p38 mitogen-activated protein (MAP) kinase but not p44/p42 MAP kinase is involved in prostaglandin E1-induced vascular endothelial growth factor synthesis in osteoblasts

We investigated the mechanism underlying vascular endothelial growth factor (VEGF) synthesis stimulated by prostaglandin E1 (PGE1) in osteoblast-like MC3T3-E1 cells. PGE1 induced the phosphorylation of both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. SB203580, a specific inhibitor of p38 MAP kinase, inhibited the PGE1-stimulated VEGF synthesis as well as PGE1-induced phosphorylation of p38 MAP kinase. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, which reduced the PGE1-induced phosphorylation of p44/p42 MAP kinase, had little effect on the VEGF synthesis stimulated by PGE1. AH-6809, an antagonist of the subtypes of the PGE receptor, EP1 and EP2, or SC-19220, an antagonist of EP1 receptor, did not inhibit the PGE1-induced VEGF synthesis. H-89, an inhibitor of cAMP-dependent protein kinase, and SQ22536, an inhibitor of adenylate cyclase, reduced the VEGF synthesis induced by PGE1. Cholera toxin, an activator of G(s), and forskolin, an activator of adenylate cyclase, induced VEGF synthesis. SB203580 and PD169316, another specific inhibitor of p38 MAP kinase, reduced the cholera toxin-, forskolin- or 8bromo-cAMP-stimulated VEGF synthesis. However, PD98059 failed to affect the VEGF synthesis stimulated by cholera toxin, forskolin or 8-bromoadenosine-3',5'-cyclic monophosphate (8bromo-cAMP). SB203580 reduced the phosphorylation of p38 MAP kinase induced by forskolin or 8bromo-cAMP. These results strongly suggest that p44/p42 MAP kinase activation is not involved in the PGE1-stimulated VEGF synthesis in osteoblasts but that p38 MAP kinase activation is involved.     

Platelet factor 4 inhibits FGF2-induced endothelial cell proliferation via the extracellular signal-regulated kinase pathway but not by the phosphatidylinositol 3-kinase pathway

Platelet factor 4 (PF-4) is a member of the chemokine family with powerful antiangiogenic properties. The mechanism by which PF-4 inhibits endothelial cell proliferation is unclear. We investigated the effects of PF-4 on the intracellular signal transduction induced by basic fibroblast growth factor (FGF2). We found that PF-4 (10 microg/mL) inhibited the FGF2-induced proliferation of adrenal cortex capillary endothelial (ACE) cells. The inhibition of MEK1/2 (mitogen-activated protein kinase kinase) by PD98059 or of PI3K (phosphatidylinositol 3-kinase) by Ly294002 abolished the proliferation induced by FGF2, suggesting that ACE cell proliferation required dual signaling through both the extracellular signal-regulated kinase (ERK) and PI3K pathways. Ly294002 had no significant effect on ERK phosphorylation, whereas PD98059 had a weak effect on the phosphorylation of Akt, suggesting that 2 separate cascades are required for ACE cell proliferation. The addition of PF-4 (10 microg/mL) significantly inhibited ERK phosphorylation (95%), showing that PF-4 acted directly on or upstream from this kinase. Surprisingly, PF-4 did not affect FGF2-induced Akt phosphorylation. This suggests that PF-4 disrupts FGF2 signaling via an intracellular mechanism of inhibition. To exclude the possibility that PF-4 inhibited the binding of FGF2 to only one FGF receptor, preferentially activating the ERK pathway, we investigated the effect of PF-4 on FGF2-induced ERK and Akt phosphorylation, using mutant heparan sulfate-deficient Chinese hamster ovary cells transfected with the FGF-R1 cDNA. The addition of PF-4 (1 microg/mL) significantly inhibited ERK phosphorylation (90%), with no effect on Akt phosphorylation, suggesting that PF-4 acts downstream from the FGF-R1 receptor. In conclusion, this is the first report showing that PF-4 inhibits FGF2 activity downstream from its receptor.     

