Differential effects of the phosphatidylinositol 4-kinases, PI4KIIα and PI4KIIIß, on Akt activation and apoptosis.

In this study, we investigated the role of PI4P synthesis by the phosphatidylinositol 4-kinases, PI4KIIα and PI4KIIIß, in epidermal growth factor (EGF)-stimulated phosphoinositide signaling and cell survival. In COS-7 cells, knockdown of either isozyme by RNA interference reduced basal levels of PI4P and PI(4,5)P(2), without affecting receptor activation. Only knockdown of PI4KIIα inhibited EGF-stimulated Akt phosphorylation, indicating that decreased PI(4,5)P(2) synthesis observed by loss of either isoform could not account for this PI4KIIα-specific effect. Phospholipase Cγ activation was also differentially affected by knockdown of either PI4K isozyme. Overexpression of kinase-inactive PI4KIIα, which induces defective endosomal trafficking without reducing PI(4,5)P(2) levels, also reduced Akt activation. Furthermore, PI4KIIα knockdown profoundly inhibited cell proliferation and induced apoptosis as evidenced by the cleavage of caspase-3 and its substrate poly(ADP-ribose) polymerase. However, in MDA-MB-231 breast cancer cells, apoptosis was observed subsequent to knockdown of either PI4KIIα or PI4KIIIß and this correlated with enhanced proapoptotic Akt phosphorylation. The differential effects of phosphatidylinositol 4-kinase knockdown in the two cell lines lead to the conclusion that phosphoinositide turnover is inhibited through PI4P substrate depletion, whereas impaired antiapoptotic Akt signaling is an indirect consequence of dysfunctional endosomal trafficking.

Taxanes synergize with the bispecific antibody MDXH447 to enhance antibody-dependent cell-mediated cytotoxicity.

The interaction between antibody based therapy and cytotoxic chemotherapy is complex. To explore these interactions we investigated, in vitro, the effects of IC20 growth inhibitory concentrations of taxanes on bispecific antibody-mediated tumor cell cytotoxicity. MDXH447 is a bispecific antibody with specificity for the high affinity IgG receptor (CD64) and the type I epidermal growth factor receptor type (EGF-R). A431 cells, an epidermoid carcinoma cell line that over expresses EGF-R, were exposed to a range of IC20 growth inhibitory concentrations of paclitaxel or docetaxel. Interferon gamma activated monocytes were armed with MDXH447 and a standard chromium release antibody-dependent cell-mediated cytotoxicity (ADCC) assay was performed. Using the Chou and Talalay median effect analysis, we found that MDXH447-mediated ADCC was enhanced when A431 target cells were pretreated with paclitaxel or docetaxel. Median effect analysis of these interactions supported a synergistic interaction (CI < 1). Pretreatment of A431 cells with taxanes did not increase EGF-R expression compared to untreated controls. A431 epidermoid carcinoma cells pretreated with IC20 growth inhibitory concentrations of taxanes enhanced interferon gamma activated monocyte mediated ADCC killing through MDXH447.

EGF receptors as transcription factors: ridiculous or sublime?

The notion that a transmembrane receptor at the cell surface can somehow reappear as a transcription factor in the nucleus is bound to be controversial. However, there are two reported examples of this. If this hypothesis can withstand the inevitable and necessary battery of additional empirical tests, then our understanding of signal transduction needs to move in a new direction.

Signalling and non-caveolar rafts.

Rafts are small membrane domains containing discrete subsets of lipids and proteins. Although microscopic raft structures termed 'caveolae' were described nearly 50 years ago, the importance of rafts, particularly signalling within rafts, is only beginning to be understood. Our studies focus on receptor-dependent phosphoinositide signalling. Using their characteristic buoyancy in density gradients, we and others found that the epidermal growth factor (EGF) receptor, phosphatidylinositol 4-kinase and phosphoinositides are localized within a caveolin-rich fraction of A431 carcinoma cells. We subsequently found that membrane fragments containing the EGF receptor and most cellular phosphoinositides can be separated from caveolae. Consequently, components of EGF-dependent phosphoinositide signalling localize to one or more novel types of raft, the composition of which we are currently determining. A key component is the type II phosphatidylinositol 4-kinase, which, for many years, has proven difficult to purify and clone. We describe our recent purification from rafts and cloning of this elusive enzyme, and discuss how the structure sheds light on the rafting of this enzyme.

