Raymond Yu

Regulation of Transcription Factor NFAT by ADP-Ribosylation

ABSTRACT ADP-ribosylation is a reversible posttranslational modification mediated by poly-ADP-ribose polymerase (PARP). The results of recent studies demonstrate that ADP-ribosylation contributes to transcription regulation. Here, we report that transcription factor NFAT binds to and is ADP-ribosylated by PARP-1 in an activation-dependent manner. Mechanistically, ADP-ribosylation increases NFAT DNA binding. Functionally, NFAT-mediated interleukin-2 (IL-2) expression was reduced in T cells upon genetic ablation or pharmacological inhibition of PARP-1. Parp-1−/− T cells also exhibit reduced expression of other NFAT-dependent cytokines, such as IL-4. Together, these results demonstrate that ADP-ribosylation mediated by PARP-1 provides a molecular switch to positively regulate NFAT-dependent cytokine gene transcription. These results also imply that, similar to the effect of calcineurin inhibition, PARP-1 inhibition may be beneficial in modulating immune functions.

BCL-6 Negatively Regulates Expression of the NF-κB1 p105/p50 Subunit

BCL-6 is a transcription repressor frequently deregulated in non-Hodgkin’s B cell lymphomas. Its activity is also critical to germinal center development and balanced Th1/Th2 differentiation. Previous studies have suggested that NF-κB activity is suppressed in germinal center and lymphoma B cells that express high levels of BCL-6, and yet the reason for this is unknown. We report in this study that BCL-6 can bind to three sequence motifs in the 5′ regulatory region of NF-κB1 in vitro and in vivo, and repress NF-κB1 transcription both in reporter assays and in lymphoma B cell lines. BCL-6−/− mice further confirm the biological relevance of BCL-6-dependent regulation of NF-κB1 because BCL-6 inactivation caused notable increase in p105/p50 proteins in several cell types. Among these, BCL-6−/− macrophage cell lines displayed a hyperproliferation phenotype that can be reversed by NF-κB inhibitors, e.g., N-tosyl-l-phenylalanine chloromethyl ketone and SN50, a result that is consistent with increased nuclear κB-binding activity of p50 homodimer and p50/p65 heterodimer. Our results demonstrate that BCL-6 can negatively regulate NF-κB1 expression, thereby inhibiting NF-κB-mediated cellular functions.

Role of Transcription Factor NFAT in Glucose and Insulin Homeostasis

ABSTRACT Compromised immunoregulation contributes to obesity and complications in metabolic pathogenesis. Here, we demonstrate that the nuclear factor of activated T cell (NFAT) group of transcription factors contributes to glucose and insulin homeostasis. Expression of two members of the NFAT family (NFATc2 and NFATc4) is induced upon adipogenesis and in obese mice. Mice with the Nfatc2−/−Nfatc4−/− compound disruption exhibit defects in fat accumulation and are lean. Nfatc2−/−Nfatc4−/− mice are also protected from diet-induced obesity. Ablation of NFATc2 and NFATc4 increases insulin sensitivity, in part, by sustained activation of the insulin signaling pathway. Nfatc2−/−Nfatc4−/− mice also exhibit an altered adipokine profile, with reduced resistin and leptin levels. Mechanistically, NFAT is recruited to the transcription loci and regulates resistin gene expression upon insulin stimulation. Together, these results establish a role for NFAT in glucose/insulin homeostasis and expand the repertoire of NFAT function to metabolic pathogenesis and adipokine gene transcription.

BCL6 suppresses RhoA activity to alter macrophage morphology and motility

BCL6 is a potent transcriptional repressor that plays important roles in germinal center formation, T helper cell differentiation and lymphomagenesis and regulates expression of several chemokine genes in macrophages. In a further investigation of its role in macrophages, we show that BCL6 inactivation in primary bone marrow-derived macrophages leads to decreased polarization, motility and cell spreading accompanied by an increase in peripheral focal complexes, anchored F-actin bundles and cortical F-actin density. These changes were associated with excess RhoA activation. C3 transferase inhibition of RhoA activity reverted the adhesion structure phenotype, which was not affected by Rho kinase inhibitors, suggesting that other downstream effectors of Rho maintain this Bcl6–/– phenotype. Excess RhoA activation in BCL6-deficient macrophages is associated with a decrease in the p120RasGAP (RASA1)-mediated translocation of p190RhoGAP (GRLF1) to active RhoA at the plasma membrane and a reduction in cell surface expression of the CSF1R that has been reported to recruit RasGAP to the plasma membrane. Reconstitution of BCL6 expression in Bcl6–/– macrophages results in complete reversion of the morphological phenotype and a significant increase in cell surface CSF1R expression whereas overexpression of the CSF1R corrects the polarization and adhesion structure defects. These results demonstrate that BCL6 suppresses RhoA activity, largely through upregulation of surface CSF1R expression, to modulate cytoskeletal and adhesion structures and increase the motility of macrophages.

