Faculty Bibliography

Akt (= protein kinase B), a subfamily of the AGC serine/threonine kinases, plays critical roles in survival, proliferation, glucose metabolism, and other cellular functions. Akt activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of phosphatidylinositol 3-kinase. Membrane-bound Akt is then phosphorylated at two sites for its full activation; Thr-308 in the activation loop of the kinase domain is phosphorylated by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 in the C-terminal hydrophobic motif by a putative kinase PDK2. The identity of PDK2 has been elusive. Here we present evidence that conventional isoforms of protein kinase C (PKC), particularly PKCbetaII, can regulate Akt activity by directly phosphorylating Ser-473 in vitro and in IgE/antigen-stimulated mast cells. By contrast, PKCbeta is not required for Ser-473 phosphorylation in mast cells stimulated with stem cell factor or interleukin-3, in serum-stimulated fibroblasts, or in antigen receptor-stimulated T or B lymphocytes. Therefore, PKCbetaII appears to work as a cell type- and stimulus-specific PDK2

The chemokine receptor CXCR4 is expressed in B cells at multiple stages of their development. CXCR4 function in humoral immunity has not been fully investigated. We have generated gene-targeted mice in which CXCR4 can be selectively inactivated in B cells and have shown that it is required for retention of B cell precursors in the bone marrow. CXCR4-deficient B cell precursors that migrated prematurely became localized in splenic follicles despite their unresponsiveness to CXCL13. Concomitantly, mature B cell populations were reduced in the splenic marginal zone and primary follicles, and in the peritoneal cavity in the mutant animals, as were T-independent antibody responses. In addition, aberrant B cell follicles formed ectopically in intestinal lamina propria around Peyer's patches. These findings establish an important role for CXCR4 in regulating homeostasis of B cell compartmentalization and humoral immunity

Development of a mouse model for human immunodeficiency virus type 1 (HIV-1) infection has advanced through the progressive identification of host cell factors required for HIV-1 replication. Murine cells lack HIV-1 receptor molecules, do not support efficient viral gene expression, and lack factors necessary for the assembly and release of virions. Many of these blocks have been described using mouse fibroblast cell lines. Here we identify a postentry block to HIV-1 infection in mouse T-cell lines that has not been detected in mouse fibroblasts. While murine fibroblastic lines are comparable to human T-cell lines in permissivity to HIV-1 transduction, infection of murine T cells is 100-fold less efficient. Virus entry occurs efficiently in murine T cells. However, reduced efficiency of the completion of reverse transcription and nuclear transfer of the viral preintegration complex are observed. Although this block has similarities to the restriction of murine retroviruses by Fv1, there is no correlation of HIV-1 susceptibility with cellular Fv1 genotypes. In addition, the block to HIV-1 infection in murine T-cell lines cannot be saturated by a high virus dose. Further studies of this newly identified block may lend insight into the early events of retroviral replication and reveal new targets for antiretroviral interventions

Obesity and stress inhibit insulin action by activating protein kinases that enhance serine phosphorylation of IRS1 and have been thus associated to insulin resistance and the development of type II diabetes. The protein kinase C (PKC) is activated by free-fatty acids, and its activity is higher in muscle from obese diabetic patients. However, a molecular link between PKC and insulin resistance has not been defined yet. Here we show that PKC phosphorylates IRS1 at serine 1101 blocking IRS1 tyrosine phosphorylation and downstream activation of the Akt pathway. Mutation of Ser(1101) to alanine makes IRS1 insensitive to the effect of PKC and restores insulin signaling in culture cells. These results provide a novel mechanism linking the activation of PKC to the inhibition of insulin signaling

Insulin resistance plays a primary role in the development of type 2 diabetes and may be related to alterations in fat metabolism. Recent studies have suggested that local accumulation of fat metabolites inside skeletal muscle may activate a serine kinase cascade involving protein kinase C-theta (PKC-theta), leading to defects in insulin signaling and glucose transport in skeletal muscle. To test this hypothesis, we examined whether mice with inactivation of PKC-theta are protected from fat-induced insulin resistance in skeletal muscle. Skeletal muscle and hepatic insulin action as assessed during hyperinsulinemic-euglycemic clamps did not differ between WT and PKC-theta KO mice following saline infusion. A 5-hour lipid infusion decreased insulin-stimulated skeletal muscle glucose uptake in the WT mice that was associated with 40-50% decreases in insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-1-associated PI3K activity. In contrast, PKC-theta inactivation prevented fat-induced defects in insulin signaling and glucose transport in skeletal muscle. In conclusion, our findings demonstrate that PKC-theta is a crucial component mediating fat-induced insulin resistance in skeletal muscle and suggest that PKC-theta is a potential therapeutic target for the treatment of type 2 diabetes

