Cheol-Hee Yoon

PKR, a p53 target gene, plays a crucial role in the tumor-suppressor function of p53.

Type I IFN-induced expression of dsRNA-activated protein kinase (PKR) during viral infection is a well-established antiviral mechanism. However, little is known about the expression of PKR in the context of p53 and about PKR involvement in p53-mediated tumor suppression. Here, we report that PKR is a p53 target gene and plays an important role in the tumor-suppressor function of p53. Activation of p53 by genotoxic stress induces a significant level of PKR expression by acting on the newly identified cis-acting element (ISRE), which is separated from the IFN-stimulated responsive element on the PKR promoter, resulting in translational inhibition and cell apoptosis. The genotoxin-mediated inhibition of translation is associated with the p53/PKR/elF2a (eukaryotic initiation factor-2α) pathway. To some extent, p53 activation induced by DNA damage facilitates cell apoptosis by activating PKR. PKR-knockdown human colon cancer cells grew rapidly in nude mice and proved resistant to anti-cancer drugs. These data indicate that p53-mediated tumor suppression can be attributed at least in part to the biological functions of PKR induced by p53 in genotoxic conditions.

The double-strand RNA-dependent protein kinase PKR plays a significant role in a sustained ER stress-induced apoptosis

Sustained ER stress leads to apoptosis. However, the exact mechanism still remains to be elucidated. Here, we demonstrate that the double strand RNA‐dependent protein kinase (PKR) is involved in the ER stress‐mediated signaling pathway. ER stress rapidly activated PKR, inducing the phosphorylation of eIF2α, followed by the activation of the ATF4/CHOP pathway. ER‐stress‐mediated eIF2α/ATF4/CHOP signaling and associated cell death was markedly reduced by PKR knockdown. We also found that PKR activation was mediated by PACT, the expression of which was elevated by ER‐stress. These results indicate that the ER‐stress‐mediated eIF2α/ATF4/CHOP/cell death pathway is, to some degree, dependent on PACT‐mediated PKR activation apart from the PERK pathway.

DC immunotherapy is highly effective for the inhibition of tumor metastasis or recurrence, although it is not efficient for the eradication of established solid tumors.

Dendritic cell (DC)-based immunotherapy has not been as effective as expected in most solid tumors even in the murine model, particularly in renal cell carcinoma (RCC). Our investigation was initiated to identify what causes the limitations of DC-based immunotherapy in solid RCC. We have investigated immunosuppressive factors from tumors and their effects on DC migration, as well as cytotoxic T lymphocyte (CTL) response and lymphocyte infiltration into the tumor mass upon vaccination with mouse renal adenocarcinoma (Renca) cell lysate-pulsed bone marrow (Bm)-derived DC in tumor-bearing mice. We also investigated pulmonary metastasis- and tumor recurrence-inhibitory effects of DC-vaccination in the solid tumor-bearing mice. In these experiments, we found that the limitations of DC-based immunotherapy to solid RCC likely result from tumor-mediated TGF-β hindrance of immune attack rather than insufficient immune induction by DC therapy. In fact, the CTL response induced by DC therapy was quite sufficient and functional for the inhibition of tumor recurrence after surgery or of tumor metastasis induced by additional tumor-challenge to the tumor-bearing mice. Taken together, our present results obtained in mouse model suggest the potential of DC immunotherapy in tumor patients for hindering or blocking disease progression by inhibition of tumor metastasis and/or tumor recurrence after surgery.

Hdm2 negatively regulates telomerase activity by functioning as an E3 ligase of hTERT.

In this study, we identified posttranslational regulation of human telomerase reverse-transcriptase (hTERT) by the E3 ligase Hdm2. The telomerase activity generated by exogenous hTERT in U2OS cells was reduced on adriamycin treatment. The overexpressed levels of hTERT were also decreased under the same conditions. These processes were reversed by treatment with a proteasome inhibitor or depletion of Hdm2. Furthermore, intrinsic telomerase activity was increased in HCT116 cells with ablation of Hdm2. Immunoprecipitation analyses showed that hTERT and Hdm2 bound to each other in multiple domains. Ubiquitination analyses showed that Hdm2 could polyubiquitinate hTERT principally at the N-terminus, which was further degraded in a proteasome-dependent manner. An hTERT mutant with all five lysine residues at the N-terminus of hTERT that mutated to arginine became resistant to Hdm2-mediated ubiquitination and degradation. In U2OS cells, depletion of Hdm2 or addition of the Hdm2-resistant hTERT mutant strengthened the cellular protective effects against apoptosis. Similar results were obtained with the Hdm2-stable H1299 cell line. These observations indicate that Hdm2 is an E3 ligase of hTERT.

