Nucleotide receptor P2RX7 stimulation enhances LPS-induced interferon-ß production in murine macrophages.

Stimulation of P2RX(7) with extracellular ATP potentiates numerous LPS-induced proinflammatory events, including cytokine induction in macrophages, but the molecular mechanisms underlying this process are not well defined. Although P2RX(7) ligation has been proposed to activate several transcription factors, many of the LPS-induced mediators affected by P2RX(7) activation are not induced by P2RX(7) agonists alone, suggesting a complementary role for P2RX(7) in transcriptional regulation. Type I IFN production, whose expression is tightly controlled by multiple transcription factors that form an enhanceosome, is critical for resistance against LPS-containing bacteria. The effect of purinergic receptor signaling on LPS-dependent type I IFN is unknown and would be of great relevance to a diverse array of inflammatory conditions. The present study demonstrates that stimulation of macrophages with P2RX(7) agonists substantially enhances LPS-induced IFN-ß expression, and this enhancement is ablated in macrophages that do not express functional P2RX(7) or when the MAPK MEK1/2 pathways are inhibited. Potentiation of LPS-induced IFN-ß expression following P2RX(7) stimulation is likely transcriptionally regulated, as this enhancement is observed at the IFN-ß promoter level. Furthermore, P2RX(7) stimulation is able to increase the phosphorylation and subsequent IFN-ß promoter occupancy of IRF-3, a transcription factor that is critical for IFN-ß transcription by TLR agonists. This newly discovered role for P2RX(7) in IFN regulation may have implications in antimicrobial defense, which has been linked to P2RX(7) activation in other studies.

Segmental allergen challenge enhances chitinase activity and levels of CCL18 in mild atopic asthma.

BACKGROUND: Allergic airway inflammation contributes to the airway remodelling that has been linked to increased obstruction and morbidity in asthma. However, the mechanisms by which allergens contribute to airway remodelling in humans are not fully established. CCL18, chitotriosidase (CHIT1) and YKL-40 are readily detectable in the lungs and contribute to remodelling in other fibrotic diseases, but their involvement in allergic asthma is unclear. OBJECTIVE: We hypothesized that CCL18, YKL-40 and CHIT1 bioactivity are enhanced in allergic asthma subjects after segmental allergen challenge and are related to increased pro-fibrotic and Th2-associated mediators in the lungs. METHODS: Levels of CCL18 and YKL-40 protein and chitotriosidase (CHIT1) bioactivity in bronchoalveolar lavage (BAL) fluid, as well as CCL18, YKL-40 and CHIT1 mRNA levels in BAL cells were evaluated in patients with asthma at baseline and 48 h after segmental allergen challenge. We also examined the correlation between CCL18 and YKL-40 levels and CHIT1 activity with the levels of other pro-fibrotic factors and chemokines previously shown to be up-regulated after allergen challenge. RESULTS: Chitotriosidase activity and YKL-40 and CCL18 levels were elevated after segmental allergen challenge and these levels correlated with those of other pro-fibrotic factors, T cell chemokines, and inflammatory cells after allergen challenge. CCL18 and YKL-40 mRNA levels also increased in BAL cells after allergen challenge. CONCLUSIONS AND CLINICAL RELEVANCE: Our results suggest that CCL18 and YKL-40 levels and CHIT1 activity are enhanced in allergic airway inflammation and thus may contribute to airway remodelling in asthma.

Human monocytic cells direct the robust release of CXCL10 by bronchial epithelial cells during rhinovirus infection.