Macrophage glucocorticoid receptors regulate Toll-like receptor 4-mediated inflammatory responses by selective inhibition of p38 MAP kinase

To explore the role of glucocorticoids in regulation of kinase pathways during innate immune responses, we generated mice with conditional deletion of glucocorticoid receptor (GR) in macrophages (MGRKO). Activation of toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) caused greater mortality and cytokine production in MGRKO mice than in controls. Ex vivo, treatment with dexamethasone (Dex) markedly inhibited LPS-mediated induction of inflammatory genes in control but not GR-deficient macrophages. We show that Dex inhibits p38 MAPK, but not PI3K/Akt, ERK, or JNK, in control macrophages. Associated with p38 inhibition, Dex induced MAP kinase phosphatase-1 (MKP-1) in control, but not MGRKO, macrophages. Consistent with the ex vivo studies, treatment with a p38 MAPK-specific inhibitor resulted in rescue of MGRKO mice from LPS-induced lethality. Taken together, we identify p38 MAPK and its downstream targets as essential for GR-mediated immunosuppression in macrophages.     

A protein factor for ras p21-dependent activation of mitogen-activated protein (MAP) kinase through MAP kinase kinase.

To identify the direct target molecule of ras p21 in higher eukaryotes, we have recently developed the cell-free system in which ras p21 activates mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK). In this cell-free system, the guanosine 5'-[gamma-thio]triphosphate- bound form of Ki-ras p21, but not the GDP-bound form, activates endogenous Xenopus MAP kinase as well as recombinant ERK2 in the presence of the cytosol fraction of Xenopus oocytes. We separated two protein factors from the cytosol fraction of Xenopus oocytes by column chromatography: one was the inactive form of MAP kinase kinase and the other was a factor tentatively named ras p21-dependent ERK-kinase stimulator (REKS). The former and latter showed M(r) values of approximately 45,000 and 150,000-200,000, respectively, as estimated by gel filtration. Both factors were necessary for Ki-ras p21-dependent activation of MAP kinase/ERK2. These results indicate that an additional protein factor (REKS) is essential for Ki-ras p21 to activate MAP kinase through MAP kinase kinase.     

CD38-mediated growth suppression of B-cell progenitors requires activation of phosphatidylinositol 3-kinase and involves its association with the protein product of the c-cbl proto-oncogene

The signalling pathways that arrest the cell cycle and trigger cell death are only partially known. Dimerization of CD38, a 45-kD transmembrane type II glycoprotein highly expressed in immature B cells, inhibits cell growth and causes apoptosis in normal and leukemic B-cell progenitors, but the molecular mechanisms underlying these cellular responses are unknown. In the present study, we found that CD38 ligation in the immature B-cell lines 380, REH, and RS4;11 caused rapid tyrosine phosphorylation of the protein product of the proto- oncogene c-cbl. Dimerization of CD38 was accompanied by the association of cbl with the p85 subunit of phosphatidylinositol 3-kinase (Pl 3-K), resulting in markedly increased Pl 3-K activity in antiphosphotyrosine and anti-cbl immunoprecipitates. Wortmannin and LY294002, two potent inhibitors of Pl 3-K, rescued immature B cells from CD38-mediated growth suppression. This effect was observed not only in model B-cell lines, but also in cultures of leukemic lymphoblasts from patients, and in normal bone marrow B-cell progenitors as well. Concentrations of inhibitors that reversed cellular responses to CD38 significantly decreased Pl 3-K activity. By contrast, rapamycin, a p70 S6-kinase inhibitor, did not rescue immature B cells from CD38-mediated suppression. These results suggest that Pl 3-K activity is essential for CD38-mediated inhibition of lymphopoiesis and that cbl and Pl 3-K are regulatory molecules whose activation can result in suppression of cell proliferation and apoptosis in immature lymphoid cells.     

