Gerald M. Feldman

Protein Kinase Cδ Is Required for p47phox Phosphorylation and Translocation in Activated Human Monocytes

Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKCδ, -ε, -θ, and -η) and conventional PKC (including PKCα and -β), inhibited both phosphorylation and translocation of p47phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKCε, did not affect the phosphorylation or translocation of p47phox, suggesting that PKCδ, -θ, or -η is required. Furthermore, rottlerin (at doses that inhibit PKCδ activity) inhibited the phosphorylation and translocation of p47phox. Rottlerin also inhibited O⨪2 production at similar doses. In addition to pharmacological inhibitors, PKCδ-specific antisense oligodeoxyribonucleotides were used. PKCδ antisense oligodeoxyribonucleotides inhibited the phosphorylation and translocation of p47phox in activated human monocytes. We also show, using the recombinant p47phox-GST fusion protein, that p47phox can serve as a substrate for PKCδ in vitro. Furthermore, lysate-derived PKCδ from activated monocytes phosphorylated p47phox in a rottlerin-sensitive manner. Together, these data suggest that PKCδ plays a pivotal role in stimulating monocyte NADPH oxidase activity through its regulation of the phosphorylation and translocation of p47phox.

Mapping of a Cytoplasmic Domain of the Human Growth Hormone Receptor That Regulates Rates of Inactivation of Jak2 and Stat Proteins

It has been previously demonstrated that growth hormone (GH)-stimulated tyrosine phosphorylation of Jak2 and Stat5a and Stat5b occurs in FDP-C1 cells expressing either the entire GH receptor or truncations of the cytoplasmic domain expressing only the membrane-proximal 80 amino acids. However, other receptor domains that might modulate rates of GH activation and inactivation of this cascade have not been examined. Here we have defined a region in the human GH receptor between amino acids 520 and 540 in the cytoplasmic domain that is required for attenuation of GH-activated Jak/Stat signaling. Immunoprecipitations with antibodies to Jak2 indicate that the protein tyrosine phosphatase SHP-1 is associated with this kinase in cells exposed to GH. To address the possibility that SHP-1 could function as a negative regulator of GH signaling, liver extracts from motheaten mice deficient in SHP-1 or unaffected littermates were analyzed for activation of Stats and Jak2. Extracts from motheaten mice displayed prolonged activation of the Stat proteins as measured by their ability to interact with DNA and prolonged tyrosine phosphorylation of Jak2. These results delineate a novel domain in the GH receptor that regulates the inactivation of the Jak/Stat pathway and appears to be modulated by SHP-1.

Nucleoporation of dendritic cells: efficient gene transfer by electroporation into human monocyte‐derived dendritic cells1

Dendritic cells (DCs) are ideal accessory cells in the developing field of gene therapy. Although viral transfection of DCs has become widespread, non‐viral transfection of DCs has shown disappointing results. Recently, a new technique for transfecting primary cells has become available – the Amaxa Nucleofector™. Here, we describe the use of this device in the successful non‐viral transfection of human monocyte‐derived DCs. Using enhanced green fluorescent protein as a reporter gene DCs were transfectable with efficiencies approaching 60%, remaining responsive to lipopolysaccharide‐stimulated cytokine production in short‐term experiments (though long‐term functional assays were hampered by loss of viability). Although these data demonstrate the ease and efficiency with which human monocyte‐derived DCs can now be non‐virally transfected, they also suggest the limitations of this technology due to the gradual loss of cell viability. The potential use of this system in the development of DC‐based cell and gene therapies will be hampered until cell viability can be maintained.

Protein kinase Cδ regulates p67phox phosphorylation in human monocytes

Phosphorylation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components p67phox and p47phox accompanies the assembly and activation of this enzyme complex. We have previously reported that activation of human monocytes with opsonized zymosan (ZOP), a potent stimulator of NADPH oxidase activity, results in the phosphorylation of p67phox and p47phox. In this study, we investigated the regulation of p67phox phosphorylation. Although protein kinase C (PKC)α has previously been shown to regulate NADPH oxidase activity, we found that inhibition of PKCα had no effect on p67phox phosphorylation. Our studies demonstrate that pretreatment of monocytes with antisense oligodeoxyribonucleotides specific for PKCδ or rottlerin, a selective inhibitor for PKCδ, inhibited the phosphorylation of p67phox in monocytes, and Go6976, a specific inhibitor for conventional PKCs, PKCα and PKCβ, had no such inhibitory effect. Additional studies indicate that ZOP stimulation of monocytes induces PKCδ and p67phox to form a complex. We also demonstrate that lysates from activated monocytes as well as PKCδ immunoprecipitates from activated monocytes can phosphorylate p67phox in vitro and that pretreatment of monocytes with rottlerin blocked the phosphorylation in each case. We further show that recombinant PKCδ can phosphorylate p67phox in vitro. Finally, we show that PKCδ‐deficient monocytes produce significantly less superoxide anion in response to ZOP stimulation, thus emphasizing the functional significance of the PKCδ regulation of p67phox phosphorylation. Taken together, this is the first report to describe the requirement of PKCδ in regulating the phosphorylation of p67phox and the related NADPH oxidase activity in primary human monocytes.