BACKGROUND: Human rhinovirus (HRV) infections are a major cause of exacerbations in chronic respiratory conditions such as asthma and chronic obstructive pulmonary disease, but HRV-induced immune responses of the lower airway are poorly understood. Earlier work examining cytokine release following HRV infection has focused on epithelial cells because they serve as the principal site of viral replication, and internalization and replication of viral RNA appear necessary for epithelial cell mediator release. However, during HRV infection, only a small proportion of epithelial cells become infected. As HRV-induced cytokine levels in vivo are markedly elevated, this observation suggests that other mechanisms independent of direct viral infection may induce epithelial cell cytokine release. OBJECTIVE: Our aim was to test for the importance of interactions between human bronchial epithelial cells (HBECs) and monocytic cells in the control of mediator release during HRV exposure. METHODS: In vitro models of HRV serotype-16 (HRV16) infection of primary HBECs and human monocytic cells, in mono or co-culture, were used. We assessed HRV16-induced CXCL10 and CCL2 protein release via ELISA. RESULTS: Co-culture of human monocytic and bronchial epithelial cells promoted a synergistic augmentation of CXCL10 and CCL2 protein release following HRV16 challenge. Transfer of conditioned media from HRV16-treated monocytic cells to epithelial cultures induced a robust release of CXCL10 by the epithelial cells. This effect was greatly attenuated by type I IFN receptor blocking antibodies, and could be recapitulated by IFN-alpha addition. CONCLUSIONS: Our data indicate that epithelial CXCL10 release during HRV infection is augmented by a monocytic cell-dependent mechanism involving type I IFN(s). Our findings support a key role for monocytic cells in the amplification of epithelial cell chemokine production during HRV infection, and help to explain how an inflammatory milieu is created in the lower airways even in the absence of extensive viral replication and epithelial infection.

Activity and cellular localization of an oncogenic glioblastoma multiforme-associated EGF receptor mutant possessing a duplicated kinase domain.

A mutation of the epidermal growth factor receptor (EGFR) that results in a tandem kinase domain duplication (TKD-EGFR) has been described in glioblastoma multiforme biopsies and cell lines. Although the TKD-EGFR confers tumorigenicity, little is known about the molecular underpinnings of receptor dysregulation. Therefore, we transfected B82L mouse fibroblast cells devoid of endogenous EGFR to determine the molecular mechanisms of receptor activation when expressed in cells as well as the contribution of each duplicated kinase domain to receptor phosphorylation. The TKD-EGFR displayed chronically elevated basal autophosphorylation at five known phosphotyrosine sites. The chronically phosphorylated TKD-EGFR was also resistant to competitive inhibition of ligand-binding compared with wild-type EGFR (WT-EGFR) and showed undetectable levels of basal dimerization, suggesting the TKD-EGFR escapes known mechanisms of receptor downregulation. Immunofluorescence analyses revealed a substantial portion of the TKD-EGFR resides in the cytosol in an activated state, although surface-localized subsets of the receptor retain ligand responsiveness. Kinase activity-deficient knockouts of the N-terminal or the C-terminal kinase domains generated TKD-EGFRs that recapitulate the autophosphorylation/localization patterns of a constitutively activated receptor versus a WT-like EGFR, respectively. Investigation of the molecular activity of the TKD-EGFR yields evidence for a unique mechanism of constitutive activity and dual kinase domain activation.