CISH is induced during DC development and regulates DC-mediated CTL activation

The cytokine inducible SH2‐domain protein (CISH) is a well‐known STAT5 target gene, but its role in the immune system remains uncertain. In this study, we found that CISH is predominantly induced during dendritic cell (DC) development from mouse bone marrow (BM) cells and plays a crucial role in type 1 DC development and DC‐mediated CTL activation. CISH knockdown reduced the expression of MHC class I, co‐stimulatory molecules and pro‐inflammatory cytokines in BMDCs. Meanwhile, the DC yield was markedly enhanced by CISH knockdown via cell‐cycle activation and reduction of cell apoptosis. Down‐regulation of cell proliferation at the later stage of DC development was found to be associated with CISH‐mediated negative feedback regulation of STAT5 activation. In T‐cell immunity, OT‐1 T‐cell proliferation was significantly reduced by CISH knockdown in DCs, whereas OT‐2 T‐cell proliferation was not affected by CISH knockdown. CTLs generated by DC vaccination were also markedly reduced by CISH knockdown, followed by significant impairment of DC‐based tumor immunotherapy. Taken together, our data suggest that CISH expression at the later stage of DC development triggers the shutdown of DC progenitor cell proliferation and facilitates DC differentiation into a potent stimulator of CTLs.

New Cdc2 Tyr 4 phosphorylation by dsRNA‐activated protein kinase triggers Cdc2 polyubiquitination and G2 arrest under genotoxic stresses

Cell division cycle 2 (Cdc2) protein is an essential subunit of M‐phase kinase (MPK), which has a key role in G2/M transition. Even though the control of MPK activity has been well established with regard to the phosphorylation of Cdc2 at Thr 14 and/or Tyr 15 and Thr 161, little is known about the proteolytic control of Cdc2. In this study, we observed that Cdc2 was downregulated under genotoxic stresses and that double‐stranded RNA‐activated protein kinase (PKR) was involved in the process. The PKR‐mediated Tyr4 phosphorylation triggered Cdc2 ubiquitination. Phospho‐mimic mutations at the Tyr 4 residue (Y4D or Y4E) caused significant ubiquitination of Cdc2 even in the absence of PKR. Our findings demonstrate that (i) PKR, Ser/Thr kinase, phosphorylates its new substrate Cdc2 at the Tyr 4 residue, (ii) PKR‐mediated Tyr 4‐phosphorylation facilitates Cdc2 ubiquitination and proteosomal degradation, (iii) unphosphorylated Tyr 4 prevents Cdc2 ubiquitination, and (iv) downstream from p53, PKR has a crucial role in G2 arrest and triggers Cdc2 downregulation under genotoxic conditions.

Tumor‐mediated down‐regulation of MHC class II in DC development is attributable to the epigenetic control of the CIITA type I promoter

In patients with cancer, DC express significantly lower amounts of MHC class II compared with those of normal individuals. However, the underlying mechanisms for this have not yet been fully defined. In the present study, we found that IL‐10 plays a major role in the tumor‐conditioned medium (TCM)‐mediated decrease of MHC class II expression on BM‐derived DC. IL‐10 inhibited the expression of type I CIITA during DC differentiation. GM‐CSF‐mediated histone (H3 and H4) acetylation at the type I promoter (pI) locus of the CIITA gene was markedly increased during DC differentiation and this increase was blocked by IL‐10. We also found that STAT5 bound to the CIITA pI locus during DC differentiation, and the binding was markedly attenuated by TCM or IL‐10. Altogether, these findings suggest that (i) the down‐regulation of MHC class II in tumor microenvironments is most likely attributable to IL‐10 in the TCM and (ii) IL‐10‐mediated MHC class II down‐regulation results from the inhibition of type I CIITA expression. This inhibition is most likely due to blocking of the STAT5‐associated epigenetic modifications of the CIITA pI locus during the entire period of DC differentiation from BM cells, as opposed to a simple inhibition of MHC class II expression at the DC stage.

p53-Derived Host Restriction of HIV-1 Replication by Protein Kinase R-Mediated Tat Phosphorylation and Inactivation

