The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), has been shown to markedly reduce metastasis of numerous tumors. The current study was focused on further elucidating the molecular mechanisms behind the antitumor function of NDRG1. We have identified for the first time that NDRG1 upregulates the potent cyclin-dependent kinase inhibitor, p21. This effect was observed in three different cancer cell types, including PC3MM and DU145 prostate cancer cells and H1299 lung carcinoma cells, and occurred independently of p53. In addition, reducing NDRG1 expression using short hairpin RNA in PC3MM and DU145 cells resulted in significantly reduced p21 protein levels. Hence, p21 is closely correlated with NDRG1 expression in these latter cell types. Examining the mechanisms behind the effect of NDRG1 on p21 expression, we found that NDRG1 upregulated p21 via transcriptional and posttranscriptional mechanisms in prostate cancer cells, although its effect on H1299 cells was posttranscriptional only. Further studies identified two additional NDRG1 protein targets. The dominant-negative p63 isoform, ΔNp63, which has been found to inhibit p21 transcription, was downregulated by NDRG1. On the other hand, a truncated 50 kDa MDM2 isoform (p50(MDM2)), which may protect p21 from proteasomal degradation, was upregulated by NDRG1. The downregulation of ΔNp63 and upregulation of p50(MDM2) are potential mechanisms by which NDRG1 increases p21 expression in these cells. Additional functional studies identified that NDRG1 inhibits cancer cell migration, suggesting that p21 is a molecular player in its antimetastatic activity.

Production of renin is critically dependent on modulation of REN mRNA stability. Here we sought to elucidate the molecular mechanisms involved. Transfections of renin-expressing Calu-6 cells with reporter constructs showed that a cis-acting 34-nucleotide AU-rich "renin stability regulatory element" in the REN 3-untranslated region (3-UTR) contributes to basal REN mRNA instability. Yeast three-hybrid screening with the REN 3-UTR as bait isolated HADHB (hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein) ␤-subunit) as a novel REN mRNA-binding protein. Recombinant HADHB bound specifically to the 3-UTR of REN mRNA, as did the known mRNA stabilizers HuR and CP1 (poly(C)-binding protein-1). This required the renin stability regulatory element. Forskolin, which augments REN mRNA stability in Calu-6 cells, increased binding of several proteins, including HuR and CP1, to the REN 3-UTR, whereas 4-bromocrotonic acid, a specific thiolase inhibitor, decreased binding and elevated renin protein levels. Upon decreasing HADHB mRNA with RNA interference, renin protein and mRNA stability increased, whereas RNA interference against HuR caused these to decrease. Immunoprecipitation and reverse transcription-PCR of Calu-6 extracts confirmed that HADHB, HuR, and CP1 each associate with REN mRNA in vivo. Intracellular imaging revealed distinct localization of HADHB to mitochondria, HuR to nuclei, and CP1 throughout the cell. Immunohistochemistry demonstrated enrichment of HADHB in renin-producing renal juxtaglomerular cells. In conclusion, HADHB, HuR, and CP1 are novel REN mRNA-binding proteins that target a cis-element in the 3-UTR of REN mRNA and regulate renin production. cAMP-mediated increased REN mRNA stability may involve stimulation of HuR and CP1, whereas REN mRNA decay may involve thiolasedependent pathways.

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It is now recognized that post-transcriptional mechanisms are pivotal to renin production. These involve factors that modulate renin mRNA stability. In 2003 new data has emerged from work in Australia and Germany that has identified several of the, as many as, 20 or so proteins involved. These include CP1 (hnRNP E1), HuR, HADHB, dynamin, nucleolin, YP-1, hnRNP K and MINT-homologous protein. Cyclic AMP (cAMP) is a crucial regulator of renin secretion as well as transcriptional and post-transcriptional control of expression. Many of the RNA-binding proteins that were identified responded to forskolin, increasing in amount by two to 10-fold. The cAMP mechanisms that regulate renin mRNA target, at least in large part, other genes that presumably encode some of these proteins. The increase in the expression of these then facilitates, sequentially, renin mRNA stabilization and destabilization. Our data, using a battery of different techniques, confirm that CP1 and HuR stabilize renin mRNA, whereas HADHB causes destabilization. These proteins target cis-acting C-rich sequences (in the case of CP1) and AU-rich sequences (HuR) in the distal region of the 3'-untranslated region of renin mRNA. We found HADHB was enriched in juxtaglomerular cells and that that within Calu-6 cells HADHB, HuR and CP1 all localized in nuclear subregions, as well as cytoplasm (HADHB and CP1) and mitochondria (HADHB) commensurate with the role each plays in control of renin mRNA stability. The specific proteins that bind to human renin mRNA have begun to be revealed. Cyclic AMP upregulates the binding of several of these proteins, which in turn affect renin mRNA stability and thus overall expression of renin.