Nitric Oxide-releasing Agents and cGMP Analogues Inhibit Murine Erythroleukemia Cell Differentiation and Suppress Erythroid-specific Gene Expression: Correlation with Decreased DNA Binding of NF-E2 and Altered

Differentiation of murine erythroleukemia (MEL) cells induced by hexamethyhene bisacetamide (HMBA) and DMSO was inhibited by several structurally unrelated nitric oxide (NO)-reheasing agents and two membranepermeable cGMP analogues. Since the effect of the NOreleasing agents was augmented by a cGMP phosphodiesterase inhibitor, at least some of their effect appeared to be mediated by activation of cytosohic guanylate cychase. The drugs did not globally block differentiation since hemin-induced differentiation was undisturbed. In HMBA-treated cells, the NO-releasing agents and cGMP analogues reduced fi-globin and &aminolevuhinate synthetase mRNA expression and inhibited the late down-regulation of c-myb mRNA that is required for HMBA-induced differentiation of MEL cells; the regulation of c-myc mRNA was not changed by the drugs. Nuclear run-off analyses showed that the drugs inhibited HMBA-induced changes in J-globin and c-myb transcription rates, and transient transfection of a reporter gene construct demonstrated that the drugs inhibited the HMBA-inducible enhancer function of the a-globin control region, which contains binding sites for the erythroid transcription factors NF-E2 and GATA-1. The NO-releasing agents and cGMP analogues largely prevented HMBA-induced increases in DNA binding of NF-E2, whereas DNA binding of GATA-1 and SP-1 was not affected. The inhibition of erythroid gene expression by NO and cGMP analogues may be physiologically important under conditions of high NO production by endothehial cells and macrophages, i.e., during acute or chronic inflammation. Introduction When exposed to a variety of agents including HMBA3 and DMSO, MEL cells transcniptionally activate erythroid-specific genes and differentiate into nonmoblast-hike cells (1 , 2). The transcriptional activation of erythroid-specific genes appears to require the cooperation of several transcription factors, including NF-E2 and GATA-i (3-6). Differentiation induced by HMBA or DMSO is accompanied by biphasic changes in mRNA levels of c-myc and c-myb; the mRNAs decrease within 1 h of adding the inducing agent, return to preinduction levels by 24 h, and decrease again at 48-72 h to remain at how levels for the duration of the differentiation period (7, 8). It is clear from experiments in which MEL cells were transfected with a c-myb construct under an inducible promoter that the late down-regulation of c-myb mRNA is critical for MEL cells to differentiate (9). Several drugs, most notably glucocorticoids, phonbol estens, and cAMP analogues, inhibit differentiation of MEL cells, and study of their mechanism of action has elucidated some of the signal transduction pathways involved in regulating erythroid cell differentiation (1 0-i 2). We now show, in a completely defined serum-free medium, that three NO-generating agents, SNP, SIN-i , and GS-NO, inhibit HMBAand DMSO-induced differentiation of MEL cells and reduce the differentiation-associated increase in mRNA for 3-globin and erythroid-specific &.ALA synthetase, the first committed enzyme of the heme synthetic pathway. The three NO-releasing drugs appeared to openate, at least in part, through activation of heme-contamning cytosohic guanyhate cyclase since an inhibitor of the cGMPspecific phosphodiesterase augmented their effect and two membrane-permeable cGMP analogues, 8-Br-cGMP and 8-pCPT-cGMP, also inhibited differentiation of MEL cells and reduced HMBA-induced increases in f3-ghobin and &-ALA synthetase mRNA. SNP and 8-Br-cGMP inhibited HMBA-induced transcriptional activation of the endogenous 3-ghobmn gene and of a transfected reporter gene under control of the a-globin promoter and enhancer. In an attempt to further characterize the mechanism by which differentiation was inhibited, we examined regulation of the c-myb and c-myc proto-oncogenes as well as DNA binding of several erythroid transcription factors. We found that Received 6/7/95; revised 9/22/95; accepted 9/27/95. I This work was supported in part by USPHS Grants K08 CAO1 548 (to R. B. P.) and ROi GM49360 (to G. R. B.), grants from the UCSD Academic Senate, the Cancer Research Coordinating Committee ofthe University of California, and the Charles H. Stern and Anna S. Stern Foundation. M. S. was supported in part by a Josephine Especial Fellowship Grant and F. E. P. by an NIH Summer Fellowship Award. 2 To whom requests for reprints should be addressed. Phone: (6i9) 5348805; Fax: (6i9) 534-i42i. 3 The abbreviations used are: HMBA, N,N’-hexamethylene bisacetamide; MEL cells, Friend virus-infected munine erythroleukemia cells; NO, nitric oxide; SNP, sodium nitroprusside; SIN-i , linsidomine; GS-NO, S-nitrosoglutathione; &-ALA, 8-aminolevulinate; 8-Br, 8-bromo; 8-pCPT-cGMP, 8-( p. chlorophenylthio)-cGMP; LCR, locus control region; RSV, Rous sarcoma virus; EMSA, electrophoretic mobility shift assay; oligodNT, oligodeoxynucleotide; TPA, 1 2-0-tetradecanoyl-phorbol 1 3-acetate; CAT, chloramphenicol acetyltransferase.