ABSTRACT Tumor suppressor p53 has been suggested to be a host restriction factor against HIV-1 replication, but the detailed molecular mechanism has remained elusive for decades. Here, we demonstrate that p53-mediated HIV-1 suppression is attributed to double-stranded RNA (dsRNA)-dependent protein kinase (PKR)-mediated HIV-1 trans-activator (Tat) phosphorylation and inactivation. p53 silencing significantly enhanced HIV-1 replication in infected cells. Ectopic expression of p53 suppressed Tat activity, which was rescued by PKR silencing. In addition, ectopic expression of PKR abolished Tat activity in p53−/− and eIF2αCA cells. Finally, we found that HIV-1 infection activates p53, followed by the induction and activation of PKR. PKR directly interacted with HIV-1 Tat and phosphorylates the first exon of Tat exclusively at five Ser/Thr residues (T23, T40, S46, S62, and S68), which inhibits Tat-mediated provirus transcription in three critical steps: (i) phosphorylation near the arginine-rich motif (ARM) inhibits Tat translocation into the nucleus, (ii) accumulation of Tat phosphorylation abolishes Tat–Tat-responsive region (TAR) binding, and (iii) Tat phosphorylation at T23 and/or T40 obliterates the Tat-cyclin T1 interaction. These five Ser/Thr sites on Tat were highly conserved in HIV-1 strains prevalent in Europe and the United States. Taken together, our findings indicate that p53-derived host restriction of HIV-1 replication is likely attributable, at least in part, to a noncanonical p53/PKR/Tat phosphorylation and inactivation pathway in HIV-1 infection and AIDS pathogenesis. IMPORTANCE HIV-1-mediated disease progression to AIDS lasts for years to decades after primary infection. Host restriction and associated viral latency have been studied for several decades. p53 has been suggested as an important host restriction factor against HIV-1 replication. However, the detailed molecular mechanism is still unclear. In the present study, we found that the p53-mediated HIV-1 restriction is attributed to a p53/PKR/Tat-inactivation pathway. HIV-1 infection activated p53, which subsequently induced PKR expression and activation. PKR directly phosphorylated Tat exclusively at five specific Ser/Thr residues, which was accompanied by significant suppression of HIV-1 replication. Accumulation of Tat phosphorylation at these sites inhibited Tat function by blocking Tat nuclear localization, Tat binding to TAR, and Tat-cyclin T1 interaction. Our findings provide a better understanding of the p53-derived host restriction mechanism against HIV-1 replication in AIDS pathogenesis and may contribute to further research focusing on the investigation of potential therapeutic targets for HIV-1.

Egr2 induced during DC development acts as an intrinsic negative regulator of DC immunogenicity

Early growth response gene 2 (Egr2), which encodes a zinc finger transcription factor, is rapidly and transiently induced in various cell types independently of de novo protein synthesis. Although a role for Egr2 is well established in T‐cell development, Egr2 expression and its biological function in dendritic cells (DCs) have not yet been described. Here, we demonstrate Egr2 expression during DC development, and its role in DC‐mediated immune responses. Egr2 is expressed in the later stage of DC development from BM precursor cells. Even at steady state, Egr2 is highly expressed in mouse splenic DCs. Egr2‐knockdown (Egr2‐KD) DCs showed increased levels of major histocompatability complex (MHC) class I and II and co‐stimulatory molecules, and enhanced antigen uptake and migratory capacities. Furthermore, Egr2‐KD abolished SOCS1 expression and signal transducer and activator of transcription 5 (STAT5) activation during DC development, probably resulting in the enhancement of IL‐12 expression and Th1 immunogenicity of a DC vaccine. DC‐mediated cytotoxic T lymphocyte (CTL) activation and antitumor immunity were significantly enhanced by Egr2‐KD, and impaired by Egr2 overexpression in antigen‐pulsed DC vaccines. These data suggest that Egr2 acts as an intrinsic negative regulator of DC immunogenicity and can be an attractive molecular target for DC vaccine development.

NUCKS1, a novel Tat coactivator, plays a crucial role in HIV-1 replication by increasing Tat-mediated viral transcription on the HIV-1 LTR promoter

BackgroundHuman immunodeficiency virus-1 (HIV-1) Tat protein plays an essential role in HIV gene transcription from the HIV-1 long terminal repeat (LTR) and replication. Transcriptional activity of Tat is modulated by several host factors, but the mechanism responsible for Tat regulation by host factors is not understood fully.ResultsUsing a yeast two-hybrid screening system, we identified Nuclear ubiquitous casein and cyclin-dependent kinase substrate 1 (NUCKS1) as a novel Tat-interacting partner. Here, we report its function as a positive regulator of Tat. In a coimmunoprecipitation assay, HIV-1 Tat interacted sufficiently with both endogenous and ectopically expressed NUCKS1. In a reporter assay, ectopic expression of NUCKS1 significantly increased Tat-mediated transcription of the HIV-1 LTR, whereas knockdown of NUCKS1 by small interfering RNA diminished Tat-mediated transcription of the HIV-1 LTR. We also investigated which mechanism contributes to NUCKS1-mediated Tat activation. In a chromatin immunoprecipitation assay (ChIP), knockdown of NUCKS1 interrupted the accumulation of Tat in the transactivation-responsive (TAR) region on the LTR, which then led to suppression of viral replication. However, NUCKS1 expression did not increase Tat nuclear localization and interaction with Cyclin T1. Interestingly, the NUCKS1 expression level was lower in latently HIV-1-infected cells than in uninfected parent cells. Besides, expression level of NUCKS1 was markedly induced, which then facilitated HIV-1 reactivation in latently infected cells.ConclusionTaken together, our data demonstrate clearly that NUCKS1 is a novel Tat coactivator that is required for Tat-mediated HIV-1 transcription and replication, and that it may contribute to HIV-1 reactivation in latently HIV-1 infected cells.