ACTH receptor: Ectopic expression, activity and signaling
ACTH receptor: Ectopic expression, activity and signaling
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DOI：
10.1007/s11010-006-9237-0
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发表时间：
2006-12-01
影响因子：
4.3
通讯作者：
Armelin, Hugo Aguirre
中科院分区：
文献类型：
--
作者：
Forti, Fabio Luis;Dias, Matheus H. S.;Armelin, Hugo Aguirre
在非肾上腺细胞中不能获得功能性促肾上腺皮质激素受体（ACTHR，或黑皮质素2受体，MC 2 R）的表达阻碍了ACTH信号通路的分子分析。在这里，我们在Balb/c小鼠3 T3成纤维细胞中异位表达小鼠ACTHR，以分析参与诱导fos和jun基因的ACTH信号通路。MC 2 R辅助蛋白（MRAP）在Balb 3 T3和其他小鼠3 T3成纤维细胞（NIH、Swiss和3 T3-L1）中的天然组成型表达使这些成纤维细胞系适合于异位表达以其活性形式正确插入质膜的ACTHR，其水平与在小鼠Y1肾上腺皮质肿瘤细胞中发现的水平相似。Y1细胞系是一种培养的细胞系统，已知其稳定显示正常肾上腺特异性代谢途径、ACTH表达和ACTH功能反应。选择39个表达ACTHR的亚系（3 T3-AR转染子）用于遗传霉素抗性，并在将ACTHR-cDNA（在pSVK 3哺乳动物质粒载体中）转染到Balb 3 T3成纤维细胞中后克隆分离。此外，同样分离了携带pSVK 3空载体的Balb 3 T3（3 T3 -0转染子）的16个克隆亚系。与Y1肾上腺皮质细胞相比，筛选了14个3 T3-AR和4个3 T3 -0克隆对ACTH的反应（39）。8个3 T3-AR克隆对ACTH（39）产生应答，激活腺苷酸环化酶并诱导c-Fos蛋白，但激活和诱导的水平分别不严格相关。在3 T3-AR转染子中，其他fos和jun基因也被ACTH诱导（39），其表达的ACTHR蛋白水平与亲本Y1细胞相似。在3个克隆中广泛研究了与c-Fos诱导相关的信号通路：3 T3-AR 01和-07和3 T3 -04。在Y1细胞中，特异性抑制剂（H89/PKA; PD 98059/MEK; Go 6983/PKC和SP 600125/JNK）显示，在PD 98059 ACTH/ACTH系统中启动的信号激活4条途径以诱导c-fos基因，即：（a）cAMP/PKA/CREB;（B）MEK/ERK 1/2;（c）PKC和d）JNK 1/2。在3 T3-AR转染子中，两种抑制剂PD 98059和Go 6983被证明完全不能抑制ACTH对PD 98059 c-Fos的诱导（39），这意味着MEK/ERK和PKC途径不参与该过程。另一方面，SP 600125对ACTH诱导的c-Fos诱导产生85%的抑制（39），此外，ACTH（39）促进JNK 1/2磷酸化，表明JNK是这些细胞中ACTH（39）介导c-Fos诱导的主要信号通路。在成瘾中，PKA抑制剂H89也抑制ACTH对3 T3-AR 7细胞中c-Fos的诱导（39），这表明ACTH对c-Fos诱导中cAMP/PKA/CREB通路的激活（39）。然而，cAMP衍生物db-cAMP和8Br-cAMP在亲本Balb 3 T3和3 T3-AR转染子中不促进CREB磷酸化和c-Fos诱导，证实了其他人先前的报道。总之，Balb 3 T3成纤维细胞中活性ACTH的表达使这些细胞对ACTH有反应，并激活cAMP/PKA/CREB和JNK通路，同时诱导fos和jun家族的基因。这些结果表明Balb 3 T3-AR亚系是用于ACTH信号通路的遗传分析的有用细胞系统。然而，cAMP/PKA/CREB和JNK途径的激活以及fos和jun基因的诱导尚不足以使ACTH能够像在Y1肾上腺皮质细胞中那样干扰Balb 3 T3成纤维细胞的形态、迁移和增殖。
Failure in obtaining expression of functional adrenocorticotropic hormone receptor (ACTHR, or melanocortin 2 receptor, MC2R) in non-adrenal cells has hindered molecular analysis of ACTH signaling pathways. Here, we ectopically expressed the mouse ACTHR in Balb/c mouse 3T3 fibroblasts to analyze ACTH signaling pathways involved in induction of fos and jun genes. Natural constitutive expression of the MC2R accessory protein (MRAP) in Balb3T3 and other mouse 3T3 fibroblasts (NIH, Swiss and 3T3-L1) renders these fibroblastic lines suitable for ectopic expression of ACTHR in its active form properly inserted into the plasma membrane at levels similar to those found in mouse Y1 adrenocortical tumor cells. The Y1 cell line is a cultured cell system well known for stably displaying normal adrenal specific metabolic pathways, ACTHR