内被Cre重组酶以组织特异性的方式删除，以产生条件性HO-1基因敲除小鼠。到目前为止，已经获得了两个不同的ES细胞克隆，其中发生了适当的同源重组，嵌合小鼠…为了寻找新的CO靶向分子，我们用CE-MS检测了醋氨酚(AAP)处理后小鼠肝脏代谢产物的变化，通过这种变化，肝内HO活性和随后的CO产生增加。我们发现，经AAP治疗后，血清S-腺苷蛋氨酸、半胱氨酸、谷胱甘肽和牛磺酸水平显著降低。另一方面，甲硫氨酸在病变肝脏中大量积聚。这些结果表明，CO可能通过结合和抑制进入硫磺途径的限速酶-胱硫醚β-合成酶来抑制进入该途径的通量。接下来，我们研究了CO如何在体外使用部分纯化的酶来抑制CBS的活性。我们的发现表明，毫摩尔水平的CO可以裂解血红素-半胱氨酸键的铁，形成羧基-血红素络合物，从而抑制酶的活性。为了阐明CO和NO在体内调节可溶性鸟苷环化酶(SGC)的作用，我们进行了免疫组织化学分析，分别通过抑制内源这些气体分子来显示气体介导的sGC激活。在一氧化碳被抑制的条件下，所有视网膜细胞层均表现出NO依赖的sGC激活增强。另一方面，在无抑制条件下，去除CO以一种特定的方式取消了基础sGC的激活。这些结果表明，内源性CO通过抑制sGC功能的最大反应和适度上调视网膜特定层的最小反应，在微调sGC功能依赖的激活中发挥作用。较少

Heme oxygenase (HO), an enzyme capable to degrade protoheme IX into carbon monoxide (CO), free divalent iron, and biliverdin-IXα, plays important roles in the regulation of many cellular functions. However, the detailed molecular mechanisms of HO-dependent biological functions remain elusive. In this study, we aimed to establish new HO gene-targeting mice to address isozyme-specific roles, to find new targets of endogenous CO, and to examine effects of CO and other gaseous molecules such as nitric oxide (NO) on the same targeting molecules in vivo.To circumvent embryonic lethality and infertility caused by an inactivation of the HO-1 gene, we constructed a new targeting vector containing exon2 of the gene flanked by loxP sequences, which can be deleted by Cre recombinase in a tissue-specific manner in vivo, in order to generate conditional HO-1 knockout mice. Two distinct ES cell clones, in which an appropriate homologous recombination occurred, were obtained so far, and chimeric mice … More have been generated.To find novel targeting molecules of CO, we examined alterations of metabolites in mouse liver treated with acetaminophen (AAP), by which intrahepatic HO activity and consequent CO production were increased, using CE-MS. We found that the levels of S-adenosylmethionine, cysteine, glutathione, and taurine were significantly reduced by AAP treatment. On the other hand, great accumulation of methionine occurred in the affected liver. These results suggest that CO might suppress the flux into a transsulfuration pathway by binding to and inhibiting cystathionine β-synthase (CBS), a rate-limiting enzyme of the pathway. We next examined how CO inhibits CBS activity using the partially purified enzyme in vitro. Our findings showed that mili molar levels of CO could cleave the Fe of heme-cysteine bond and form a carboxy-heme complex, resulting in an inhibition of the enzyme activity.To clarify the roles of CO and NO to regulate soluble guanylate cyclase (sGC) in vivo, we carried out immunohistochemical analyses to visualize the gas-mediated sGC activation by suppressing endogenous these gaseous molecules separately. Under conditions where CO was suppressed by ZnPP, all retinal cell layers homogenously exhibited enhancement of NO-dependent activation of sGC. On the other hand, under NO-suppressing conditions, eliminating CO abrogated the basal sGC activation in a layer-specific manner. These results suggest that endogenous CO plays a role in fine tuning the NO-dependent activation of sGC function by suppressing the maximum response as well as by modestly elevating the minimum response in particular layer of retina. Less