Cloning of a human type II phosphatidylinositol 4-kinase reveals a novel lipid kinase family.

Phosphoinositide lipids regulate numerous cellular processes in all eukaryotes. The versatility of this phospholipid is provided by combinations of phosphorylation on the 3', 4', and 5' positions of the inositol head group. Two distinct structural families of phosphoinositide (PI) kinases have so far been identified and named after their prototypic members, the PI 3-kinase and phosphatidylinositol (PtdIns) phosphate kinase families, both of which have been found to contain structural homologues possessing PI 4-kinase activity. Nevertheless, the prevalent PtdIns 4-kinase activity in many mammalian cell types is conferred by the widespread type II PtdIns 4-kinase, which has so far resisted molecular characterization. We have partially purified the human type II isoform from plasma membrane rafts of human A431 epidermoid carcinoma cells and obtained peptide mass and sequence data. The results allowed the cDNA containing the full open reading frame to be cloned. The predicted amino acid sequence revealed that the type II enzyme is the prototypic member of a novel, third family of PI kinases. We have named the purified protein type IIalpha and a second human isoform, type IIbeta. The type IIalpha mRNA appears to be expressed ubiquitously in human tissues, and homologues appear to be expressed in all eukaryotes.

Bispecific antibody-targeted phagocytosis of HER-2/neu expressing tumor cells by myeloid cells activated in vivo.

Studies from our laboratory and others have established that both mononuclear phagocytes and neutrophils mediate very efficient cytotoxicity when targeted through Fc receptors using a suitable monoclonal or bispecific antibody (BsAb). Cross-linking an Fc receptor for IgG (FcgammaR) triggers multiple anti-tumor activities including superoxide generation, cytokine and enzyme release, phagocytosis and antibody-dependent cellular cytotoxicity (ADCC). In this report, using unfractionated leukocytes and two color flow cytometric analysis, we describe the phagocytic capacity of peripheral blood polymorphonuclear cells (PMN) and monocytes isolated from patients enrolled in a phase I clinical trial of MDX-H210 given in combination with IFNgamma. MDX-H210 is a BsAb targeting the myeloid trigger molecule FcgammaRI and the HER-2/neu proto-oncogene product overexpressed on a variety of adenocarcinomas. In this trial, cohorts of patients received escalating doses of MDX-H210 3 times per week for 3 weeks. Interferon-gamma (IFNgamma) was given 24 h prior to each BsAb infusion. Our results demonstrate that monocytes from these patients were inherently capable of phagocytosing the HER-2/neu positive SK-BR-3 cell line and that addition of MDX-H210 into the assay significantly enhanced the number of targets phagocytosed. Two days after administration of an immunologically active dose of MDX-H210 (10 mg/m2), monocytes from these patients were able to phagocytose greater amounts of target cell material, indicating that these cells remained armed with functionally sufficient BsAb for at least 48 h. PMN from these patients very efficiently mediated phagocytosis through FcgammaRI after being treated with IFNgamma, but not before. We conclude that phagocytosis is not only an efficient mechanism of myeloid cell-mediated cytotoxicity, but may also be a mechanism by which antigens from phagocytosed cells can enter a professional antigen presenting cell for processing and presentation.

Characterization of RNA binding proteins associated with CD40 ligand (CD154) mRNA turnover in human T lymphocytes.

CD154 (CD40 ligand (CD40L)) has been demonstrated to play an essential role in the development of humoral and cellular immunity through its interaction with CD40. While earlier studies have examined the regulation of CD154 expression by transcriptional and posttranslational pathways, scant data exist on its regulation at a posttranscriptional level. In this report we demonstrate that CD154 mRNA is rapidly turned over in primary culture of activated human T lymphocytes. Moreover, we demonstrate that CD154 mRNA is unstable, but can be stabilized by treatment with either phorbol esters or calcium ionophores. To address this lability of CD154 mRNA, we examined the ability of cytoplasmic proteins to bind to its 3' untranslated region (3'UTR). Two major proteins (p25 and p50) capable of binding the 3'UTR of CD154 were identified. The p25 binding activity was associated with polysomes and appeared to correlate with CD154 mRNA instability. Intriguingly, these proteins did not appear to bind to the AU-rich elements present in the 3'UTR of CD154. Rather, their binding was localized to unique sites between nt 471-811 of the 3'UTR, which lack any classical AU-rich elements. These data suggest that these proteins interact with distinct cis-acting elements that are important in the posttranscriptional regulation of CD154 expression. As such, identifying these proteins will help us understand the signals that are necessary for CD154 expression by activated T cells.

