Pseudorabies Virus Infection Results in a Broad Inhibition of Host Gene Transcription.

Pseudorabies virus (PRV) is a porcine alphaherpesvirus that belongs to the Herpesviridae family. We showed earlier that infection of porcine epithelial cells with PRV triggers activation of the nuclear factor κB (NF-κB) pathway, a pivotal signaling axis in the early immune response. However, PRV-induced NF-κB activation does not lead to NF-κB-dependent gene expression. Here, using electrophoretic mobility shift assays (EMSAs), we show that PRV does not disrupt the ability of NF-κB to interact with its κB target sites. Assessing basal cellular transcriptional activity in PRV-infected cells by quantitation of prespliced transcripts of constitutively expressed genes uncovered a broad suppression of cellular transcription by PRV, which also affects the inducible expression of NF-κB target genes. Host cell transcription inhibition was rescued when viral genome replication was blocked using phosphonoacetic acid (PAA). Remarkably, we found that host gene expression shutoff in PRV-infected cells correlated with a substantial retention of the NF-κB subunit p65, the TATA box binding protein, and RNA polymerase II-essential factors required for (NF-κB-dependent) gene transcription-in expanding PRV replication centers in the nucleus and thereby away from the host chromatin. This study reveals a potent mechanism used by the alphaherpesvirus PRV to steer the protein production capacity of infected cells to viral proteins by preventing expression of host genes, including inducible genes involved in mounting antiviral responses. IMPORTANCE Herpesviruses are highly successful pathogens that cause lifelong persistent infections of their host. Modulation of the intracellular environment of infected cells is imperative for the success of virus infections. We reported earlier that a DNA damage response in epithelial cells infected with the alphaherpesvirus pseudorabies virus (PRV) results in activation of the hallmark proinflammatory NF-κB signaling axis but, remarkably, that this activation does not lead to NF-κB-induced (proinflammatory) gene expression. Here, we report that PRV-mediated inhibition of host gene expression stretches beyond NF-κB-dependent gene expression and in fact reflects a broad inhibition of host gene transcription, which correlates with a substantial recruitment of essential host transcription factors in viral replication compartments in the nucleus, away from the host chromatin. These data uncover a potent alphaherpesvirus mechanism to interfere with production of host proteins, including proteins involved in antiviral responses.

IκBα Nuclear Export Enables 4-1BB-Induced cRel Activation and IL-2 Production to Promote CD8 T Cell Immunity.

Optimal CD8 T cell immunity is orchestrated by signaling events initiated by TCR recognition of peptide Ag in concert with signals from molecules such as CD28 and 4-1BB. The molecular mechanisms underlying the temporal and spatial signaling dynamics in CD8 T cells remain incompletely understood. In this study, we show that stimulation of naive CD8 T cells with agonistic CD3 and CD28 Abs, mimicking TCR and costimulatory signals, coordinately induces 4-1BB and cRel to enable elevated cytosolic cRel:IκBα complex formation and subsequent 4-1BB-induced IκBα degradation, sustained cRel activation, heightened IL-2 production and T cell expansion. NfkbiaNES/NES CD8 T cells harboring a mutated IκBα nuclear export sequence abnormally accumulate inactive cRel:IκBα complexes in the nucleus following stimulation with agonistic anti-CD3 and anti-CD28 Abs, rendering them resistant to 4-1BB induced signaling and a disrupted chain of events necessary for efficient T cell expansion. Consequently, CD8 T cells in NfkbiaNES/NES mice poorly expand during viral infection, and this can be overcome by exogenous IL-2 administration. Consistent with cell-based data, adoptive transfer experiments demonstrated that the antiviral CD8 T cell defect in NfkbiaNES/NES mice was cell intrinsic. Thus, these results reveal that IκBα, via its unique nuclear export function, enables, rather than inhibits 4-1BB-induced cRel activation and IL-2 production to facilitate optimal CD8 T cell immunity.

Nuclear export of the NF-κB inhibitor IκBα is required for proper B cell and secondary lymphoid tissue formation.

