Role of 5‐IP 7 in the Regulation of Gene Expression

Inositol pyrophosphates (PP‐IPs) are unique, evolutionarily conserved signaling molecules, which act at the interface between signal transduction and cellular metabolism. 5‐IP 7 (5‐diphosphoinositol 1,2,3,4,6‐pentakisphosphate) is the most physiologically abundant PP‐IP (cytoplasmic concentration: 1–2 μM). 5‐IP 7 is a highly electronegative molecule with five monophosphate groups and one bisphosphate group, all packed around a six‐carbon inositol ring. Reported functions of 5‐IP 7 include non‐enzymatic pyrophosphorylation of proteins and non‐covalent binding to the highly electropositive domains in certain proteins (e.g., pleckstrin homology domains). Here, we provide evidence of a new mechanism of action for a PP‐IP, namely, as an enzyme inhibitor. The enzyme in question, encoded by NUDT3 , catalyzes mRNA 5′‐decapping activity. NUDT3 knockdown in MCF7 breast cancer cells cause an increase in the mRNA stability of several ‘migration marker proteins’, namely, β 6 ‐integrin, fibronectin, lipocalin and S100A8, which promote an elevated migratory phenotype compared to the wild type (WT) MCF7 cells. The NUDT3 gene product also hydrolyzes 5‐IP 7 , so we hypothesized that a persistent elevation of cellular levels of that PP‐IP would competitively inhibit mRNA decapping, and hence promote the expression of migration marker proteins. To pursue this idea, we used HCT116 colon cancer cells and HEK293 kidney epithelial cells in which we knocked out the IP 7 Ks that metabolize 5‐IP 7 , thereby increasing levels of this PP‐IP by 2 to 3‐fold. In the current study, qRT‐PCR analysis of IP 7 K −/− HCT116 cells showed an increase in the mRNA levels of β 6 ‐integrin by 2 ± 0.2‐fold, fibronectin by 2.5 ± 0.4‐fold, lipocalin by 5 ± 0.4‐fold, and S100A8 by 3 ± 0.4‐fold compared to WT cells (data are expressed as mean ± SE, n=5, p ≤ 0.01). Knockout of IP 7 K in HEK293 cells resulted in elevation of mRNA levels of β 6 ‐integrin by 6 ± 0.5‐fold, fibronectin by 3 ± 0.6‐fold, lipocalin by 7.5 ± 0.4‐fold, and S100A8 by 3.7 ± 0.35‐fold vs. WT HEK293 cells (data are expressed as mean ± SE, n=5, p ≤ 0.01). We are now developing techniques to load WT cells with 5‐IP 7 to more directly test our idea that this PP‐IP has a novel, enzymatic function as a regulator of expression of pro‐migratory genes that may have relevance to tumor metastasis. Support or Funding Information Intramural Research Program Funding for NIEHS/NIH. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .