Protein Phosphatase-1 Regulates Expression of Neuregulin-1.

Protein phosphatase 1 (PP1), a cellular serine/threonine phosphatase, is targeted to cellular promoters by its major regulatory subunits, PP1 nuclear targeting subunit, nuclear inhibitor of PP1 (NIPP1) and RepoMan. PP1 is also targeted to RNA polymerase II (RNAPII) by NIPP1 where it can dephosphorylate RNAPII and cycle-dependent kinase 9 (CDK9). Here, we show that treatment of cells with a small molecule activator of PP1 increases the abundance of a neuregulin-1 (NRG-1)-derived peptide. NRG-1 mRNA and protein levels were increased in the cells stably or transiently expressing mutant NIPP1 (mNIPP1) that does not bind PP1, but not in the cells expressing NIPP1. Expression of mNIPP1 also activated the NRG-1 promoter in an NF-κB-dependent manner. Analysis of extracts from mNIPP1 expressing cells by glycerol gradient centrifugation showed a redistribution of PP1 and CDK9 between large and small molecular weight complexes, and increased CDK9 Thr-186 phosphorylation. This correlated with the increased CDK9 activity. Further, RNAPII co-precipitated with mNIPP1, and phosphorylation of RNAPII C-terminal domain (CTD) Ser-2 residues was greater in cells expressing mNIPP1. In mNIPP1 expressing cells, okadaic acid, a cell-permeable inhibitor of PP1, did not increase Ser-2 CTD phosphorylation inhibited by flavopiridol, in contrast to the NIPP1 expressing cells, suggesting that PP1 was no longer involved in RNAPII dephosphorylation. Finally, media conditioned with mNIPP1 cells induced the proliferation of wild type 84-31 cells, consistent with a role of neuregulin-1 as a growth promoting factor. Our study indicates that deregulation of PP1/NIPP1 holoenzyme activates NRG-1 expression through RNAPII and CDK9 phosphorylation in a NF-κB dependent manner.

Hepcidin induces HIV-1 transcription inhibited by ferroportin.

BACKGROUND: Physiological regulation of cellular iron involves iron export by the membrane protein, ferroportin, the expression of which is induced by iron and negatively modulated by hepcidin. We previously showed that iron chelation is associated with decreased HIV-1 transcription. We hypothesized that increased iron export by ferroportin might be associated with decreased HIV-1 transcription, and degradation of ferroportin by hepcidin might in turn induce HIV-1 transcription and replication. Here, we analyzed the effect of ferroportin and hepcidin on HIV-1 transcription. RESULTS: Expression of ferroportin was associated with reduced HIV-1 transcription in 293T cells and addition of hepcidin to ferroportin-expressing cells counteracted this effect. Furthermore, exposure of promonocytic THP-1 cells to hepcidin was associated with decreased ferroportin expression, increased intracellular iron and induction of reporter luciferase gene expression. Finally, exposure of human primary macrophages and CD4+ T cells to hepcidin and iron was also associated with induction of viral production. CONCLUSION: Our results suggest that the interplay between ferroportin-mediated iron export and hepcidin-mediated degradation of ferroportin might play a role in the regulation of HIV-1 transcription and may be important for understanding of HIV-1 pathogenesis.

Regulation of HIV-1 transcription at 3% versus 21% oxygen concentration.

HIV transcription is induced by the HIV-1 Tat protein, in concert with cellular co-factors including CDK9, CDK2, NF-kappaB, and others. The cells of most of the body's organs are exposed to approximately 3-6% oxygen, but most in vitro studies of HIV replication are conducted at 21% oxygen. We hypothesized that activities of host cell factors involved in HIV-1 replication may differ at 3% versus 21% O(2), and that such differences may affect HIV-1 replication. Here we show that Tat-induced HIV-1 transcription was reduced at 3% O(2) compared to 21% O(2). HIV-1 replication was also reduced in acutely or chronically infected cells cultured at 3% O(2) compared to 21% O(2). This reduction was not due the decreased cell growth or increased cellular toxicity and also not due to the induction of hypoxic response. At 3% O(2), the activity of CDK9/cyclin T1 was inhibited and Sp1 activity was reduced, whereas the activity of other host cell factors such as CDK2 or NF-kappaB was not affected. CDK9-specific inhibitor ARC was much less efficient at 3% compared to 21% O(2) and also expression of CDK9/cyclin T1-dependent IkappaB inhibitor alpha was repressed. Our results suggest that lower HIV-1 transcription at 3% O(2) compared to 21% O(2) may be mediated by lower activity of CDK9/cyclin T1 and Sp1 at 3% O(2) and that additional host cell factors such as CDK2 and NF-kappaB might be major regulators of HIV-1 transcription at low O(2) concentrations.