Shattuck R L, Wood L D, Jaffe G J, Richmond A
Veterans Affairs Medical Center, Nashville, Tennessee 37212-2637.
Mol Cell Biol. 1994 Jan;14(1):791-802. doi: 10.1128/mcb.14.1.791-802.1994.
We have characterized constitutive and cytokine-regulated MGSA/GRO alpha, -beta, and -gamma gene expression in normal retinal pigment epithelial (RPE) cells and a malignant melanoma cell line (Hs294T) to discern the mechanism for MGSA/GRO constitutive expression in melanoma. In RPE cells, constitutive MGSA/GRO alpha, -beta, and -gamma mRNAs are not detected by Northern (RNA) blot analysis although nuclear runoff experiments show that all three genes are transcribed. In Hs294T cells, constitutive MGSA/GRO alpha expression is detectable by Northern blot analysis, and the level of basal MGSA/GRO alpha transcription is 8- to 30-fold higher than in RPE cells. In contrast, in Hs294T cells, basal MGSA/GRO beta and -gamma transcription is only twofold higher than in RPE cells and no beta or gamma mRNA is detected by Northern blot. These data suggest that the constitutive MGSA/GRO alpha mRNA in Hs294T cells is due to increased basal MGSA/GRO alpha gene transcription. The cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) significantly increase the mRNA levels for all three MGSA/GRO isoforms in Hs294T and RPE cells, and both transcriptional and posttranscriptional mechanisms are operational. Nuclear runoff assays indicate that in RPE cells, a 1-h IL-1 treatment induces a 10- to 20-fold increase in transcription of MGSA/GRO alpha, -beta and -gamma but only a 2-fold increase in Hs294T cells. Similarly, chloramphenicol acetyltransferase (CAT) reporter gene analysis using the MGSA/GRO alpha, -beta, and -gamma promoter regions demonstrates that IL-1 treatment induces an 8- to 14-fold increase in CAT activity in RPE cells but only a 2-fold increase in Hs294T cells. The effect of deletion or mutation of the MGSA/GRO alpha NF-kappa B element, combined with data from gel mobility shift analyses, indicates that the NF-kappa B p50/p65 heterodimer in RPE cells plays an important role in IL-1- and TNF alpha-enhanced gene transcription. In Hs294T cells, gel shift analyses indicate that IL-1 and TNF alpha induce NF-kappa B complex formation; however, transactivation does not occur, suggesting that subtle differences in the NF-kappa B complexes may result in the inability of the cytokines IL-1 and TNF alpha to activate transcription of the MGSA/GRO genes. IL-1 and TNF alpha posttranscriptionally regulate MGSA/GRO mRNA levels in both cell types. In Hs294T cells, IL-1 increases the half-life of MGSA/GRO alpha from 15 min to 6 h (a 24-fold increase in half-life). These data indicate that IL-1 and TNF alpha transcriptionally and posttranscriptionally regulate MGSA/GRO alpha, -beta, and -gamma mRNA levels in RPE cells, while in Hs294T cells, the major effect of IL-1 and TNF alpha is on mRNA stability.
我们已对正常视网膜色素上皮（RPE）细胞和恶性黑色素瘤细胞系（Hs294T）中组成型和细胞因子调节的MGSA/GROα、β和γ基因表达进行了表征，以探究黑色素瘤中MGSA/GRO组成型表达的机制。在RPE细胞中，通过Northern（RNA）印迹分析未检测到组成型MGSA/GROα、β和γ mRNA，尽管核转录实验表明这三个基因均被转录。在Hs294T细胞中，通过Northern印迹分析可检测到组成型MGSA/GROα表达，且基础MGSA/GROα转录水平比RPE细胞高8至30倍。相比之下，在Hs294T细胞中，基础MGSA/GROβ和γ转录仅比RPE细胞高两倍，且通过Northern印迹未检测到β或γ mRNA。这些数据表明，Hs294T细胞中组成型MGSA/GROα mRNA是由于基础MGSA/GROα基因转录增加所致。细胞因子白细胞介素-1（IL-1）和肿瘤坏死因子α（TNFα）显著增加了Hs294T和RPE细胞中所有三种MGSA/GRO异构体的mRNA水平，且转录和转录后机制均起作用。核转录分析表明，在RPE细胞中，1小时的IL-1处理可诱导MGSA/GROα、β和γ转录增加10至20倍，但在Hs294T细胞中仅增加2倍。同样，使用MGSA/GROα、β和γ启动子区域的氯霉素乙酰转移酶（CAT）报告基因分析表明，IL-1处理可诱导RPE细胞中CAT活性增加8至14倍，但在Hs294T细胞中仅增加2倍。MGSA/GROα NF-κB元件缺失或突变的影响，结合凝胶迁移率变动分析的数据表明，RPE细胞中的NF-κB p50/p65异二聚体在IL-1和TNFα增强的基因转录中起重要作用。在Hs294T细胞中，凝胶迁移分析表明IL-1和TNFα诱导NF-κB复合物形成；然而，转录激活并未发生，这表明NF-κB复合物中的细微差异可能导致细胞因子IL-1和TNFα无法激活MGSA/GRO基因的转录。IL-1和TNFα在两种细胞类型中均对MGSA/GRO mRNA水平进行转录后调节。在Hs294T细胞中，IL-1将MGSA/GROα的半衰期从15分钟延长至6小时（半衰期增加24倍）。这些数据表明，IL-1和TNFα在转录和转录后水平调节RPE细胞中MGSA/GROα、β和γ mRNA水平，而在Hs294T细胞中，IL-1和TNFα的主要作用是影响mRNA稳定性。
