IKKα represses a network of inflammation and proliferation pathways and elevates c-Myc antagonists and differentiation in a dose-dependent manner in the skin.

Inhibitor of nuclear factor κB kinase-α (IKKα) is required for maintaining skin homeostasis and preventing skin tumorigenesis. However, its signaling has not been extensively investigated. In the present study, we generated two mouse lines that expressed different levels of transgenic IKKα in the basal epidermis under the control of keratin-5 promoter and further evaluated their effects on the major pathways of inflammation, proliferation, and differentiation in the skin. Regardless of the transgenic IKKα levels, the mice develop normally. Because IKKα deletion in keratinocytes blocks terminal differentiation and induces epidermal hyperplasia and skin inflammation, we depleted the endogenous IKKα in these transgenic mice and found that the transgenic IKKα represses epidermal thickness and induces terminal differentiation in a dose-dependent manner. Also, transgenic IKKα was found to elevate expression of Max dimer protein 1 (Mad1) and ovo-like 1, c-Myc antagonists, but repress activities of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK), Jun-amino-terminal kinases, c-Jun, signal transducer and activator of transcription 3 (Stat3), and growth factor levels in a dose-dependent fashion in the skin. Moreover, EGFR reduction represses IKKα deletion-induced excessive ERK, Stat3 and c-Jun activities, and skin inflammation. These new findings indicate that elevated IKKα expression not only represses epidermal thickness and induces terminal differentiation, but also suppresses skin inflammation by an integrated loop. Thus, IKKα maintains skin homeostasis through a broad range of signaling pathways.

Interaction of Ly-49D+ NK cells with H-2Dd target cells leads to Dap-12 phosphorylation and IFN-gamma secretion.

Murine Ly-49D augments NK cell function upon recognition of target cells expressing H-2Dd. Ly-49D activation is mediated by the immunoreceptor tyrosine-based activation motif-containing signaling moiety Dap-12. In this report we demonstrate that Ly-49D receptor ligation can lead to the rapid and potent secretion of IFN-gamma. Cytokine secretion can be induced from Ly-49D+ NK cells after receptor ligation with Ab or after interaction with target cells expressing their H-2Dd ligand. Consistent with the dominant inhibitory function of Ly-49G, NK cells coexpressing Ly-49D and Ly-49G show a profound reduction in IFN-gamma secretion after interaction with targets expressing their common ligand, H-2Dd. Importantly, we are able to demonstrate for the first time that effector/target cell interactions using Ly-49D+ NK cells and H-2Dd targets result in the rapid phosphorylation of Dap-12. However, Dap-12 is not phosphorylated when Ly-49D+ NK cells coexpress the inhibitory receptor, Ly-49G. These studies are novel in describing Ly-49 activation vs inhibition, where two Ly-49 receptors recognize the same class I ligand, with the dominant inhibitory receptor down-regulating phosphorylation of Dap-12, cytokine secretion, and cytotoxicity in NK cells.

Structure/function relationship of activating Ly-49D and inhibitory Ly-49G2 NK receptors.

Murine NK cells express Ly-49 family receptors capable of either inhibiting or activating lytic function. The overlapping patterns of expression of the various receptors have complicated their precise biochemical characterization. Here we describe the use of the Jurkat T cell line as the model for the study of Ly-49s. We demonstrate that Ly-49D is capable of delivering activation signals to Jurkat T cells even in the absence of the recently described Ly-49D-associated chain, DAP-12. Ly-49D signaling in Jurkat leads to tyrosine phosphorylation of TCRzeta and requires Syk/Zap70 family kinases and arginine 54 of Ly-49D, suggesting that Ly-49D signals via association with TCRzeta. Coexpression studies in 293-T cells confirmed the ability of Ly-49D to associate with TCRzeta. In addition, we have used this model to study the functional interactions between an inhibitory Ly-49 (Ly-49G2) and an activating Ly-49 (Ly-49D). Ly-49G2 blocks activation mediated by Ly-49D in an immunoreceptor tyrosine-based inhibitory motif (ITIM)-dependent manner. In contrast, Ly-49G2 was incapable of inhibiting activation by the TCR even though human killer cell inhibitory receptor (KIR) (KIR3DL2(GL183)) effectively inhibits TCR. Both the ability of Ly-49G2 to block Ly-49D activation and the failure of Ly-49G2 to inhibit TCR signaling were confirmed in primary murine NK cells and NK/T cells, respectively. These data demonstrate the dominant effects of the inhibitory receptors over those that activate and suggest an inability of the Ly-49 type II inhibitory receptors to efficiently inhibit type I transmembrane receptor signaling in T cells and NK cells.