Down-regulation of epidermal growth factor receptor-dependent signaling by Porphyromonas gingivalis lipopolysaccharide in life-expanded human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Human gingival fibroblasts exhibit proliferative responses following epidermal growth factor exposure, which are thought to enhance periodontal regeneration in the absence of bacterial products such as lipopolysacharide. However, lipopolysaccharide challenge activates human gingival fibroblasts to release several inflammatory mediators that contribute to the immune response associated with periodontitis and attenuate wound repair. We tested the hypothesis that Porphyromonas gingivalis lipopolysaccharide-activated signaling pathways down-regulate epidermal growth factor receptor-dependent events. MATERIAL AND METHODS: To study lipopolysaccharide/epidermal growth factor interactions in human gingival fibroblasts, we introduced the catalytic subunit of human telomerase into human gingival fibroblasts, thereby generating a more long-lived cellular model. These cells were characterized and evaluated for lipopolysaccharide/epidermal growth factor responsiveness and regulation of epidermal growth factor-dependent pathways. RESULTS: Comparison of human telomerase-transduced gingival fibroblasts with human gingival fibroblasts revealed that both cell lines exhibit a spindle-like morphology and express similar levels of epidermal growth factor receptor, CD14 and Toll-like receptors 2 and 4. Importantly, human telomerase-transduced gingival fibroblasts proliferation rates are increased 5-9 fold over human gingival fibroblasts and exhibit a longer life span in culture. In addition, human telomerase-transduced gingival fibroblasts and human gingival fibroblasts exhibit comparable profiles of mitogen-activated protein kinase kinase (extracellular signal-regulated kinase 1/2) activation upon epidermal growth factor or P. gingivalis lipopolysaccharide administration. Interestingly, treatment with P. gingivalis lipopolysaccharide leads to a down-regulation of epidermal growth factor-dependent extracellular signal-regulated kinase 1/2, p38 and cyclic-AMP response element binding protein phosphorylation in both cell types. CONCLUSION: These studies demonstrate that human telomerase-transduced gingival fibroblasts exhibit an extended life span and recapitulate human gingival fibroblasts biology. Moreover, this system has allowed for the first demonstration of lipopolysaccharide down-regulation of epidermal growth factor activated pathways in human gingival fibroblasts and should facilitate the analysis of signaling events relevant to the pathogenesis and treatment of periodontitis.

Transduction of a dominant-negative H-Ras into human eosinophils attenuates extracellular signal-regulated kinase activation and interleukin-5-mediated cell viability.

Inhibition of eosinophil apoptosis by exposure to interleukin-5 (IL-5) is associated with the development of tissue eosinophilia and may contribute to the inflammation characteristic of asthma. Analysis of the signaling events associated with this process has been hampered by the inability to efficiently manipulate eosinophils by the introduction of active or inhibitory effector molecules. Evidence is provided, using a dominant-negative N17 H-Ras protein (dn-H-Ras) and MEK inhibitor U0126, that activation of the Ras-Raf-MEK-ERK pathway plays a determining role in the prolongation of eosinophil survival by IL-5. For these studies, a small region of the human immunodeficiency virus Tat protein, a protein transduction domain known to enter mammalian cells efficiently, was fused to the N-terminus of dn-H-Ras. The Tat-dn-H-Ras protein generated from this construct transduced isolated human blood eosinophils at more than 95% efficiency. When Tat-dn-H-Ras-transduced eosinophils were treated with IL-5, they exhibited a time- and dosage-dependent reduction in extracellular regulated kinase 1 and 2 activation and an inhibition of p90 Rsk1 phosphorylation and IL-5-mediated eosinophil survival in vitro. In contrast, Tat-dn-H-Ras did not inhibit CD11b up-regulation or STAT5 tyrosine phosphorylation. These data demonstrate that Tat dominant-negative protein transduction can serve as an important and novel tool in studying primary myeloid cell signal transduction in primary leukocytes and can implicate the Ras-Raf-MEK-ERK pathway in IL-5-initiated eosinophil survival.

Cutting edge: the nucleotide receptor P2X7 contains multiple protein- and lipid-interaction motifs including a potential binding site for bacterial lipopolysaccharide.

The nucleotide receptor P2X7 has been shown to modulate LPS-induced macrophage production of numerous inflammatory mediators. Although the C-terminal portion of P2X7 is thought to be essential for multiple receptor functions, little is known regarding the structural motifs that lie within this region. We show here that the P2X7 C-terminal domain contains several apparent protein-protein and protein-lipid interaction motifs with potential importance to macrophage signaling and LPS action. Surprisingly, P2X7 also contains a conserved LPS-binding domain. In this report, we demonstrate that peptides derived from this P2X7 sequence bind LPS in vitro. Moreover, these peptides neutralize the ability of LPS to activate the extracellular signal-regulated kinases (ERK1, ERK2) and to promote the degradation of the inhibitor of kappaB-alpha isoform (IkappaB-alpha) in RAW 264.7 macrophages. Collectively, these data suggest that the C-terminal domain of P2X7 may directly coordinate several signal transduction events related to macrophage function and LPS action.