Agonist-induced desensitization and phosphorylation of m1-muscarinic receptors.

Pre-stimulation of Chinese hamster ovary (CHO) cells expressing the human m1-muscarinic receptor (CHO-m1 cells) with a maximally effective concentration of the muscarinic agonist methacholine resulted in desensitization of Ins(1,4,5)P3 accumulation, apparent as a approximately 4-fold shift in the agonist dose-response curve. Agonist-induced desensitization was rapid (detectable by 10 s) and concentration dependent (EC50=8.2+/-2.2 microM) and resulted in a complete loss of receptor reserve for the agonist-stimulated Ins(1,4, 5)P3 response. An investigation of the possible mechanisms involved in m1-muscarinic receptor desensitization indicated that agonist-induced receptor internalization, PtdIns-(4,5)P2 depletion or an increased rate of Ins(1,4,5)P3 metabolism were not involved. m1-Muscarinic receptors did, however, undergo rapid agonist-induced phosphorylation with a time course that was consistent with an involvement in receptor desensitization. Characterization studies indicated that the receptor-specific kinase involved was distinct from protein kinase C and other second-messenger-dependent protein kinases. Since previous studies have suggested that the m3-muscarinic receptor subtype undergoes agonist-dependent phosphorylation via casein kinase 1alpha (CK1alpha) [Tobin, Totty, Sterlin and Nahorski (1997) J. Biol. Chem. 272, 20844-20849], we examined the ability of m1-muscarinic receptors to be phosphorylated by this kinase. In reconstitution experiments, CK1alpha was able to phosphorylate purified, soluble m1-muscarinic receptors in an agonist-dependent manner.

Epidermal growth factor receptor activation is localized within low-buoyant density, non-caveolar membrane domains.

Increasing evidence for the organization of cell-surface proteins and lipids into different detergent-insoluble rafts led us to investigate epidermal growth factor (EGF) receptor activation in the plasma membranes of A431 carcinoma cells, using a combination of cell fractionation and immunoprecipitation techniques. Density-gradient centrifugation of sodium carbonate cell extracts revealed that the vast majority of both stimulated and unstimulated EGF receptors were concentrated in a caveolin-rich light membrane (CLM) fraction, with the biochemical characteristics of detergent-insoluble glycolipid-rich domains (DIGs). However, ultrastructural analysis of the CLM fraction revealed that it contained a heterogeneous collection of vesicles, some with sizes greater than that expected for individual caveolae. Experiments with detergent-solubilized cells and isolated CLMs indicated that, in contrast with caveolin, EGF receptors were unlikely to be localized to DIG domains. Furthermore, immunoisolation of caveolin from CLMs revealed that EGF receptor activation occurs in a compartment distinct from caveolae. Similarly, using an anti-(EGF receptor) antibody, the bulk of the cellular caveolin was not co-immunoprecipitated from CLMs, thereby confirming that these two proteins reside in separate membrane domains. The deduction that caveolar signalling and EGF receptor activation occur in separable rafts argues for a multiplicity of signal transduction compartments within the plasma membrane. In addition, by demonstrating that EGF receptor activation is compartmentalized within low-density, non-caveolar regions of the plasma membrane, it is also shown that the co-localization of proteins in a CLM fraction is insufficient to prove caveolar localization.

Phosphatidylinositol 4-phosphate synthesis in immunoisolated caveolae-like vesicles and low buoyant density non-caveolar membranes.