The serine/threonine-specific protein kinase C (PKC)-theta is predominantly expressed in T cells and localizes to the center of the immunological synapse upon T cell receptor (TCR) and CD28 signaling. T cells deficient in PKC-theta exhibit reduced interleukin (IL)-2 production and proliferative responses in vitro, however, its significance in vivo remains unclear. We found that pkc-theta(-/-) mice were protected from pulmonary allergic hypersensitivity responses such as airway hyperresponsiveness, eosinophilia, and immunoglobulin E production to inhaled allergen. Furthermore, T helper (Th)2 cell immune responses against Nippostrongylus brasiliensis were severely impaired in pkc-theta(-/-) mice. In striking contrast, pkc-theta(-/-) mice on both the C57BL/6 background and the normally susceptible BALB/c background mounted protective Th1 immune responses and were resistant against infection with Leishmania major. Using in vitro TCR transgenic T cell-dendritic cell coculture systems and antigen concentration-dependent Th polarization, PKC-theta-deficient T cells were found to differentiate into Th1 cells after activation with high concentrations of specific peptide, but to have compromised Th2 development at low antigen concentration. The addition of IL-2 partially reconstituted Th2 development in pkc-theta(-/-) T cells, consistent with an important role for this cytokine in Th2 polarization. Taken together, our results reveal a central role for PKC-theta signaling during Th2 responses

Intestinal intraepithelial T lymphocytes (IELs) are likely to play a key role in host mucosal immunity and, unlike other T cells, have been proposed to differentiate from local precursors rather than from thymocytes. We show here that IELs expressing the alphabeta T cell receptor are derived from precursors that express RORgammat, an orphan nuclear hormone receptor detected only in immature CD4+CD8+ thymocytes, fetal lymphoid tissue-inducer (LTi) cells, and LTi-like cells in cryptopatches within the adult intestinal lamina propria. Using cell fate mapping, we found that all intestinal alphabeta T cells are progeny of CD4+CD8+ thymocytes, indicating that the adult intestine is not a significant site for alphabeta T cell development. Our results suggest that intestinal RORgammat+ cells are local organizers of mucosal lymphoid tissue

Runx family proteins have the potential for either activating or suppressing gene expression in a context-dependent manner. There are several mechanisms by which transcriptional repression can occur. A wide range of locus inactivation, that is often called gene silencing, is thought to be achieved by chromatin modifications. Recently, Runx family proteins were found to have an essential role in either temporal transcriptional repression or irreversible epigenetic silencing at the CD4 locus through binding to a CD4 silencer at different stages of development. These findings link Runx function to epigenetic gene regulation, and shed new light on the mechanisms by which Runx represses target gene expression

In this study, we analyzed the phenotypic and physiological consequences of the interaction of plasmacytoid dendritic cells (pDCs) with human immunodeficiency virus type 1 (HIV-1). pDCs are one cellular target of HIV-1 and respond to the virus by producing alpha/beta interferon (IFN-alpha/beta) and chemokines. The outcome of this interaction, notably on the function of bystander myeloid DC (CD11c+ DCs), remains unclear. We therefore evaluated the effects of HIV-1 exposure on these two DC subsets under various conditions. Blood-purified pDCs and CD11c+ DCs were exposed in vitro to HIV-1, after which maturation markers, cytokine production, migratory capacity, and CD4 T-cell stimulatory capacity were analyzed. pDCs exposed to different strains of infectious or even chemically inactivated, nonreplicating HIV-1 strongly upregulated the expression of maturation markers, such as CD83 and functional CCR7, analogous to exposure to R-848, a synthetic agonist of toll-like receptor-7 and -8. In addition, HIV-1-activated pDCs produced cytokines (IFN-alpha and tumor necrosis factor alpha), migrated in response to CCL19 and, in coculture, matured CD11c+ DCs, which are not directly activated by HIV. pDCs also acquired the ability to stimulate naive CD4+ T cells, albeit less efficiently than CD11c+ DCs. This HIV-1-induced maturation of both DC subsets may explain their disappearance from the blood of patients with high viral loads and may have important consequences on HIV-1 cellular transmission and HIV-1-specific T-cell responses