A PI-3 kinase-dependent, Stat1-independent signaling pathway regulates interferon-stimulated monocyte adhesion

Type I interferon (IFN)‐α/β and type II IFN‐γ induce the expression of early response genes through activation of the Janus tyrosine kinase/signal transducer and activator of transcription (Stat) pathway. Although IFNs regulate a variety of other signaling cascades, little is known about how they contribute to the biological activities of these cytokines. In this study, we demonstrate that IFN‐β or IFN‐γ induces the phosphorylation of the serine/threonine kinase Akt in primary human peripheral blood monocytes. Abrogation of the IFN‐stimulated Akt activation by phosphatidylinositol‐3 kinase (PI‐3K) inhibitors prevents IFN‐induced adhesion in these cells, and IFN activation of the Stat1‐dependent guanylate‐binding protein (GBP) gene is not affected. Importantly, Stat1‐deficient bone marrow macrophages displayed a similar level of IFN‐γ‐stimulated adhesion compared with macrophages derived from wild‐type littermates. These findings demonstrate for the first time that IFN stimulation of a PI‐3K signaling cascade modulates the ability of these cytokines to regulate monocyte adhesion, and this process does not require the expression of Stat1, a primary mediator of IFN‐γ signaling.

Activation by prion peptide PrP106-126 induces a NF-κB-driven proinflammatory response in human monocyte-derived dendritic cells

Specific prion peptides have been shown to mimic the pathologic isoform of the prion protein (PrP) and to induce a neurotoxic effect in vitro and in vivo. As monocytic cells are thought to play a role in the transmission and pathogenesis of prion disease, the use of these peptides in regulating monocytic cell function is under intense investigation. In the current study, we characterize the ability of prion peptide PrP106–126 to activate specific signaling pathways in human monocyte‐derived dendritic cells (DCs). Electrophoretic mobility shift assays establish the activation of transcription factor nuclear factor‐κB within 15 min of exposure, with as little as 25 μM peptide. This signaling cascade results in the up‐regulation of inflammatory cytokines interleukin (IL)‐1β, IL‐6, and tumor necrosis factor α (TNF‐α) at the mRNA and protein levels. Phenotypic activation of DCs exposed to PrP106–126 is partly a result of an autocrine TNF‐α response and results in an increased ability of these cells to induce lymphocyte proliferation. The effects of PrP106–126 on DCs were elicited through a receptor complex distinct from that used by human monocytes, demonstrating the ability of this peptide to interact with a multiplicity of receptors on various cell types. Together, these data suggest an involvement of DCs in prion disease pathogenesis.

NF-κB elements contribute to junB inducibility by lipopolysaccharide in the murine macrophage cell line RAW264.7

Macrophages respond to bacterial lipopolysaccharide (LPS) by activating latent cis‐acting factors that initiate transcription of immediate early genes. One such immediate early gene, junB, is induced by LPS in macrophages within 30 min. To identify elements that mediate the induction of junB by LPS, upstream and downstream sequences flanking the junB gene were examined by transient expression in the RAW264.7 murine macrophage cell line using a luciferase reporter gene vector containing the junB minimal promoter. A >10‐fold enhancement was associated with a 222 bp region downstream of the junB promoter in response to LPS. Transient reporter assays demonstrated that multiple nuclear factor (NF) κB sites are required for inducibility of junB by LPS in RAW264.7 cells. Electrophoretic mobility shift assays confirmed binding of LPS‐induced nuclear proteins included p50/p65 heterodimers at these NF‐κB sites.