This study examined phosphatidylinositol 4-phosphate (PtdIns4P) synthesis in caveolae that have been suggested to be discrete signaling microdomains of the plasma membrane and are enriched in the marker protein caveolin. Caveolin-rich light membranes (CLMs) were isolated from A431 cells by detergent-free, discontinuous density-gradient centrifugation method. The CLM fraction was separated from the bulk of the cellular protein and was greatly enriched in PtdIns, PtdIns4P, and phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) and an adenosine-sensitive type II PtdIns 4-kinase activity. Preparation of CLMs by an OptiPrep-based cell fractionation procedure confirmed the co-localization of PtdIns 4-kinase and caveolin. Electron microscopy confirmed that an anti-caveolin antiserum immunopurified vesicles from CLMs that were within the size range described for caveolae in other systems. Co-immunoprecipitated PtdIns 4-kinase activity could utilize endogenous PtdIns, present within the caveolae-like vesicles, to produce PtdIns4P. The addition of recombinant phosphatidylinositol transfer protein increased PtdIns 4-kinase activity both in immunoisolated caveolae and CLMs. However, less than 1% of the total cellular PtdIns and PtdIns 4-kinase activity was present in caveolae-like vesicles, indicating that non-caveolar light membrane rafts are the main site for cellular PtdIns4P production.

Decreased accessory cell function and costimulatory activity by 1,25-dihydroxyvitamin D3-treated monocytes.

To characterize the mechanism(s) by which 1,25-dihydroxyvitamin D3 (calcitriol) modulates the costimulatory capacity of monocytes, we examined the effect of calcitriol pretreatment of monocytes on their capacity to promote T cell proliferation (accessory cell function). Correlation of calcitriol-dependent changes in monocyte accessory cell function and alterations in phenotype and cytokine production, and the dependence of these changes on cell viability, were studied. Calcitriol pretreatment induced a defect in accessory cell function that was evident with fixed monocytes, suggesting a cell-surface-associated mechanism. Altered accessory cell function did not correlate with changes in HLA-DR antigen expression and was unaffected by concurrent treatment with interferon-gamma. Calcitriol treatment did not alter either the expression of adhesion molecules or monocytic production of interleukin-1 beta (IL-1 beta) or IL-6. Exogenous IL-1 or IL-6 did not overcome the impaired costimulatory activity of calcitriol-treated monocytes. Thus, calcitriol treatment reduces the capacity of monocytes to promote lectin-induced T cell activation at the level of the plasma membrane, perhaps through altered expression of an uncharacterized molecule important in monocyte-T cell interactions. At chronically inflamed sites, elaboration of calcitriol by activated macrophages may regulate the ability of monocytes to induce both antigen-dependent and antigen-independent T cell proliferation.

1,25-Dihydroxyvitamin D3 modulates the effects of interleukin 2 independent of IL-2 receptor binding.

Previous studies have shown that 1,25-dihydroxyvitamin D3 (calcitriol) is a macrophage-derived cytokine and a potent inhibitor of IL-2 and interferon-gamma (IFN-gamma) production and T lymphocyte proliferation. The growth inhibitory effect of calcitriol is only partially reversed by IL-2 addition, suggesting IL-2 independent effects. In this report we characterize the IL-2-independent effects of calcitriol on lymphocyte activation. Calcitriol inhibited cellular transition from early to late G1 (G1A-G1B transition) in both the absence and presence of IL-2. Exogenous IL-2 did not increase either IFN-gamma production or transferrin receptor (TfR) expression in the presence of calcitriol despite increases in cell entry into late G1 and proliferation. Calcitriol treatment reduced TfR expression by activated T lymphocytes independent of their location in the cell cycle, further suggesting its independence from IL-2-mediated events. Combinations of rIL-2 and rIL-4 did not reverse calcitriol-dependent inhibition of proliferation and TfR expression to any greater degree than rIL-2 alone. Northern blot analysis demonstrated the decrease in IFN-gamma and TfR mRNA accumulation with calcitriol treatment was unaffected by exogenous IL-2. In contrast, IL-2R mRNA and protein were increased by IL-2, with superinduction in the presence of calcitriol, demonstrating that the lack of effect on IFN-gamma and TfR was not due to IL-2 insensitivity. Moreover, equivalent numbers of high-affinity IL-2R were expressed by both control and calcitriol-treated T lymphoblasts. Thus, lectin-activated T lymphocyte responsiveness to IL-2, as measured by IL-2R expression and proliferation, can be partly to completely dissociated from IFN-gamma production and TfR expression in the presence of calcitriol. Finally, IL-2-induced proliferation of unstimulated mononuclear cells and purified T lymphocytes was inhibited by calcitriol. These data indicate that local production of calcitriol by activated macrophages is capable of regulating T lymphocyte activation not only through suppression of IL-2 production, but also through additional mechanism(s), that are mediated at a post-IL-2R level.