Granulocyte macrophage colony-stimulating factor and interleukin-5 activate STAT5 and induce CIS1 mRNA in human peripheral blood eosinophils.

In these studies, we examined signaling through the transcription factor STAT5 in human peripheral blood eosinophils after treatment with granulocyte macrophage colony-stimulating factor (GM-CSF) or interleukin (IL)-5. In response to either cytokine, STAT5 was rapidly tyrosine phosphorylated and acquired interferon gamma activation site (GAS) DNA binding activity. Tyrosine-phosphorylated STAT5 was associated with both cytosolic and nuclear cell fractions. Consistent with activation, the transcription of a STAT5-dependent gene, cytokine inducible, SH2-containing protein (CIS1), was enhanced after cytokine stimulation. This is the first report of IL-5 regulation of CIS1 gene expression in any cell type. Given its role in cytokine signaling, CIS1 upregulation may serve to attenuate IL-5 and GM-CSF modulation of eosinophil function. These data suggest that active nuclear STAT5 participates in the regulation of IL-5 and GM-CSF--inducible genes in stimulated human peripheral blood eosinophils.

CD14(+) cells are necessary for increased survival of eosinophils in response to lipopolysaccharide.

There has been considerable interest in the effect that gram-negative bacterial endotoxin (lipopolysaccharide [LPS]) can have in asthma, given that inhalation of LPS has been shown to cause bronchial hyperresponsiveness. Further, there is evidence that the endotoxin-binding protein CD14 may be a marker for asthma. Inhaled LPS has been shown to cause an influx of eosinophils into the nasal airway and to increase the survival of CD16-negatively selected eosinophils in vitro. In this study, we compared survival of eosinophils isolated via CD16-negative selection with eosinophils that were isolated using both CD16- and CD14-negative selection criteria. Survival of CD16-negatively selected eosinophils was enhanced by LPS in a dose-dependent manner and was inhibited by the endotoxin antagonists polymyxin B or lipid X. In contrast, depletion of CD14(+) cells within the eosinophil preparations (CD14/CD16-negatively selected eosinophils) decreased the effect of LPS on survival. Preincubation of CD16-negatively selected eosinophils with antibody 60bd, which blocks LPS binding to CD14, prevented the survival-enhancing effect of LPS. However, CD14 was not detected on eosinophils by flow cytometry, even after incubation with LPS for up to 24 h. These results suggest that the survival-enhancing effect of LPS on eosinophils requires the presence of CD14(+) cells in the population. It is our hypothesis that enhanced eosinophil survival with LPS involves the contribution of another cell type.

Estrogen modulation of prolactin gene expression requires an intact mitogen-activated protein kinase signal transduction pathway in cultured rat pituitary cells.

Expression of the PRL gene is regulated by many factors, including cAMP, estradiol (E2), phorbol esters, epidermal growth factor (EGF), and TRH. The promoter region of the rat PRL gene has been shown to contain DNA sequences that are thought to support the direct interaction of estrogen receptors (ERs) with DNA. It is by this direct ER/DNA interaction that estrogen is thought to modulate expression of PRL. We report here that estrogeninduced PRL expression requires an intact mitogen-activated protein kinase (MAPK) signal transduction pathway in cultured rat pituitary cells (PR1 lactotroph and GH3 somatolactotroph cell lines). Interfering with the MAPK signaling cascade by inhibiting the activity of MAPK kinase (MEK) ablates the ability of estrogen to induce PRL mRNA and protein. In these cell lines, estrogen activates extracellular regulated protein kinases ERK-1 and ERK-2 enzyme activities maximally within 10 min of 1 nM E2 treatment. This activity is blocked by pretreatment of the cells with the MEK inhibitors PD98059 and UO126. The mechanism by which ERKs-1 and -2 are activated by estrogen appears to be independent of c-Src since the effects of estrogen on PRL gene expression are not affected by herbimycin A or PP1 administration. c-Raf-1 may be involved in the effects of E2 because estrogen causes the rapid and transient tyrosine phosphorylation of c-Raf-1. The ER antagonist ICI 182,780 blocks both ERK-1 and ERK-2 activation in addition to PRL protein and mRNA, implying a central role for the classical ER in the activation of the MAPK pathway resulting in PRL gene expression.

