Kim H, Hawley T S, Hawley R G, Baumann H
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Mol Cell Biol. 1998 Mar;18(3):1525-33. doi: 10.1128/MCB.18.3.1525.
Signals propagated via the gp130 subunit of the interleukin-6 (IL-6)-type cytokine receptors mediate, among various cellular responses, proliferation of hematopoietic cells and induction of acute-phase plasma protein (APP) genes in hepatic cells. Hematopoietic growth control by gp130 is critically dependent on activation of both STAT3 and protein tyrosine phosphatase 2 (SHP-2). To investigate whether induction of APP genes has a similar requirement for SHP-2, we constructed two chimeric receptors, G-gp130 and G-gp130(Y2F), consisting of the transmembrane and cytoplasmic domains of gp130 harboring either a wild-type or a mutated SHP-2 binding site, respectively, fused to the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor. Rat hepatoma H-35 cells stably expressing the chimeric receptors were generated by retroviral transduction. Both chimeric receptors transmitted a G-CSF-induced signal characteristic of that triggered by IL-6 through the endogenous gp130 receptor; i.e., both activated the appropriate JAK, induced DNA binding activity by STAT1 and STAT3, and up-regulated expression of the target APP genes, those for alpha-fibrinogen and haptoglobin. Notwithstanding these similarities in the patterns of signaling responses elicited, mutation of the SHP-2 interaction site in G-gp130(Y2F) abrogated ligand-activated receptor recruitment of SHP-2 as expected. Moreover, the tyrosine phosphorylation state of the chimeric receptor, the associated JAK activity, and the induced DNA binding activity of STAT1 and STAT3 were maintained at elevated levels and for an extended period of time in G-gp130(Y2F)-expressing cells following G-CSF treatment compared to that in cells displaying the G-gp130 receptor. H-35 cells ectopically expressing G-gp130(Y2F) were also found to display an enhanced sensitivity to G-CSF and a higher level of induction of APP genes. Overexpression of the enzymatically inactive SHP-2 enhanced the signaling by the wild-type but not by the Y2F mutant G-gp130 receptor. These results indicate that gp130 signaling for APP gene induction in hepatic cells differs qualitatively from that controlling the proliferative response in hematopoietic cells in not being strictly dependent on SHP-2. The data further suggest that SHP-2 functions normally to attenuate gp130-mediated signaling in hepatic (and, perhaps, other) cells by moderating JAK action.
通过白细胞介素6（IL-6）型细胞因子受体的gp130亚基传播的信号，在各种细胞反应中，介导造血细胞的增殖以及肝细胞中急性期血浆蛋白（APP）基因的诱导。gp130对造血生长的控制严重依赖于STAT3和蛋白酪氨酸磷酸酶2（SHP-2）的激活。为了研究APP基因的诱导是否对SHP-2有类似的需求，我们构建了两种嵌合受体，G-gp130和G-gp130(Y2F)，它们分别由带有野生型或突变型SHP-2结合位点的gp130的跨膜和胞质结构域与粒细胞集落刺激因子（G-CSF）受体的胞外结构域融合而成。通过逆转录病毒转导产生稳定表达嵌合受体的大鼠肝癌H-35细胞。两种嵌合受体都通过内源性gp130受体传递了由G-CSF诱导的、与IL-6触发的信号特征相同的信号；即两者都激活了相应的JAK，诱导了STAT1和STAT3的DNA结合活性，并上调了靶APP基因（α-纤维蛋白原和触珠蛋白的基因）的表达。尽管在引发的信号反应模式上存在这些相似之处，但正如预期的那样，G-gp130(Y-2F)中SHP-2相互作用位点的突变消除了配体激活的受体对SHP-2的招募。此外，与显示G-gp130受体细胞相比，在G-CSF处理后，G-gp130(Y2F)表达细胞中嵌合受体的酪氨酸磷酸化状态、相关的JAK活性以及诱导的STAT1和STAT3的DNA结合活性在升高水平上维持了更长时间。还发现异位表达G-gp130(Y2F)的H-35细胞对G-CSF的敏感性增强，APP基因的诱导水平更高。酶活性无活性的SHP-2的过表达增强了野生型G-gp130受体的信号传导，但没有增强Y2F突变型G-gp130受体的信号传导。这些结果表明，肝细胞中APP基因诱导的gp130信号传导在性质上不同于控制造血细胞增殖反应的信号传导，因为它不严格依赖于SHP-2。数据进一步表明，SHP-2通常通过调节JAK作用来减弱肝（也许还有其他）细胞中gp130介导的信号传导。