Integration of protein kinases mTOR and extracellular signal-regulated kinase 5 in regulating nucleocytoplasmic localization of NFATc4.

ABSTRACT The target of rapamycin (TOR) signaling regulates the nucleocytoplasmic shuttling of transcription factors in yeast. Whether the mammalian counterpart of TOR (mTOR) also regulates nucleocytoplasmic shuttling is not known. Using a phospho-specific monoclonal antibody, we demonstrate that mTOR phosphorylates Ser168,170 of endogenous NFATc4, which are conserved gate-keeping Ser residues that control NFAT subcellular distribution. The mTOR acts as a basal kinase during the resting state to maintain NFATc4 in the cytosol. Inactivation and nuclear export of NFATc4 are mediated by rephosphorylation of Ser168,170, which can be a nuclear event. Kinetic analyses demonstrate that rephosphorylation of Ser168,170 of endogenous NFATc4 is mediated by mTOR and, surprisingly, by extracellular signal-regulated kinase 5 (ERK5) mitogen-activated protein kinase as well. Ablation of ERK5 in the Erk5−/− cells ascertains defects in NFATc4 rephosphorylation and nucleocytoplasmic shuttling. In addition, phosphorylation of NFATc4 by ERK5 primes subsequent phosphorylation mediated by CK1α. These results demonstrate that distinct protein kinases are integrated to phosphorylate the gate-keeping residues Ser168,170 of NFATc4, to regulate subcellular distribution. These data also expand the repertoire of physiological substrates of mTOR and ERK5.

A Naturally Occurring, Soluble Antagonist of Human IL-23 Inhibits the Development and In Vitro Function of Human Th17 Cells

Th17 CD4 cells are critical to inflammation. Their secretion of IL-17 drives inflammation in human diseases, including inflammatory bowel disease. Differentiation of mature Th17 cells depends on stimulation with IL-6, TGF-β, and IL-21 and the induction of RORγt, but IL-23 is essential to Th17 phenotype, stability, and function. Induction of Th17 cells can be antagonized by IL-4 or IFN-γ, but mechanisms through which terminal differentiation can be inhibited have not been identified. Human IL-23Rα (HuIL23Rα)-chain mRNA transcripts exist that lack exon 9 (“Δ9”); these are translated to a truncated receptor containing the entire external domain. This soluble variant of the HuIL23Rα-chain antagonizes Th17 maturation. It is secreted and present at low levels in the blood. It represents 10% of HuIL23Rα-chain mRNA, binds IL-23 in solution, and inhibits the phosphorylation of STAT3 caused by IL-23. In in vitro Th17 cell differentiation experiments, Δ9 inhibits the production of the Th17-associated cytokines IL-17A and IL-17F. Δ9 does not bind IL-12; thus, it is a specific inhibitor of IL-23 and a modulator of Th17 cells. Our results indicate that this soluble form of HuIL23Rα likely functions to regulate Th17 activity.

The lambda interferons: guardians of the immune-epithelial interface and the T-helper 2 response.

The type-III interferons (IFNs) are the most recently discovered IFNs in the human immune system and have important, but as yet poorly characterized, functions in innate and adaptive immunity that complement their antiviral functions. It is now becoming clear that these type-III IFNs have a functional niche where epithelial surfaces interact with the adaptive immune system, that their antiviral capability is not as highly developed as that of the type-I IFNs, and that they have their own profile of immunomodulatory functions; specifically, they are key modulators of the T-helper (Th)2 response.

Evolutionarily conserved role of calcineurin in phosphodegron-dependent degradation of phosphodiesterase 4D.