The N-terminal nuclear export sequence (NES) of inhibitor of nuclear factor kappa B (NF-κB) alpha (IκBα) promotes NF-κB export from the cell nucleus to the cytoplasm, but the physiological role of this export regulation remains unknown. Here we report the derivation and analysis of genetically targeted mice harboring a germline mutation in IκBα NES. Mature B cells in the mutant mice displayed nuclear accumulation of inactive IκBα complexes containing a NF-κB family member, cRel, causing their spatial separation from the cytoplasmic IκB kinase. This resulted in severe reductions in constitutive and canonical NF-κB activities, synthesis of p100 and RelB NF-κB members, noncanonical NF-κB activity, NF-κB target gene induction, and proliferation and survival responses in B cells. Consequently, mice displayed defective B cell maturation, antibody production, and formation of secondary lymphoid organs and tissues. Thus, IκBα nuclear export is essential to maintain constitutive, canonical, and noncanonical NF-κB activation potentials in mature B cells in vivo.

Hyaluronan and proteoglycan link protein 1 (HAPLN1) activates bortezomib-resistant NF-κB activity and increases drug resistance in multiple myeloma.

Nuclear factor-κB (NF-κB) is a family of transcription factors that play a key role in cell survival and proliferation in many hematological malignancies, including multiple myeloma (MM). Bortezomib, a proteasome inhibitor used in the management of MM, can inhibit both canonical and noncanonical activation of NF-κB in MM cells. However, we previously reported that a significant fraction of freshly isolated MM cells harbor bortezomib-resistant NF-κB activity. Here, we report that hyaluronan and proteoglycan link protein 1 (HAPLN1) is produced in bone marrow stromal cells from MM patients, is detected in patients' bone marrow plasma, and can activate an atypical bortezomib-resistant NF-κB pathway in MM cells. We found that this pathway involves bortezomib-resistant degradation of the inhibitor of NF-κB (IκBα), despite efficient bortezomib-mediated inhibition of proteasome activity. Moreover, HAPLN1 can also confer bortezomib-resistant survival of MM cells. We propose that HAPLN1 is a novel pathogenic factor in MM that induces an atypical NF-κB activation and thereby promotes bortezomib resistance in MM cells.

IPO3-mediated Nonclassical Nuclear Import of NF-κB Essential Modulator (NEMO) Drives DNA Damage-dependent NF-κB Activation.

Activation of IκB kinase (IKK) and NF-κB by genotoxic stresses modulates apoptotic responses and production of inflammatory mediators, thereby contributing to therapy resistance and premature aging. We previously reported that genotoxic agents induce nuclear localization of NF-κB essential modulator (NEMO) via an undefined mechanism to arbitrate subsequent DNA damage-dependent IKK/NF-κB signaling. Here we show that a nonclassical nuclear import pathway via IPO3 (importin 3, transportin 2) mediates stress-induced NEMO nuclear translocation. We found putative nuclear localization signals in NEMO whose mutations disrupted stress-inducible nuclear translocation of NEMO and IKK/NF-κB activation in stably reconstituted NEMO-deficient cells. RNAi screening of both importin α and ß family members, as well as co-immunoprecipitation analyses, revealed that a nonclassical importin ß family member, IPO3, was the only importin that was able to associate with NEMO and whose reduced expression prevented genotoxic stress-induced NEMO nuclear translocation, IKK/NF-κB activation, and inflammatory cytokine transcription. Recombinant IPO3 interacted with recombinant NEMO but not the nuclear localization signal mutant version and induced nuclear import of NEMO in digitonin-permeabilized cells. We also provide evidence that NEMO is disengaged from IKK complex following genotoxic stress induction. Thus, the IPO3 nuclear import pathway is an early and crucial determinant of the IKK/NF-κB signaling arm of the mammalian DNA damage response.

Withaferin A disrupts ubiquitin-based NEMO reorganization induced by canonical NF-κB signaling.

The NF-κB family of transcription factors regulates numerous cellular processes, including cell proliferation and survival responses. The constitutive activation of NF-κB has also emerged as an important oncogenic driver in many malignancies, such as activated B-cell like diffuse large B cell lymphoma, among others. In this study, we investigated the impact and mechanisms of action of Withaferin A, a naturally produced steroidal lactone, against both signal-inducible as well as constitutive NF-κB activities. We found that Withaferin A is a robust inhibitor of canonical and constitutive NF-κB activities, leading to apoptosis of certain lymphoma lines. In the canonical pathway induced by TNF, Withaferin A did not disrupt RIP1 polyubiquitination or NEMO-IKKß interaction and was a poor direct IKKß inhibitor, but prevented the formation of TNF-induced NEMO foci which colocalized with TNF ligand. While GFP-NEMO efficiently formed TNF-induced foci, a GFP-NEMO(Y308S) mutant that is defective in binding to polyubiquitin chains did not form foci. Our study reveals that Withaferin A is a novel type of IKK inhibitor which acts by disrupting NEMO reorganization into ubiquitin-based signaling structures in vivo.