expression and ACTH functional responses. Thirty-nine sub-lines expressing ACTHR (3T3-AR transfectants) were selected for geneticin-resistance and clonally isolated after transfection of ACTHR-cDNA (in the pSVK3 mammalian plasmidial vector) into Balb3T3 fibroblasts. In addition, sixteen clonal sub-lines of Balb3T3 (3T3-0 transfectants) carrying the pSVK3 empty vector were likewise isolated. Fourteen 3T3-AR and four 3T3-0 clones were screened for response to ACTH(39) in comparison with Y1 adrenocortical cells. Eight 3T3-AR clones responded to ACTH(39) with activation of adenylate cyclase and induction of c-Fos protein, but the levels of, respectively, activation and induction were not strictly correlated. Other fos and jun genes were also induced by ACTH(39) in 3T3-AR transfectants, which express levels of ACTHR protein similar to parental Y1 cells. Signaling pathways relevant to c-Fos induction was extensively investigated in 3 clones: 3T3-AR01 and -07 and 3T3-04. In Y1 cells, specific inhibitors (H89/PKA; PD98059/MEK; Go6983/PKC and SP600125/JNK) show that signals initiated in the PD98059 ACTH/ACTHR-system activate 4 pathways to induce the c-fos gene, namely: (a) cAMP/PKA/CREB; (b) MEK/ERK1/2; (c) PKC and d) JNK1/2. In 3T3-AR transfectants, both inhibitors PD98059 and Go6983 proved completely ineffective to inhibit PD98059 c-Fos induction by ACTH(39), implying that MEK/ERK and PKC pathways are not involved in this process. On the other hand, SP600125 caused 85% inhibition of c-Fos induction by ACTH(39) and, in addition, ACTH(39) promotes JNK1/2 phosphorylation, suggesting that JNK is a major signaling pathway mediating c-Fos induction by ACTH(39) in these cells. In addiction, PKA inhibitor H89 also inhibits c-Fos induction in 3T3-AR7 cells by ACTH(39), implicating activation of the cAMP/PKA/CREB pathway in c-Fos induction by ACTH(39). However, the cAMP derivatives db-cAMP and 8Br-cAMP, do not promote CREB phosphorylation and c-Fos induction in parental Balb3T3 and 3T3-AR transfectants, confirming previous report by others. In conclusion, expression of active ACTHR in Balb3T3 fibroblasts renders these cells responsive to ACTH with activation of cAMP/PKA/CREB and JNK pathways and, also, induction of genes from the fos and jun families. These results show that Balb 3T3-AR sublines are useful cellular systems for genetic analysis of ACTH-signaling pathways. However, activation of cAMP/PKA/CREB and JNK pathways and induction of fos and jun genes are not yet sufficient to enable ACTH for interference in morphology, migration and proliferation of Balb3T3 fibroblasts as it does in Y1 adrenocortical cells.