James L R, Ingram A, Ly H, Thai K, Cai L, Scholey J W
Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
Am J Physiol Renal Physiol. 2001 Jul;281(1):F151-62. doi: 10.1152/ajprenal.2001.281.1.F151.
Expression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for glucose entry into the hexosamine pathway, is transcriptionally regulated. Immunohistochemical studies of human kidney biopsies demonstrate increased GFAT expression in diabetic glomeruli, but the mechanism responsible for this overexpression is unknown. Given the role of ANG II in diabetic kidney disease, we chose to study the effect of ANG II on GFAT promoter activity in mesangial cells (MC). Exposure of MC to ANG II (10(-7) M) increased GFAT promoter activity (2.5-fold), mRNA (3-fold), and protein (1.6-fold). ANG II-mediated GFAT promoter activation was inhibited by the ANG II type I receptor antagonist candesartan (10(-8) M) but was unaffected by the ANG II type II receptor antagonist PD-123319 (10(-8) M). The intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (10(-6) M), protein kinase C (PKC) inhibitors bisindoylmaleimide-4 (10(-6) M) and calphostin C (10(-7) M), protein tyrosine kinase (PTK) inhibitor genistein (10(-4) M), Src family kinase inhibitor PP2 (2.5 x 10(-7) M), p42/44 mitogen-activated protein kinase (MAPK) inhibitor PD-98059 (10(-5) M), and the epidermal growth factor (EGF) inhibitor AG-1478 all attenuated GFAT promoter activation by ANG II. We conclude that the GFAT promoter is activated by ANG II via the AT1 receptor. Promoter activation is calcium dependent and PKC dependent but also involves PTK signaling pathways including Src, the EGF receptor, and p42/44 MAPK.
果糖-6-磷酸酰胺转移酶（GFAT）是葡萄糖进入己糖胺途径的限速酶，其表达受转录调控。对人肾活检组织的免疫组织化学研究表明，糖尿病肾小球中GFAT表达增加，但这种过表达的机制尚不清楚。鉴于血管紧张素II（ANG II）在糖尿病肾病中的作用，我们选择研究ANG II对系膜细胞（MC）中GFAT启动子活性的影响。将MC暴露于ANG II（10⁻⁷ M）可使GFAT启动子活性增加（2.5倍）、mRNA增加（3倍）和蛋白质增加（1.6倍）。ANG II介导的GFAT启动子激活被ANG II 1型受体拮抗剂坎地沙坦（10⁻⁸ M）抑制，但不受ANG II 2型受体拮抗剂PD-123319（10⁻⁸ M）影响。细胞内钙螯合剂1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（10⁻⁶ M）、蛋白激酶C（PKC）抑制剂双吲哚马来酰胺-4（10⁻⁶ M）和钙磷蛋白C（10⁻⁷ M）、蛋白酪氨酸激酶（PTK）抑制剂染料木黄酮（10⁻⁴ M）、Src家族激酶抑制剂PP2（2.5×10⁻⁷ M）、p42/44丝裂原活化蛋白激酶（MAPK）抑制剂PD-98059（10⁻⁵ M）以及表皮生长因子（EGF）抑制剂AG-1478均减弱了ANG II对GFAT启动子的激活作用。我们得出结论，ANG II通过AT1受体激活GFAT启动子。启动子激活依赖于钙和PKC，但也涉及包括Src受体、EGF受体和p42/44 MAPK在内的PTK信号通路。