PIASy mediates NEMO sumoylation and NF-kappaB activation in response to genotoxic stress.

Protein modification by SUMO (small ubiquitin-like modifier) is an important regulatory mechanism for multiple cellular processes. SUMO-1 modification of NEMO (NF-kappaB essential modulator), the IkappaB kinase (IKK) regulatory subunit, is critical for activation of NF-kappaB by genotoxic agents. However, the SUMO ligase, and the mechanisms involved in NEMO sumoylation, remain unknown. Here, we demonstrate that although small interfering RNAs (siRNAs) against PIASy (protein inhibitor of activated STATy) inhibit NEMO sumoylation and NF-kappaB activation in response to genotoxic agents, overexpression of PIASy enhances these events. PIASy preferentially stimulates site-selective modification of NEMO by SUMO-1, but not SUMO-2 and SUMO-3, in vitro. PIASy-NEMO interaction is increased by genotoxic stress and occurs in the nucleus in a manner mutually exclusive with IKK interaction. In addition, hydrogen peroxide (H2O2) also increases PIASy-NEMO interaction and NEMO sumoylation, whereas antioxidants prevent these events induced by DNA-damaging agents. Our findings demonstrate that PIASy is the first SUMO ligase for NEMO whose substrate specificity seems to be controlled by IKK interaction, subcellular targeting and oxidative stress conditions.

Abstraction of core measure data: creating a process for interrater reliability.

Health care facilities are challenged with an ever-increasing demand for producing accurate quality data to be used for guiding internal improvement initiatives as well as for reimbursement. It is essential that data abstraction be reliable and valid. In this article, we describe an interrater reliability process of data abstraction using the Centers for Medicare and Medicaid Services core measures that successfully reduced variability between abstractors and produced higher quality data.

Several Alphaherpesviruses Interact Similarly with the NF-κB Pathway and Suppress NF-κB-Dependent Gene Expression.

Alphaherpesvirus infection is associated with attenuation of different aspects of the host innate immune response that is elicited to confine primary infections at the mucosal epithelia. Here, we report that infection of epithelial cells with several alphaherpesviruses of different species, including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), feline alphaherpesvirus 1 (FHV-1), and bovine alphaherpesvirus 1 (BoHV-1) results in the inactivation of the responses driven by the nuclear factor kappa B (NF-κB) pathway, considered a pillar of the innate immune response. The mode to interact with and circumvent NF-κB-driven responses in infected epithelial cells is seemingly conserved in human, feline, and porcine alphaherpesviruses, consisting of a persistent activation of the NF-κB cascade but a potent repression of NF-κB-dependent transcription activity, which relies on replication of viral genomes. However, BoHV-1 apparently deviates from the other investigated members of the taxon in this respect, as BoHV-1-infected epithelial cells do not display the persistent NF-κB activation observed for the other alphaherpesviruses. In conclusion, this study suggests that inhibition of NF-κB transcription activity is a strategy used by several alphaherpesviruses to prevent NF-κB-driven responses in infected epithelial cells. IMPORTANCE The current study provides a side-by-side comparison of the interaction of different alphaherpesviruses with NF-κB, a key and central player in the (proinflammatory) innate host response, in infected nontransformed epithelial cell lines. We report that all studied viruses prevent expression of the hallmark NF-κB-dependent gene IκB, often but not always via similar strategies, pointing to suppression of NF-κB-dependent host gene expression in infected epithelial cells as a common and therefore likely important aspect of alphaherpesviruses.

Ectopic CH60 mediates HAPLN1-induced cell survival signaling in multiple myeloma.

Multiple myeloma (MM), the second most common hematological malignancy, is generally considered incurable because of the development of drug resistance. We previously reported that hyaluronan and proteoglycan link protein 1 (HAPLN1) produced by stromal cells induces activation of NF-κB, a tumor-supportive transcription factor, and promotes drug resistance in MM cells. However, the identity of the cell surface receptor that detects HAPLN1 and thereby engenders pro-tumorigenic signaling in MM cells remains unknown. Here, we performed an unbiased cell surface biotinylation assay and identified chaperonin 60 (CH60) as the direct binding partner of HAPLN1 on MM cells. Cell surface CH60 specifically interacted with TLR4 to evoke HAPLN1-induced NF-κB signaling, transcription of anti-apoptotic genes, and drug resistance in MM cells. Collectively, our findings identify a cell surface CH60-TLR4 complex as a HAPLN1 receptor and a potential molecular target to overcome drug resistance in MM cells.

CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein.

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wild-type NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU-423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.

Exercise and calcium supplementation: effects on calcium homeostasis in sportswomen.

PURPOSE: Exercise-induced sweat calcium losses have been reported as substantial in male athletes. The first aim of the study was to quantify the increase in 24-h total dermal calcium losses and the net changes in calcium retention in active sportswomen after a 1-h strenuous exercise session. A second aim was to determine the effectiveness of calcium supplementation to offset any calcium loss. METHODS: Twenty-six premenopausal sportswomen completed three 8-d intervention phases in a randomized-order, crossover design. The three phases were placebo+no exercise (control), placebo+exercise, and 400 mg of calcium as calcium carbonate (TUMS Ultra) twice daily+exercise. The supervised exercise was 1 h.d(-1) cycling at 65-70% of heart rate reserve. A controlled diet of approximately 450 mg.d(-1) of calcium and 24-h pooled urine and fecal collections allowed determination of calcium balance on days 5-8 of each phase. Twenty-four-hour dermal collections were made at the end of each phase using a whole-body washdown procedure. RESULTS: Exercise increased (P<0.05) dermal calcium losses (means+/-SD, 92+/-49 vs 79+/-31 mg.d(-1) in the nonexercise intervention period), which was no longer significant (P=0.14) when calcium supplementation was provided (83+/-49 mg.d(-1)). Higher (P<0.01) urinary calcium excretion during calcium supplementation is suggestive of higher net calcium absorption. Exercise did not affect urinary calcium excretion indicating lack of compensation for dermal losses. Net calcium retention was positive only during the exercise+calcium supplementation intervention period. CONCLUSIONS: Calcium supplementation can correct for negative calcium balance attributable to low calcium dietary intake and additional dermal losses from exercise.

Enhanced G2-M arrest by nuclear factor-{kappa}B-dependent p21waf1/cip1 induction.

The transcription factor nuclear factor-kappaB (NF-kappaB) regulates cell survival pathways, but the molecular mechanisms involved are not completely understood. Here, we developed a NF-kappaB reporter cell system derived from CEM T leukemic cells to monitor the consequences of NF-kappaB activation following DNA damage insults. Cells that activated NF-kappaB in response to ionizing radiation or etoposide arrested in the G2-M phase for a prolonged time, which was followed by increased cell cycle reentry and survival. In contrast, those that failed to activate NF-kappaB underwent transient G2-M arrest and extensive cell death. Importantly, p21waf1/cip1 was induced in S-G2-M phases in a NF-kappaB-dependent manner, and RNA interference of this cell cycle regulator reduced the observed NF-kappaB-dependent phenotypes. Thus, cell cycle-coupled induction of p21waf1/cip1 by NF-kappaB represents a resistance mechanism in certain cancer cells.

Mu-calpain activation in beta-lapachone-mediated apoptosis.

Beta-lapachone (beta-Lap) triggers apoptosis in a number of human breast and prostate cancer cell lines through a unique apoptotic pathway that is dependent upon NQO1, a two-electron reductase. Recently, our laboratory showed that beta-lap-exposed MCF-7 cells exhibited an early increase in intracellular cytosolic Ca(2+) from endoplasmic reticulum stores, and that BAPTA-AM (an intracellular Ca(2+) chelator) blocked these early increases and partially inhibited all aspects of beta-lap-induced apoptosis. We now show that exposure of NQO1-expressing breast cancer cells to beta-lap stimulates a unique proteolytic apoptotic pathway involving mu-calpain activation. No apparent activation of m-calpain was noted. Upon activation, mu-calpain translocated to the nucleus concomitant with specific nuclear proteolytic events. Apoptotic responses in beta-lap-exposed NQO1-expressing cells were significantly delayed and survival enhanced by exogenous over-expression of calpastatin, a natural inhibitor of mu- and m-calpains. Furthermore, purified mu-calpain cleaved PARP to a unique fragment (approximately 60 kDa), not previously reported for calpains. We provide evidence that beta-lap-induced, mu-calpain-stimulated apoptosis does not involve any known apoptotic caspases; the activated fragments of caspases were not observed after beta-lap exposures, nor were there any changes in the pro-enzyme forms as measured by Western blot analyses. The ability of beta-lap to trigger an apparently novel, p53-independent, calpain-mediated apoptotic cell death further support the development of this drug for improved breast cancer therapy.