Epidermal growth factor-stimulated tyrosine phosphorylation of caveolin-1. Enhanced caveolin-1 tyrosine phosphorylation following aberrant epidermal growth factor receptor status.

Caveolin-1 is the major coat protein of caveolae and has been reported to interact with various intracellular signaling molecules including the epidermal growth factor (EGF) receptor. To investigate the involvement of caveolin-1 in EGF receptor action, we used mouse B82L fibroblasts transfected with (a) wild type EGF receptor, (b) a C-terminally truncated EGF receptor at residue 1022, (c) a C-terminally truncated EGF receptor at residue 973, or (d) a kinase-inactive EGF receptor (K721M). Following EGF treatment, there was a distinct electrophoretic mobility shift of the caveolin-1 present in cells expressing the truncated forms of the EGF receptor, but this shift was not detectable in cells bearing either normal levels of the wild type EGF receptor or a kinase-inactive receptor. This mobility shift was also not observed following the addition of other cell stimuli, such as platelet-derived growth factor, insulin, basic fibroblast growth factor, or phorbol 12-myristate 13-acetate. Analysis of caveolin-1 immunoprecipitates from EGF-stimulated or nonstimulated cells demonstrated that the EGF-induced mobility shift of caveolin-1 was associated with its tyrosine phosphorylation in cells expressing truncated EGF receptors. Maximal caveolin-1 phosphorylation was achieved within 5 min after exposure to 10 nM EGF and remained elevated for at least 2 h. Additionally, several distinct phosphotyrosine-containing proteins (60, 45, 29, 24, and 20 kDa) were co-immunoprecipitated with caveolin-1 in an EGF-dependent manner. Furthermore, the Src family kinase inhibitor, PP1, does not affect autophosphorylation of the receptor, but it does inhibit the EGF-induced mobility shift and phosphorylation of caveolin-1. Conversely, the MEK inhibitors PD98059 and UO126 could attenuate EGF-induced mitogen-activated protein kinase activation, they do not affect the EGF-induced mobility shift of caveolin-1. Because truncation and overexpression of the EGF receptor have been linked to cell transformation, these results provide the first evidence that the tyrosine phosphorylation of caveolin-1 occurs via an EGF-sensitive signaling pathway that can be potentiated by an aberrant activity or expression of various forms of the EGF receptor.

Platelet-derived growth factor-stimulated migration of murine fibroblasts is associated with epidermal growth factor receptor expression and tyrosine phosphorylation.

Previous studies have shown that epidermal growth factor (EGF) synergizes with various extracellular matrix components in promoting the migration of B82L fibroblasts expressing wild-type EGF receptors and that functional EGF receptors are critical for the conversion of B82L fibroblasts to a migratory cell type (). In the present study, we examined the effects of platelet-derived growth factor (PDGF) on the motility of B82L fibroblasts using a microchemotaxis chamber. We found that PDGF can enhance fibronectin-induced migration of B82L fibroblasts expressing wild-type EGF receptors (B82L-clone B3). However, B82L cells that lack the EGF receptor (B82L-parental) or that express an EGF receptor that is kinase-inactive (B82L-K721M) or C-terminally truncated (B82L-c'973) exhibit little PDGF-stimulated migration. In addition, none of these three cell lines exhibit the capacity to migrate to fibronectin alone. These observations indicate that, similar to cell migration toward fibronectin, PDGF-induced cell migration of B82L fibroblasts is augmented by the expression of an intact EGF receptor kinase. The loss of PDGF-stimulated motility in B82L cells that do not express an intact EGF receptor does not appear to result from a gross dysfunction of PDGF receptors, because ligand-stimulated tyrosine phosphorylation of the PDGF-beta receptor and the activation of mitogen-activated protein kinases are readily detectable in these cells. Moreover, an interaction between EGF and PDGF receptor systems is supported by the observation that the EGF receptor exhibits an increase in phosphotyrosine content in a time-dependent fashion upon the addition of PDGF. Altogether, these studies demonstrate that the expression of EGF receptor is critical for PDGF-stimulated migration of murine B82L fibroblasts and suggest a role for the EGF receptor downstream of PDGF receptor activation in the signaling events that lead to PDGF-stimulated cell motility.