Monocyte 15-Lipoxygenase Expression Is Regulated by a Novel Cytosolic Signaling Complex with Protein Kinase C δ and Tyrosine-Phosphorylated Stat3

Our previous studies demonstrated that the IL-13-induced 15-lipoxygenase expression in primary human monocytes is regulated by the activation of both Stat1 and Stat3 and by protein kinase C (PKC)δ. IL-13 stimulated the phosphorylation of Stat3 on both Tyr705 and Ser727. In this study we show that IL-13 induces the association of PKCδ with Stat3, not with Stat1, and is required for Stat3 Ser727 phosphorylation. We found a novel IL-13-dependent cytosolic signaling complex of PKCδ and tyrosine-phosphorylated Stat3. A tyrosine kinase inhibitor blocked PKCδ association with Stat3 as well as Stat3 Ser727 phosphorylation. We therefore hypothesized that tyrosine phosphorylation was required for Stat3 interaction with PKCδ and subsequent PKCδ-dependent phosphorylation of Stat3 Ser727. We developed an efficient transfection protocol for human monocytes. Expression of Stat3 containing a mutation in Tyr705 inhibited the association of PKCδ with Stat3 and blocked Stat3 Ser727 phosphorylation, whereas transfection with wild-type Stat3 did not. Furthermore, by transfecting monocytes with Stat3 containing mutations in Tyr705 or Ser727 or with wild-type Stat3, we demonstrated that both Stat3 tyrosine and serine phosphorylations are required for optimal binding of Stat3 with DNA and maximal expression of 15-lipoxygenase, an important regulator of inflammation and apoptosis.

Monocyte 15-Lipoxygenase Gene Expression Requires ERK1/2 MAPK Activity

IL-13 induces profound expression of 15-lipoxygenase (15-LO) in primary human monocytes. Our studies have defined the functional IL-13R complex, association of Jaks with the receptor components, and the tyrosine phosphorylation of several Stat molecules in response to IL-13. Furthermore, we identified both p38MAPK and protein kinase Cδ as critical regulators of 15-LO expression. In this study, we report an ERK1/2-dependent signaling cascade that regulates IL-13–mediated 15-LO gene expression. We show the rapid phosphorylation/activation of ERK1/2 upon IL-13 exposure. Our results indicate that Tyk2 kinase is required for the activation of ERK1/2, which is independent of the Jak2, p38MAPK, and protein kinase Cδ pathways, suggesting bifurcating parallel regulatory pathways downstream of the receptor. To investigate the signaling mechanisms associated with the ERK1/2-dependent expression of 15-LO, we explored the involvement of transcription factors, with predicted binding sites in the 15-LO promoter, in this process including Elk1, early growth response-1 (Egr-1), and CREB. Our findings indicate that IL-13 induces Egr-1 nuclear accumulation and CREB serine phosphorylation and that both are markedly attenuated by inhibition of ERK1/2 activity. We further show that ERK1/2 activity is required for both Egr-1 and CREB DNA binding to their cognate sequences identified within the 15-LO promoter. Furthermore, by transfecting monocytes with the decoy oligodeoxyribonucleotides specific for Egr-1 and CREB, we discovered that Egr-1 and CREB are directly involved in regulating 15-LO gene expression. These studies characterize an important regulatory role for ERK1/2 in mediating IL-13–induced monocyte 15-LO expression via the transcription factors Egr-1 and CREB.

Soluble interleukin-4 receptor production by murine myeloid progenitor cells: Induction by interleukin-6 and interferon-γ

Soluble cytokine receptors, molecules that can selectively modulate the effects of a single cytokine, have generated great interest both as indicators of disease and as targeted immunotherapeutic agents. However, cellular sources for soluble cytokine receptors are not well characterized and the regulation of soluble receptor production is not clear. We recently found that interleukin 6 (IL-6) induces the expression of membrane-bound interleukin 4 (IL-4) receptors (mIL-4R) in the murine myeloid leukemia M1 cell line. In the present study we show that IL-6 induces the expression and secretion of the soluble form of the IL-4 receptor (sIL-4R) in these cells as well, with the protein accumulating for up to 72 hours in the cell supernatants. This inducible production of sIL-4R protein is accompanied by the induction of mRNA specific for sIL-4R. In mature bone marrow (BM)-derived macrophages sIL-4R expression can be induced by exposure to either IL-6 or interferon-gamma (IFN gamma). The data suggest that myeloid cells exposed to inflammatory stimuli are one possible source for sIL-4R enabling them to modulate the effects of IL-4 on the immune response by the regulated production of the soluble receptor for this cytokine.