Src-induced activation of inducible T cell kinase (ITK) requires phosphatidylinositol 3-kinase activity and the Pleckstrin homology domain of inducible T cell kinase

The Tec family of tyrosine kinases are involved in signals emanating from cytokine receptors, antigen receptors, and other lymphoid cell surface receptors. One family member, ITK (inducible T cell kinase), is involved in T cell activation and can be activated by the T cell receptor and the CD28 cell surface receptor. This stimulation of tyrosine phosphorylation and activation of ITK can be mimicked by the Src family kinase Lck. We have explored the mechanism of this requirement for Src family kinases in the activation of ITK. We found that coexpression of ITK and Src results in increased membrane association, tyrosine phosphorylation and activation of ITK, which could be blocked by inhibitors of the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase) as well as overexpression of the p85 subunit of PI 3-kinase. Removal of the Pleckstrin homology domain (PH) of ITK resulted in a kinase that could no longer be induced to localize to the membrane or be activated by Src. The PH of ITK was also able to bind inositol phosphates phosphorylated at the D3 position. Membrane targeting of ITK without the PH recovered its ability to be activated by Src. These results suggest that ITK can be activated by a combination of Src and PI 3-kinase.     

Interferon regulatory factor 3 is involved in Toll-like receptor 4 (TLR4)- and TLR3-induced IL-12p35 gene activation

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by dendritic cells (DCs) in response to Toll-like receptor (TLR) ligation. While the mechanisms regulating IL-12p40 chain gene expression are well characterized, molecular events involved in IL-12p35 chain gene activation remain to be clarified. Since IL-12p35 mRNA was induced in human DCs activated through TLR3 or TLR4 but not TLR2, we investigated the potential role of interferon regulatory factor 3 (IRF-3) in IL-12p35 gene transactivation. First, a binding site for IRF-3 named interferon-stimulated response element-1 (ISRE-1) was identified in the human IL-12p35 promoter region between nucleotides -251 and -240. The ISRE-1 site was required for IL-12p35 gene activation in RAW 264.7 cells stimulated by lipopolysaccharide (LPS) or PolyI:C. Ectopic expression of IRF-3 was found to up-regulate IL-12p35 gene activation in the same system. Furthermore, chromatin immunoprecipitation (ChIP) studies demonstrated that IRF-3 is recruited to ISRE-1 site in TLR4- or TLR3-stimulated human DCs. Finally, experiments on DCs from IRF-3-deficient mice established that TLR4-induced IL-12p35 mRNA and IL-12p70 synthesis are impaired in absence of IRF-3. We conclude that IRF-3 binds to a critical cis-acting element in the IL-12p35 gene promoter and thereby represents a key factor for the induction of IL-12p70 synthesis in DCs.     

Mitogen-activated protein (MAP) kinases are involved in interleukin-1 (IL-1)-induced IL-6 synthesis in osteoblasts: modulation not of p38 MAP kinase, but of p42/p44 MAP kinase by IL-1-activated protein kinase C.

We previously reported that interleukin-1alpha (IL-1alpha)-induced activation of protein kinase C (PKC) via phosphatidylcholine-specific phospholipase C (PC-PLC) limits IL-6 synthesis induced by IL-1alpha itself in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism behind IL-1alpha-induced IL-6 synthesis in MC3T3-E1 cells. IL-1alpha time-dependently stimulated the phosphorylation of both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase. PD98059, a specific inhibitor of the upstream kinase that activates p42/p44 MAP kinase, inhibited the IL-1alpha-induced IL-6 synthesis as well as the phosphorylation of p42/p44 MAP kinase induced by IL-1alpha. SB203580, a specific inhibitor of p38 MAP kinase, also reduced both the phosphorylation of p38 MAP kinase and the IL-6 synthesis. 1-Oleoyl-2-acetylglycerol, an activator of PKC, suppressed the IL-1alpha-induced IL-6 synthesis. Calphostin C, a specific inhibitor of PKC, or D-609, a specific inhibitor of PC-PLC, significantly enhanced the IL-1alpha-induced phosphorylation of p42/p44 MAP kinase without affecting the phosphorylation of p38 MAP kinase. The phosphorylation of p42/p44 MAP kinase by IL-1alpha was markedly increased in PKC-down-regulated MC3T3-E1 cells. Neither 12-O-tetradecanoylphorbol-13-acetate, known to be an activator of PKC, nor 1-oleoyl-2-acetylglycerol affected the phosphorylation of p38 MAP kinase induced by IL-1alpha. These results strongly suggest that IL-1alpha-induced IL-6 synthesis is mediated via activations of both p42/p44 MAP kinase and p38 MAP kinase in osteoblasts, and that PKC activated by IL-1alpha itself negatively regulates IL-6 synthesis at a point upstream from p42/p44 MAP kinase.     