Induction of DAP12 phosphorylation, calcium mobilization, and cytokine secretion by Ly49H.

The ability of several Ly49 family members to inhibit natural killer (NK) cell functions through recruitment of SHP-1 phosphatase has been reported. In contrast, the mechanisms underlying the activating signal generated by Ly49D are poorly understood. A homodimeric phosphoprotein (pp16) that physically and functionally associates with Ly49D has been described. In this study, a rabbit anti-mouse pp16 antiserum was generated and used to demonstrate that pp16 corresponds to the recently described DAP12 molecule. In addition, we show that a second Ly49 family member that lacks an immunoreceptor tyrosine-based inhibitory motif and contains a charged residue in the transmembrane domain, Ly49H, also associates with DAP12. Furthermore, we show that engagement of the Ly49H/DAP12 complex results in phosphorylation of DAP12, intracellular calcium mobilization, and tumor necrosis factor secretion in transfected cells. These results thus provide evidence that Ly49H is an activating receptor that associates with DAP12, previously described as a pp16 component of the Ly49D receptor complex.

DAP12-mediated signal transduction in natural killer cells. A dominant role for the Syk protein-tyrosine kinase.

The murine Ly49 family contains nine genes in two subgroups: the inhibitory receptors (Ly49A, B, C, E, F, G2, and I) and the noninhibitory receptors (Ly49D and H). Unlike their inhibitory counterparts, Ly49D and H do not contain immunoreceptor tyrosine-based inhibitory motifs but associate with a recently described co-receptor, DAP12, to transmit positive signals to natural killer (NK) cells. DAP12 is also expressed in myeloid cells, but the receptors coupled to it there are unknown. Here we document the signaling pathways of the Ly49D/DAP12 complex in NK cells. We show that ligation of Ly49D results in 1) tyrosine phosphorylation of several substrates, including phospholipase Cgamma1, Cbl, and p44/p42 mitogen-activated protein kinase, and 2) calcium mobilization. Moreover, we demonstrate that although human DAP12 reportedly binds the SH2 domains of both Syk and Zap-70, ligation of Ly49D leads to activation of Syk but not Zap-70. Consistent with this observation, Ly49D/DAP12-mediated calcium mobilization is blocked by dominant negative Syk but not by catalytically inactive Zap-70. These data demonstrate the dependence of DAP12-coupled receptors on Syk and suggest that the outcome of Ly49D/DAP12 engagement will be regulated by Cbl and culminate in the activation of transcription factors.

Characterization of an associated 16-kDa tyrosine phosphoprotein required for Ly-49D signal transduction.

Ly-49D is an activating receptor on NK cells that does not become tyrosine phosphorylated upon activation. This report demonstrates that immunoprecipitation of Ly-49D, following pervanadate treatment or specific Ab cross-linking, coprecipitates a 16-kDa tyrosine-phosphorylated protein (pp16). Immunoblotting experiments and data from TCR-zeta/Fc epsilonRIgamma double knockout mice confirm that pp16 is not TCR-zeta, TCR-eta, or Fc epsilonRIgamma. Association of pp16 with Ly-49D involves a transmembrane arginine since mutation to leucine (Ly-49D[R54L]) abolishes association with pp16 in transfected P815 cells. In addition, Ly-49D(R54L) transfectants fail to mediate Ca2+ mobilization following Ab cross-linking. Therefore, signaling through Ly49D on NK cells depends on association with a distinct tyrosine phosphoprotein (pp16) in a manner analogous to that of TCR and FcR. Expression of this novel signaling peptide in both the NK and myeloid lineages indicates that pp16 is likely involved in the signal transduction cascade of additional receptor families.