The juxtamembrane region of the epidermal growth factor receptor is required for phosphorylation of Galpha(s).

We have previously demonstrated that Galpha(s) associates with the juxtamembrane region of the epidermal growth factor (EGF) receptor (EGFR) and that the EGFR can phosphorylate and activate this G protein (H. Poppleton et al., 1996, J. Biol. Chem. 271, 6947-6951; H. Sun et al., 1995, Proc. Natl. Acad. Sci. USA 92, 2229-2233). In this report, we have employed peptides EGFR-13 and EGFR-14 (corresponding to amino acids 645-657 and 679-692 in the EGFR, respectively) which disrupt the association of Galpha(s) with the EGFR to investigate whether or not this region of the EGFR is required for phosphorylation of the G protein. EGFR-13 increased the tyrosine phosphorylation of G(alpha)s by two-fold whereas EGFR-14 decreased the phosphorylation of the G protein. Phosphorylation of EGFR-13 on the threonine residue corresponding to Thr654 of the EGFR obliterated the ability of the peptide to increase Galpha(s) phosphorylation. EGFR-13 and EGFR-14, but not phospho-EGFR-13, competed for the association of the EGFR with Galpha(s). A peptide betaIII-2 corresponding to amino acids Arg259-Lys273 in the beta2-adrenergic receptor which competes for association of Galpha(s) with the EGFR and increases protein tyrosine kinase activity of the EGFR could mimic the effects of EGFR-13. Among the three peptides (EGFR-13, EGFR-14, and betaIII-2) that interfere with association of Galpha(s) to the EGFR, only EGFR-13 and betaIII-2 have been shown to activate the G protein. Polylysine which increases EGFR tyrosine kinase activity but does not interfere with association of Galpha(s) and EGFR also augmented phosphorylation of Galpha(s) by the EGFR. Phosphopeptide mapping demonstrated that EGFR-13 and polylysine increased phosphorylation of Galpha(s) by the EGFR on the same additional sites. Collectively, these data suggest that the interaction of Galpha(s) with residues 645-657 of the EGFR, or a peptide corresponding to this sequence alters the conformation of the G protein and/or the EGFR such that Galpha(s) is readily phosphorylated by the EGFR. The peptide EGFR-14, which does not activate Galpha(s), does not allow for the efficient phosphorylation of the G protein even though it does elevate the intrinsic tyrosine kinase activity of the EGFR. The hyperphosphorylation of Galpha(s) by EGFR is likely to require the contact of the G protein with EGFR-13 region (aa 645-657 in the EGFR) as well as augmentation of EGFR kinase activity.

ERK1 and ERK2 activation by chemotactic factors in human eosinophils is interleukin 5-dependent and contributes to leukotriene C(4) biosynthesis.

Eosinophils, the major immune effector cells contributing to allergic inflammation and asthma, are profoundly affected by interleukin (IL) 5 with respect to their differentiation, viability, recruitment, and cytotoxic effector functions. IL-5 enhances eosinophil responsiveness to a variety of chemotactic factors via a process called priming, although the molecular mechanism is unknown. In this study, we report that, following IL-5 priming of eosinophils, chemotactic agents including fMet-Leu-Phe, IL-8, and RANTES, promote vigorous transient activation of ERK1 and ERK2. In contrast, these chemotactic factors stimulate weak or indiscernible ERK activation in unprimed eosinophils. Furthermore, this intracellular marker of priming is selective for IL-5-related cytokines, in that it is observed following exposure to IL-5 and granulocyte macrophage-colony stimulating factor but not to interferon-gamma, stem cell factor, tumor necrosis factor alpha, or IL-4. Interestingly, priming of chemoattractant-induced ERK activation is accompanied by an increase in association of tyrosine-phosphorylated proteins with the adapter protein Grb2. The biological relevance of ERK activation to IL-5 priming is supported by the observation that inhibition of ERK activity by treatment with the MEK inhibitors PD98059 or U0126 inhibited the release of leukotriene C(4) stimulated by fMet-Leu-Phe in IL-5-primed eosinophils. These data provide evidence for a previously undescribed fundamental mechanism by which stimulation of IL-5 family receptors induces a rapid phenotypic alteration in the signal transduction pathways of chemotactic receptors, enabling their activation of the ERK1 and ERK2 pathway and contributing to the capacity of these cells to synthesize LTC(4).

