de Boland A R, Norman A W
Departamento de Biologia, Bioquimica, y Farmacia, Universidad Nacional del Sur, Bahia Blanca, Argentina.
J Cell Biochem. 1998 Jun 15;69(4):470-82. doi: 10.1002/(sici)1097-4644(19980615)69:4<470::aid-jcb8>3.0.co;2-k.
The steroid hormone 1alpha,25(OH)2-vitamin D3 (1alpha,25(OH)2D3) generates biological responses in intestinal and other cells via both genomic and rapid, nongenomic signal transduction pathways. We examined the hypothesis that 1alpha,25(OH)2D3 action in chick enterocytes may be linked to pathways involving tyrosine phosphorylation. Brief exposure of isolated chick enterocytes to 1alpha,25(OH)2D3 demonstrated increased tyrosine phosphorylation of several cellular proteins (antiphosphotyrosine immunoblots of whole cell lysates) with prominent bands at 42-44, 55-60, and 105-120 Kda. The 42-44 Kda bands comigrated with mitogen-activated protein (MAP) kinase (immunoblotting with anti-MAP kinase antibody) The response occurred within 30 s, peaked at 1 min, and was dose-dependent (0.01-10 nM), with maximal stimulation at 1 nM (three- to fivefold). This effect was specific for 1alpha,25(OH)2D3 since its metabolic precursors 25(OH)D3 and vitamin D3 did not increase MAP kinase tyrosine phosphorylation. The tyrosine kinase inhibitor, genistein, blocked 1alpha,25(OH)2D3-induced tyrosine phosphorylation of MAP kinase, while staurosporine, a PKC inhibitor, attenuated the hormone's effects by 30%. We have evaluated the ability of 1alpha,25(OH)2D3 analogs, which have complete flexibility around the 6,7 carbon-carbon bond (6F) or which are locked in either the 6-s-cis (6C) or the 6-s-trans (6T) shape(s), to activate MAP kinase. Thus, two 6F and one 6C analog stimulated while one 6T analog did not stimulate MAP kinase tyrosine phosphorylation. In addition, 1beta,25(OH)2D3, a known antagonist of 1alpha,25(OH)2D3-mediated rapid responses, blocked the hormone effects on MAP kinase. We conclude that 1alpha,25(OH)2D3 and analogs which can achieve the 6-s-cis shape (6F and 6C) can increase tyrosine phosphorylation and activation of MAP kinase in chick enterocytes.
类固醇激素1α,25(OH)2 - 维生素D3（1α,25(OH)2D3）通过基因组和快速、非基因组信号转导途径在肠道及其他细胞中产生生物学反应。我们检验了这样一个假设，即1α,25(OH)2D3在鸡小肠上皮细胞中的作用可能与涉及酪氨酸磷酸化的途径有关。将分离的鸡小肠上皮细胞短暂暴露于1α,25(OH)2D3，结果显示几种细胞蛋白的酪氨酸磷酸化增加（全细胞裂解物的抗磷酸酪氨酸免疫印迹），在42 - 44 kDa、55 - 60 kDa和105 - 120 kDa处有明显条带。42 - 44 kDa的条带与丝裂原活化蛋白（MAP）激酶共迁移（用抗MAP激酶抗体进行免疫印迹）。该反应在30秒内出现，1分钟时达到峰值，且呈剂量依赖性（0.01 - 10 nM），在1 nM时刺激最大（增加三到五倍）。这种效应是1α,25(OH)2D3所特有的，因为其代谢前体25(OH)D3和维生素D3不会增加MAP激酶的酪氨酸磷酸化。酪氨酸激酶抑制剂染料木黄酮可阻断1α,25(OH)2D3诱导的MAP激酶酪氨酸磷酸化，而蛋白激酶C（PKC）抑制剂星形孢菌素可使该激素的效应减弱30%。我们评估了1α,25(OH)2D3类似物激活MAP激酶的能力，这些类似物在6,7碳 - 碳键周围具有完全的灵活性（6F），或者锁定在6 - s - 顺式（6C）或6 - s - 反式（6T）构型。因此，两种6F和一种6C类似物可刺激MAP激酶酪氨酸磷酸化，而一种6T类似物则不能。此外，1β,25(OH)2D3是1α,25(OH)2D3介导的快速反应的已知拮抗剂，可阻断该激素对MAP激酶的作用。我们得出结论，1α,25(OH)2D3以及能够形成6 - s - 顺式构型的类似物（6F和6C）可增加鸡小肠上皮细胞中酪氨酸磷酸化并激活MAP激酶。