A conformational rearrangement upon binding of IgE to its high affinity receptor.

One of the critical steps in the allergic reaction is the binding of immunoglobulin E (IgE) to its high affinity receptor (FcepsilonRI). FcepsilonRI is a tetrameric complex composed of an alpha-chain, a beta-chain, and a dimeric gamma-chain. The extracellular portion of the alpha-chain (alpha-t) is sufficient for the binding of IgE. The Fc portion of IgE contains two copies of the FcepsilonRI binding sites. In contrast, the binding stoichiometry is 1:1. Previously, it was hypothesized that the binding of FcepsilonRI to IgE results in a conformational change in IgE that precludes the binding of a second molecule (Presta, L., Shields, R., O'Connel, L., Lahr, S., Porter, J. , Gorman, C., and Jardieu, P.(1994) J. Biol. Chem. 269, 26368-26373). Here we characterize the secondary structure of IgE and alpha-t and analyze their interaction by circular dichroism spectroscopy. Binding experiments show that when IgE interacts with alpha-t there is a 15-26% decrease of the negative ellipticity at 217 nm. Together, the absence of an alpha-helix element in alpha-t and the small contribution of alpha-t to the spectra of the complex indicate that upon binding, a major conformational rearrangement must occur on IgE. In addition, we analyze the thermal unfolding of alpha-t, IgE, and their complex. Despite the several domains that constitute IgE and alpha-t, these molecules unfold cooperatively with two-state kinetics.

Glycosylation of human truncated Fc epsilon RI alpha chain is necessary for efficient folding in the endoplasmic reticulum.

The high affinity immunoglobulin E (IgE) receptor is an alpha beta gamma 2 tetrameric complex. The truncated extracellular segment (alpha t) of the heavily glycosylated alpha chain is sufficient for high affinity binding of IgE. Here we have expressed various alpha t mutants in eukaryotic and prokaryotic cells to analyze the role of glycosylation in the folding, stability, and secretion of alpha t. All seven N-linked glycosylation sites in alpha t are glycosylated and their mutations have an additive effect on the folding and secretion of alpha t. Mutation of the seven N-glycosylation sites (delta 1-7 alpha t) induces misfolding and retention of alpha t in the endoplasmic reticulum. Similarly, tunicamycin treatment reduces substantially the folding efficiency of wild-type alpha t. In contrast, no difference in folding efficiency is detected between wild-type alpha t and delta 1-7 alpha t expressed in Escherichia coli. In addition, maturation of N-linked oligosaccharides and addition of O-linked carbohydrates are not required for either the transport or the IgE-binding function of alpha t. Furthermore, complete enzymatic deglycosylation does not affect the stability and the IgE-binding capacity of alpha t. Therefore, glycosylation is not intrinsically necessary for proper folding of alpha t but is required for folding in the endoplasmic reticulum. Our data are compatible with the concept that specific interactions between N-linked oligosaccharides and the folding machinery of the endoplasmic reticulum are necessary for efficient folding of alpha t in eukaryotic cells.

Regulation of c-myc and ornithine decarboxylase expression by distinct protein kinase systems in IL-3-dependent myeloid cells.

We have investigated whether PK-C-regulated events are independent of those biochemical events related to IL-3-induced tyrosine kinase activation by 32Dcl cells. The depletion of functional PK-C isoform activity by prolonged PMA treatment reduced the proliferative response to IL-3 by half that of untreated control cells. PK-C-deficient 32Dcl cells were unable to respond to PMA for the induction of c-myc and ODC mRNA accumulation. PK-C down-regulation did not affect IL-3-induced tyrosine phosphorylation and inhibited IL-3-regulated c-myc and ODC mRNA expression by only 30%. However, PK-C down-regulation had a pronounced inhibitory effect on IL-3 regulation of ODC enzymatic activity. While a PK-C-dependent and -independent pathway for the regulation of c-myc and ODC mRNA expression could be demonstrated, the regulation of ODC enzymatic activity appeared to require an intact PK-C system. The data suggest that the optimum biological and biochemical responses to IL-3 requires both pathways intact, however, tyrosine kinase activation and significant increases in gene products associated with proliferation can be achieved in the absence of a functional PK-C system.

