MGSA/GRO transcription is differentially regulated in normal retinal pigment epithelial and melanoma cells.
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.
Shattuck
R L
RL
Veterans Affairs Medical Center, Nashville, Tennessee 37212-2637.
Wood
L D
LD
Jaffe
G J
GJ
Richmond
A
A
D001483:Base Sequence; D002460:Cell Line; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006133:Growth Substances / Q000235:genetics*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007375:Interleukin-1 / Q000494:pharmacology; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics; D015345:Oligonucleotide Probes; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
C475829:CXCL1 protein, human; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D018076:DNA, Complementary; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D007375:Interleukin-1; D016328:NF-kappa B; D009363:Neoplasm Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha