MGSA GRO TRANSCRIPTION IS DIFFERENTIALLY REGULATED IN NORMAL RETINAL-PIGMENT EPITHELIAL AND MELANOMA-CELLS

We have characterized constitutive and cytokine-regulated MGSA/GROalph a, -beta, and -gamma gene expression in normal retinal pigment epithel ial (RPE) cells and a malignant melanoma cell line (Hs294T) to discern the mechanism for MGSA/GRO constitutive expression in melanoma. In RP E cells, constitutive MGSA/GROalpha, -beta, and -gamma mRNAs are not d etected by Northern (RNA) blot analysis although nuclear runoff experi ments show that all three genes are transcribed. In Hs294T cells, cons titutive MGSA/GROalpha expression is detectable by Northern blot analy sis, and the level of basal MGSA/GROalpha transcription is 8- to 30-fo ld higher than in RPE cells. In contrast, in Hs294T cells, basal MGSA/ GRObeta and -gamma transcription is only twofold higher than in RPE ce lls and no beta or gamma mRNA is detected by Northern blot. These data suggest that the constitutive MGSA/GROalpha mRNA in Hs294T cells is d ue to increased basal MGSA/GROalpha gene transcription. The cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha) signif icantly increase the mRNA levels for all three MGSA/GRO isoforms in Hs 294T and RPE cells, and both transcriptional and posttranscriptional m echanisms are operational. Nuclear runoff assays indicate that in RPE cells, a 1-h IL-1 treatment induces a 10- to 20-fold increase in trans cription of MGSA/GROalpha -beta and -gamma but only a 2-fold increase in Hs294T cells. Similarly, chloramphenicol acetyltransferase (CAT) re porter gene analysis using the MGSA/GROalpha, -beta, and -gamma promot er regions demonstrates that IL-1 treatment induces an 8- to 14-fold i ncrease in CAT activity in RPE cells but only a 2-fold increase in Hs2 94T cells. The effect of deletion or mutation of the MGSA/GROalpha NF- kappaB element, combined with data from gel mobility shift analyses, i ndicates that the NF-kappaB p50/p65 heterodimer in RPE cells plays an important role in IL-1- and TNFalpha-enhanced gene transcription. In H s294T cells, gel shift analyses indicate that IL-1 and TNFalpha induce NF-kappaB complex formation; however, transactivation does not occur, suggesting that subtle differences in the NF-kappaB complexes may res ult in the inability of the cytokines IL-1 and TNFalpha to activate tr anscription of the MGSA/GRO genes. IL-1 and TNFalpha posttranscription ally regulate MGSA/GRO mRNA levels in both cell types. In Hs294T cells , IL-1 increases the half-life of MGSA/GROalpha from 15 min to 6 h (a 24-fold increase in half-life). These data indicate that IL-1 and TNFa lpha transcriptionally and posttranscriptionally regulate MGSA/GROalph a -beta, and -gamma mRNA levels in RPE cells, while in Hs294T cells, t he major effect of IL-1 and TNFalpha is on mRNA stability.