cGMP-dependent protein kinase and iron metabolism

铁是必需蛋白质正常功能所必需的重要金属离子，也是许多细胞功能的关键元素。铁主要由十二指肠肠上皮细胞吸收，然后通过转铁蛋白在血液中运输，并与铁蛋白结合储存在肝脏、骨髓、红细胞和骨骼肌中。血红蛋白含有人体大约一半的铁。铁的吸收在肠道中受到严格的调节，并受肝脏因子海普西丁的控制。海普西丁抑制存在于巨噬细胞和肠细胞基底外侧的铁出口蛋白铁蛋白。海普西丁的转录受促炎细胞因子如IL-6的刺激，受缺氧、促红细胞生成素和缺铁的抑制。我们已经建立了在SM22a启动子下在平滑肌细胞(救援鼠)中表达cGKIa或cGKIb的小鼠系。对这些小鼠和常规基因敲除小鼠的分析表明，cGKI-/-或cGKI救援小鼠会出现十二指肠溃疡出血、贫血和严重的铁缺乏。铁利用率的降低导致了血红蛋白浓度和红细胞含量的降低。这些动物的血浆网织红细胞和促红细胞生成素水平显著升高，支持了cGKI基因修饰的小鼠患有缺铁性贫血的观点。我们无法通过肌肉注射来挽救救援动物的表型。注入铁。CGKI-/-小鼠表现出脾内红细胞凋亡增强。然而，脾中红细胞的过早凋亡不能解释脾中铁含量非常低的原因，因为红细胞在巨噬细胞中降解，巨噬细胞储存在血红蛋白降解过程中释放的铁，并为血红蛋白的重新合成提供铁。与这些结果一致的是，未经治疗的cGKI救治小鼠的脾转铁蛋白受体和二价金属离子转运体的mRNA表达增加，表明缺铁是通过脾的网状内皮细胞感受到的。正如预期的那样，未经治疗的cGKI小鼠脾中铁储存蛋白铁蛋白降低到几乎为零的水平，并且只有在注射铁的情况下略有增加。初步数据显示，cGKI-/-和cGKI救援小鼠的贫血是由脾中严重的铁缺乏引起的。口头或即时消息。补充铁既不能恢复脾的铁缺乏，也不能恢复贫血。非常初步的数据甚至表明，对患有PPI的动物进行治疗，显著延长了cGKI-/-小鼠的子集的寿命，并不能改变贫血。本项目的目的是了解cGKI在何种水平上影响铁的代谢，因为到目前为止还没有报道铁代谢和cGKI之间的相互作用。

Iron is a vital metal ion required for the proper function of essential proteins and a critical element in many cellular functions. Iron is resorbed mostly by the duodenal enterocytes, is then transported in the blood by transferrin, and stored bound to ferritin in liver, bone marrow, erythroblasts, and skeletal muscle. Hemoglobin contains about half of the bodys iron. Iron absorption is tightly regulated in the intestine and controlled by the hepatic factor hepcidin. Hepcidin inhibits the iron exporter ferroportin present in macrophages and at the basolateral side of the enterocyte. The transcription of hepcidin is stimulated by proinflammatory cytokines such as IL-6, and inhibited by hypoxia, erythropoietin, and lack of iron. We have generated mouse lines that express cGKIa or cGKIb under the SM22a promoter in smooth muscle cells (rescue mice). Analysis of these mice and conventional knock-out mice revealed that cGKI-/- or cGKI rescue mice develop a bleeding ulcus duodeni, anemia and a severe deficit of iron. The decreased iron availability resulted in a decreased hemoglobin concentration and erythrocyte content. The same animals had significantly elevated plasma levels of reticulocytes and erythropoietin supporting the notion that the cGKI modified mice had an iron-deficiency anemia. We were unable to rescue the phenotype in the rescue animals by i.m. injection of iron. cGKI-/- mice show enhanced apoptosis of erythrocytes in the spleen. However, premature apoptosis of the erythrocytes in the spleen would not explain the very low iron content of the spleen, because erythrocytes are degraded in macrophages, which store the iron released during hemoglobin degradation and provide it for the resynthesis of hemoglobin. In agreement with these results, untreated cGKI rescue mice had elevated mRNA expression of the transferrin receptor and of the divalent metal ion transporter in the spleen suggesting the iron deficiency is sensed by the reticuloendothelial cells of the spleen. As expected, the iron storage protein ferritin is reduced to nearly zero level in the spleen of untreated cGKI mouse lines and does increase only slightly with the injection of iron. These results are indicative of an iron absorption/storage deficit of the spleen macrophages.Preliminary data show that cGKI-/- and the cGKI rescue mice have anemia caused by a severe iron deficit in the spleen. Oral or i.m. supplementation of iron has not restored the spleen iron deficit nor the anemia. Very preliminary data even suggest that treatment of the animals with PPIs that prolongs dramatically the live span of a subset of the cGKI-/- mice does not change the anemia. Goal of this project is to understand at which level cGKI affects the iron metabolism, because so far no interaction between iron metabolism and cGKI has been reported.