A novel pathway links oxidative stress to loss of insulin growth factor-2 (IGF2) imprinting through NF-κB activation.

Genomic imprinting is the allele-specific expression of a gene based on parental origin. Loss of imprinting(LOI) of Insulin-like Growth Factor 2 (IGF2) during aging is important in tumorigenesis, yet the regulatory mechanisms driving this event are largely unknown. In this study oxidative stress, measured by increased NF-κB activity, induces LOI in both cancerous and noncancerous human prostate cells. Decreased expression of the enhancer-blocking element CCCTC-binding factor(CTCF) results in reduced binding of CTCF to the H19-ICR (imprint control region), a major factor in the allelic silencing of IGF2. This ICR then develops increased DNA methylation. Assays identify a recruitment of the canonical pathway proteins NF-κB p65 and p50 to the CTCF promoter associated with the co-repressor HDAC1 explaining gene repression. An IκBα super-repressor blocks oxidative stress-induced activation of NF-κB and IGF2 imprinting is maintained. In vivo experiments using IκBα mutant mice with continuous NF-κB activation demonstrate increased IGF2 LOI further confirming a central role for canonical NF-κB signaling. We conclude CTCF plays a central role in mediating the effects of NF-κB activation that result in altered imprinting both in vitro and in vivo. This novel finding connects inflammation found in aging prostate tissues with the altered epigenetic landscape.

Chromatin-bound IκBα regulates a subset of polycomb target genes in differentiation and cancer.

IκB proteins are the primary inhibitors of NF-κB. Here, we demonstrate that sumoylated and phosphorylated IκBα accumulates in the nucleus of keratinocytes and interacts with histones H2A and H4 at the regulatory region of HOX and IRX genes. Chromatin-bound IκBα modulates Polycomb recruitment and imparts their competence to be activated by TNFα. Mutations in the Drosophila IκBα gene cactus enhance the homeotic phenotype of Polycomb mutants, which is not counteracted by mutations in dorsal/NF-κB. Oncogenic transformation of keratinocytes results in cytoplasmic IκBα translocation associated with a massive activation of Hox. Accumulation of cytoplasmic IκBα was found in squamous cell carcinoma (SCC) associated with IKK activation and HOX upregulation.

TAK-ling IKK activation: "Ub" the judge.

Investigation of the signaling events that lead to the activation of the transcription factor nuclear factor kappaB (NF-kappaB) has been a hotbed for the discovery of previously uncharacterized signaling mechanisms. The important role that nondegradative polyubiquitin chains play in these processes is now well recognized; however, precisely how they orchestrate NF-kappaB signaling is still a matter of much controversy. A recent study has challenged the dogmatic view by demonstrating that interleukin-1beta (IL-1beta), a major proinflammatory cytokine, activates two consecutive pathways, the "RING" and "zinc" pathways, to coordinate early and late activation of NF-kappaB, respectively. This study introduces a paradigm shift in the still-evolving mechanism of regulation of NF-kappaB.

Mornings with Art, lessons learned: feedback regulation, restriction threshold biology, and redundancy govern molecular stress responses.

Work from the laboratory of Dr. Arthur B. Pardee has highlighted basic principles that govern cellular and molecular biological processes in living cells. Among the most important governing principles in cellular and molecular responses are: (i) threshold "restriction" responses, wherein a level of response is reached and a "point of no return" is achieved; (ii) feedback regulation; and (iii) redundancy. Lessons learned from the molecular biology of cellular stress responses in mammalian cancer versus normal cells after ionizing radiation (IR) or chemotherapeutic agent exposures reveal similar instances of these guiding principles in mammalian cells. Among these are the: (i) induction of cell death responses by beta-lapachone (beta-lap), a naphthoquinone anti-tumor agent that kills cancer cells via an NQO1 (i.e., X-ray-inducible protein-3, xip3)-dependent mechanism; (ii) induction of secretory clusterin (sCLU) in response to TGF-beta1 exposure, and the ability of induced sCLU protein to down-regulate TGF-beta1 signaling; and (iii) induction of DNA mismatch repair-dependent G(2) cell cycle checkpoint responses after exposure to alkylating agents. We have learned these lessons and now adopted strategies to exploit them for improved therapy. These examples will be discussed and compared to the pioneering findings of researchers in the Pardee laboratory over the years.