IL-2 regulation of tyrosine kinase activity is mediated through the p70-75 beta-subunit of the IL-2 receptor.

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2. Although phorbol ester stimulated serine phosphorylation of the IL-2R alpha (p55) subunit recognized by alpha TAC mAb, IL-2 did not stimulate any detectable phosphorylation of IL-2R alpha or associated coimmune precipitated proteins. In fact, the tyrosine phosphorylated proteins did not coprecipitate with alpha Tac antibody and similar phosphoproteins were stimulated by IL-2 in IL-2R alpha- human large granular lymphocytes which express only the 70 to 75 kDa IL-2R beta subunit of the high affinity IL-2R. Anti-Tac mAb could inhibit IL-2-stimulated tyrosine phosphorylation in activated T cells, which express both IL-2R subunits that together form the high affinity receptor complex, but not in large granular lymphocytes expressing only the IL-2R beta subunit. The data suggest that IL-2 stimulation of tyrosine kinase activities requires only the IL-2R beta subunit.

Detection of low and high affinity binding sites with fluoresceinated human recombinant interleukin-2.

In this study, we describe a new methodology to detect and quantify lymphokine receptors, using interleukin-2 as a prototype. Human recombinant interleukin-2 (IL-2) was conjugated to fluorescein isothiocyanate. Binding of fluoresceinated IL-2 to different cell types was assessed by flow cytometry analysis, on a FACS 440 calibrated using fluoresceinated Sephadex G-25 beads. This calibration procedure allowed us to quantify the actual number of binding sites for IL-2. Fluoresceinated IL-2 did not bind to normal resting T cells, whereas a highly significant binding was observed on PHA-activated human T cells. The binding was inhibited by an excess of unlabeled IL-2 and by an excess of anti-IL-2 receptor p55 antibodies (anti-TAC). Dose curves of IL-2 showed a two plateau saturation, the first plateau corresponding to the saturation of high affinity binding sites, as assessed by correlation with the biological activity on IL-2-dependent T cells. Among the cell types tested, fluoresceinated IL-2 bound to IL-2-dependent mouse T cells (the binding in that case was not inhibited by anti-IL-2 receptor p55 antibodies), and to different p70 expressing cell lines or normal cells (MLA 144, normal large granular lymphocytes). Taken together, these results indicate that fluoresceinated IL-2 can be used to detect high as well as low affinity IL-2 binding sites.

Interleukin 3 and phorbol ester stimulate tyrosine phosphorylation of overlapping substrate proteins.

FDC-P1 is a murine myeloid cell line that requires interleukin 3 (IL3) for survival and proliferation. While the biological effects of IL3 have been well described, the biochemical mechanisms of IL3 actions have only recently been examined. We have investigated whether IL3 or PMA stimulates phosphorylation of proteins on tyrosine as well as on serine/threonine residues as previously described [(1986) Blood 68, 906-913; (1987) Biochem. J. 244, 683-691]. Here we report that both IL3 and PMA stimulate the tyrosine phosphorylation of at least two proteins: pp70 and pp50 in FDC-P1 cells.

Comparative analysis of IL-2 and IL-3 induced tyrosine phosphorylation.

IL2 and IL3 are polypeptide growth factors that support the survival and proliferation of, respectively, activated T lymphocytes and a range of myeloid cell types. We have examined the involvement of tyrosine phosphorylation in IL2 and IL3 mediated signal transduction. Phosphotyrosyl proteins were immunoaffinity purified and analyzed by single and two dimensional gel electrophoresis. The majority of phosphotyrosyl proteins purified from human T lymphocytes and murine myeloid cells had identical 2 D electrophoretic mobilities, suggesting a high degree of evolutionary conservation. Several proteins in both cell types increased in tyrosine phosphorylation after factor stimulation, including pp200, pp180, pp92, and pp42. The 92 kD protein was the most highly modulated phosphoprotein identified, with increases in phosphorylation greater than 18 fold after 20 min of stimulation. These results suggest that signal transduction pathways for IL2 and IL3 involve tyrosine phosphorylation of protein substrates common to both lymphoid and myeloid linages.