Stepan Vinzenz, Ramamoorthy Saravanan, Nitsche Hildegard, Zavros Yana, Merchant Juanita L, Todisco Andrea
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
J Biol Chem. 2005 Apr 22;280(16):15700-8. doi: 10.1074/jbc.M413037200. Epub 2005 Feb 3.
Shh (Sonic hedgehog) regulates gastric epithelial cell differentiation. We reported that incubation of purified canine parietal cells with epidermal growth factor (EGF) for 6-16 h, stimulates H(+)/K(+)-ATPase alpha-subunit gene expression through the activation of Akt. We explored if Shh mediates some of the actions of EGF in the parietal cells. EGF induced a 6-fold increase in Shh expression, measured by Western blots, after 5 h of incubation. This effect was inhibited by both the phosphatidylinositol 3-kinase inhibitor LY294002 and by transduction of the cells with an adenoviral vector expressing dominant negative Akt. EGF stimulated the release of Shh-like immunoreactivity from the parietal cells, after 16 h of incubation. Shh induced H(+)/K(+)-ATPase alpha-subunit gene expression, assessed by Northern blots, it stimulated a luciferase reporter plasmid containing the EGF-responsive sequence (ERE) of the canine H(+)/K(+)-ATPase alpha-subunit gene promoter, and it induced parietal cell nuclear protein binding to the ERE. Gli transcription factors mediate the intracellular actions of Shh. Co-transfection of the parietal cells with the H(+)/K(+)-luc plasmid together with one expressing Gli2, induced H(+)/K(+)-luciferase activity 5-fold, whereas co-transfection of the cells with the H(+)/K(+)-luc plasmid together with one expressing dominant negative Gli2, inhibited EGF induction of H(+)/K(+)-luciferase activity. Identical results were observed in the presence of the Shh signal transduction pathway inhibitor, cyclopamine. Transfection of the cells with dominant negative Akt inhibited EGF, but not Shh stimulation of H(+)/K(+)-ATPase-luciferase activity. Thus, EGF but not Shh signals through Akt. Preincubation of the cells for 16 h with either Shh or EGF enhanced histamine-stimulated [(14)C]aminopyrine uptake by 50%. In conclusions, some of the actions of EGF in the parietal cells are mediated by the sequential activation of the Akt and the Shh signal transduction pathways. These effects might represent novel mechanisms mediating the actions of growth factors on gastric epithelial cell differentiation.
音猬因子（Shh）调控胃上皮细胞分化。我们曾报道，将纯化的犬壁细胞与表皮生长因子（EGF）孵育6 - 16小时，可通过激活Akt刺激H⁺/K⁺ - ATP酶α亚基基因表达。我们探究了Shh是否介导EGF在壁细胞中的某些作用。孵育5小时后，通过蛋白质免疫印迹法检测发现，EGF诱导Shh表达增加了6倍。磷脂酰肌醇3激酶抑制剂LY294002以及用表达显性负性Akt的腺病毒载体转导细胞均能抑制这一效应。孵育16小时后，EGF刺激壁细胞释放Shh样免疫反应性物质。通过Northern印迹法评估，Shh诱导H⁺/K⁺ - ATP酶α亚基基因表达，它刺激了一个含有犬H⁺/K⁺ - ATP酶α亚基基因启动子的表皮生长因子反应序列（ERE）的荧光素酶报告质粒，并且它诱导壁细胞核蛋白与ERE结合。Gli转录因子介导Shh的细胞内作用。将壁细胞与H⁺/K⁺ - luc质粒共转染，同时转染一个表达Gli2的质粒，可诱导H⁺/K⁺ - 荧光素酶活性增加5倍，而将壁细胞与H⁺/K⁺ - luc质粒共转染，同时转染一个表达显性负性Gli2的质粒，则抑制EGF诱导的H⁺/K⁺ - 荧光素酶活性。在存在Shh信号转导通路抑制剂环杷明的情况下观察到相同结果。用显性负性Akt转染细胞可抑制EGF，但不抑制Shh对H⁺/K⁺ - ATP酶 - 荧光素酶活性的刺激。因此，EGF通过Akt信号转导，而Shh不通过。用Shh或EGF对细胞预孵育16小时，均可使组胺刺激的[¹⁴C]氨基比林摄取增加50%。总之，EGF在壁细胞中的某些作用是由Akt和Shh信号转导通路的顺序激活介导的。这些效应可能代表了生长因子对胃上皮细胞分化作用的新机制。
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