Growth hormone-induced alteration in ErbB-2 phosphorylation status in 3T3-F442A fibroblasts.

The growth hormone receptor (GHR), a cytokine receptor superfamily member, requires the JAK2 tyrosine kinase for signaling. We now examine functional interactions between growth hormone (GH) and epidermal growth factor (EGF) in 3T3-F442A fibroblasts. Although EGF enhanced ErbB-2 tyrosine phosphorylation, GH, while causing retardation of its migration on SDS-polyacrylamide gel electrophoresis, decreased ErbB-2's tyrosine phosphorylation. GH-induced retardation was reversed by treatment of anti-ErbB-2 precipitates with both alkaline phosphatase and protein phosphatase 2A, suggesting that GH induced serine/threonine phosphorylation of ErbB-2. Both GH-induced shift in ErbB-2 migration and GH-induced MAP kinase activation were unaffected by a protein kinase C inhibitor but were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1) inhibitor, PD98059. Notably, leukemia inhibitory factor, but not interferon-gamma, also promoted ErbB-2 shift and mitogen-activated protein kinase activation. Cotreatment with EGF and GH versus EGF alone resulted in a 35% decline in acute ErbB-2 tyrosine 1248 autophosphorylation, a marked decline (approximately 50%) in DNA synthesis, and substantially decreased cyclin D1 expression. We conclude that in 3T3-F442A cells, 1) the GH-induced decrease in ErbB-2 tyrosine phosphorylation correlates with MEK1/mitogen-activated protein kinase activity and 2) GH antagonizes EGF-induced DNA synthesis and cyclin D1 expression in a pattern consistent with its alteration in ErbB-2 phosphorylation status.

Design, combinatorial chemical synthesis and in vitro characterization of novel urea based gelatinase inhibitors.

A novel series of hydroxamate/urea-based inhibitors of gelatinases has been discovered via solid-phase combinatorial chemistry. SAR of P1', P2', and P3' has been exploited and structures different from traditional succinate-based MMP inhibitors have been found.

Calcium and phosphatidylserine stimulate the self-association of conventional protein kinase C isoforms.

Conflicting evidence exists as to whether "conventional" protein kinase C isoforms (cPKCs) function as monomers or oligomers. In this report, we demonstrate that purified cPKC isoforms can be rapidly cross-linked by the sulfhydryl-selective cross-linker bis(maleimido)hexane, but only in the presence of both Ca(2+) and phosphatidylserine; cross-linking was minimal in the presence of either of these activators alone. In addition, cross-linking of these cPKCs did not require Mg(2+) or ATP. Among the various phospholipids tested, phosphatidylserine was found to be the most effective in the promotion of cPKC self-association and for the stimulation of protein kinase activity toward the exogenous substrate histone. Phosphatidic acid and phosphatidylinositol were less effective in this regard, whereas phosphatidylcholine exhibited little ability to induce cPKC self-association or to stimulate kinase activity. An examination of the mechanism by which the cPKC isoforms self-associate in the presence of phospholipid/Ca(2+) revealed that this process occurred independently of phospholipid aggregation. Moreover, self-association was not inhibited by saturating the enzyme active site with a peptide substrate, suggesting that self-association is distinct from an enzyme-substrate interaction. Isoform-specific antibodies revealed that all cPKC isoforms (alpha, beta, and gamma) self-associate and that, in a mixture of cPKC isoforms, PKC-alpha forms primarily alpha-alpha homodimers. Besides cPKC interactions detected with purified enzyme, PKC-alpha also appeared capable of self-association in murine B82L fibroblasts that were treated with calcium ionophore, phorbol ester, or epidermal growth factor but not in untreated cells. Collectively, these data indicate that self-association occurs in parallel with cPKC activation, that self-association is not mediated by the substrate binding site, and, at least in the case of PKC-alpha, that the formation of isoform homodimers predominates.