ABSTRACT Calcineurin is a widely expressed and highly conserved Ser/Thr phosphatase. Calcineurin is inhibited by the immunosuppressant drug cyclosporine A (CsA) or tacrolimus (FK506). The critical role of CsA/FK506 as an immunosuppressant following transplantation surgery provides a strong incentive to understand the phosphatase calcineurin. Here we uncover a novel regulatory pathway for cyclic AMP (cAMP) signaling by the phosphatase calcineurin which is also evolutionarily conserved in Caenorhabditis elegans. We found that calcineurin binds directly to and inhibits the proteosomal degradation of cAMP-hydrolyzing phosphodiesterase 4D (PDE4D). We show that ubiquitin conjugation and proteosomal degradation of PDE4D are controlled by a cullin 1-containing E3 ubiquitin ligase complex upon dual phosphorylation by casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β) in a phosphodegron motif. Our findings identify a novel signaling process governing G-protein-coupled cAMP signal transduction—opposing actions of the phosphatase calcineurin and the CK1/GSK3β protein kinases on the phosphodegron-dependent degradation of PDE4D. This novel signaling system also provides unique functional insights into the complications elicited by CsA in transplant patients.

Ablation of Calcineurin Aβ Reveals Hyperlipidemia and Signaling Cross-talks with Phosphodiesterases

Background: Transplant patients treated with cyclosporine A, an inhibitor of calcineurin, frequently develop metabolic complications. Results: Mice lacking calcineurin Aβ develop metabolic complications. A cell autonomous mechanism with altered cAMP-PKA signaling is found. Conclusion: Calcineurin plays a role in metabolism via regulating phosphodiesterases. Significance: Potentiated cAMP signaling provides an alternative mechanism for the metabolic complications in transplant patients. Insulin resistance, hyperlipidemia, and cardiovascular complications are common dysregulations of metabolic syndrome. Transplant patients treated with immunosuppressant drugs such as cyclosporine A (CsA), an inhibitor of calcineurin phosphatase, frequently develop similar metabolic complications. Although calcineurin is known to mediate insulin sensitivity by regulating β-cell growth and adipokine gene transcription, its role in lipid homeostasis is poorly understood. Here, we examined lipid homeostasis in mice lacking calcineurin Aβ (CnAβ−/−). We show that mice lacking calcineurin Aβ are hyperlipidemic and develop age-dependent insulin resistance. Hyperlipidemia found in CnAβ−/− mice is, in part, due to increased lipolysis in adipose tissues, a process mediated by β-adrenergic G-protein-coupled receptor signaling pathways. CnAβ−/− mice also exhibit additional pathophysiological phenotypes caused by the potentiated GPCR signaling pathways. A cell autonomous mechanism with sustained cAMP/PKA activation is found in CnAβ−/− mice or upon CsA treatment to inhibit calcineurin. Increased PKA activation and cAMP accumulation in CnAβ−/− mice, however, are sensitive to phosphodiesterase inhibitor. Indeed, we show that calcineurin regulates degradation of phosphodiesterase 3B, in addition to phosphodiesterase 4D. These results establish a role for calcineurin in lipid homeostasis. These data also indicate that potentiated cAMP signaling pathway may provide an alternative molecular pathogenesis for the metabolic complications elicited by CsA in transplant patients.

The Human IL-23 Receptor rs11209026 A Allele Promotes the Expression of a Soluble IL-23R–Encoding mRNA Species

The human IL23R gene single nucleotide polymorphism rs11209026 A allele confers protection against inflammatory diseases. However, although this difference has been associated with reductions in IL-23–induced IL-17A production and STAT3 phosphorylation, the molecular mechanism underlying these changes remains undefined. Th17 cell maturation depends on IL-23 signaling. Multiple splice forms of the human IL23R transcript exist, and one, Δ9, encodes a soluble form of the receptor. In this study, we asked whether this protective allele was associated with mRNA splicing. Using mini-gene constructs and competitive oligonucleotide binding, we showed that the A allele alters IL-23R α-chain mRNA splicing and favors exon 9 skipping by reducing the binding of the splicing enhancer SF2. This enhances expression of the Δ9 mRNA and consequently diminishes IL-23 signaling. Thus, the presence of the A allele increases expression of the soluble form of IL23R mRNA (which then functions as a decoy receptor) and lowers the ability to develop a Th17 phenotype upon IL-23 stimulation. We further showed that antisense oligonucleotides targeting the SF2 binding site could efficiently induce exon 9 skipping in the presence of the G allele, and thereby replicate the effect of the A allele. Antisense oligonucleotide treatment caused dose-responsive induction of the IL23RΔ9 mRNA and interfered with in vitro differentiation of human Th17 cells, reducing their expression of the signature Th17 cytokines IL-17A and IL-17F. This may represent a novel approach to therapy of Th17-mediated diseases by elevating soluble IL-23R while simultaneously reducing the remaining cell surface receptor density.