Chronic exposure to lead acetate affects the development of protein kinase C activity and the distribution of the PKCgamma isozyme in the rat hippocampus.

This study has examined the effect of chronic inorganic lead exposure on phospholipid-dependent protein kinase C (PKC) activity, and the distribution of its alpha (alpha), beta II (betaII), gamma (gamma), and zeta (zeta) isozymes in subcellular fractions of the developing rat hippocampus. Dams were exposed to either 0 or 1000 ppm lead acetate in their drinking water for one week and mated. Offspring were exposed to lead in utero, via lactation, and directly in the drinking water after weaning. The offspring were sacrificed at postnatal days 1 (P1), 8 (P8), 15 (P15), and 29 (P29). PKC activity was determined in the post-synaptosomal supernatant (PSS) and synaptosomal (P-2) membrane fractions by an in vitro assay using histone as the phosphate acceptor. The selected PKC isozymes were detected by immunoblotting techniques. In control animals, PKC activity (pmole/min/mg total protein) in both subcellular fractions substantially increased between P1 and P8. In chronically exposed rats exhibiting clinically relevant blood lead concentrations, this marked increase in PKC activity on P8 was significantly attenuated in both subcellular fractions. On this postnatal day, the amount of immunodetectable PKC gamma was significantly higher in the synaptosomal membrane fraction of lead-exposed rats. Other isozymes were unaffected. These results imply that in lead-exposed animals the PKC gamma isozyme was inactive even though it was associated with the membrane. These results also suggest that prolonged exposure to the heavy metal attenuated PKC activity at an important developmental time to potentially adversely affect normal hippocampal function.

Transmodulation of epidermal growth factor receptor function by cyclic AMP-dependent protein kinase.

Binding of epidermal growth factor (EGF) to its receptor (EGFR) augments the tyrosine kinase activity of the receptor and autophosphorylation. Exposure of some tissues and cells to EGF also stimulates adenylyl cyclase activity and results in an increase in cyclic AMP (cAMP) levels. Because cAMP activates the cAMP-dependent protein kinase A (PKA), we investigated the effect of PKA on the EGFR. The purified catalytic subunit of PKA (PKAc) stoichiometrically phosphorylated the purified full-length wild type (WT) and kinase negative (K721M) forms of the EGFR. PKAc phosphorylated both WT-EGFR as well as a mutant truncated form of EGFR (Delta1022-1186) exclusively on serine residues. Moreover, PKAc also phosphorylated the cytosolic domain of the EGFR (EGFRKD). Phosphorylation of the purified WT as well as EGFRDelta1022-1186 and EGFRKD was accompanied by decreased autophosphorylation and diminished tyrosine kinase activity. Pretreatment of REF-52 cells with the nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio)-cAMP, decreased EGF-induced tyrosine phosphorylation of cellular proteins as well as activation of the WT-EGFR. Similar effects were also observed in B82L cells transfected to express the Delta1022-1186 form of EGFR. Furthermore, activation of PKAc in intact cells resulted in serine phosphorylation of the EGFR. The decreased phosphorylation of cellular proteins and diminished activation of the EGFR in cells treated with the cAMP analog was not the result of altered binding of EGF to its receptors or changes in receptor internalization. Therefore, we conclude that PKA phosphorylates the EGFR on Ser residues and decreases its tyrosine kinase activity and signal transduction both in vitro and in vivo.