Neutrophil adhesion to endothelial cells induced by platelet factor 4 requires sequential activation of Ras, Syk, and JNK MAP kinases

Signal transduction mechanisms associated with neutrophil activation by platelet factor 4 (PF4; CXCL4) are as yet poorly characterized. In a recent report, we showed that PF4-induced neutrophil functions (such as adhesion and secondary granule exocytosis) involve the activation of Src-kinases. By analyzing intracellular signals leading to adherence, we here demonstrate by several lines of evidence that in addition to Src-kinases, PF4 signaling involves the monomeric GTPase Ras, the tyrosine kinase Syk, and the MAP kinase JNK. Furthermore, on stimulation, GTPases Rac2 and RhoA were activated, and each was translocated to a different membrane compartment. As shown by inhibitor studies, Rac2 and JNK are located downstream of Syk and Ras. Most intriguingly, the latter 2 elements appear to control the activity of Rac2 and JNK independently of each other at different phases of the activation process. Although a first phase of Rac2 and JNK activation of up to 5 minutes is initiated by Ras, the second phase (5-30 minutes) depends predominantly on the activity of Syk. In summary, we describe that coordinated activity of Syk, Ras, and JNK mediates neutrophil adhesion to endothelial cells and that PF4 induces sequential activation of these elements.     

TEL/platelet-derived growth factor receptor beta activates phosphatidylinositol 3 (PI3) kinase and requires PI3 kinase to regulate the cell cycle

TEL/platelet-derived growth factor receptor beta (PDGF beta R) is the protein product of the t(5;12) translocation in chronic myelomonocytic leukemia. TEL/PDGF beta R transforms interleukin-3 (IL-3)-dependent Ba/F3 and 32D cells to IL-3 independence and induces a murine myeloproliferative disease in a bone marrow transplantation model of leukemogenesis. The fusion protein encodes a constitutively activated, cytoplasmic tyrosine kinase that activates multiple signal transduction pathways. To identify the signaling pathways that are necessary for transformation by TEL/PDGF beta R, transformed Ba/F3 and 32D cells were studied. TEL/PDGF beta R activates the kinase activity of phosphatidylinositol-3 (PI3) kinase and stimulates phosphorylation of its downstream substrates, including Akt and p70S6 kinase. Activation of this pathway requires the kinase activity of TEL/PDGF beta R and is inhibited by the PDGF beta R inhibitor, STI571. Furthermore, inhibition of PI3 kinase with the pharmacologic inhibitor, LY294002, inhibits growth of the transformed cells. Treated cells arrest in the G1 phase of the cell cycle within 16 hours but do not undergo apoptosis. To study the mechanism of cell cycle arrest by LY294002, the activity of the cdk4 complex, which regulates the transit of cells from the G1 to S phase in hematopoietic cells, was examined. Both STI571 and LY294002 lead to a decrease in the activity of cdk4 kinase activity and a decrease in expression of both Cyclin D2 and Cyclin E within several hours. These studies demonstrate the presence of a signaling pathway from TEL/PDGF beta R to PI3 kinase and subsequently to regulation of the cdk4 kinase complex. Activation of this pathway is necessary for transformation by TEL/PDGF beta R.     

MAP kinase activation by mu opioid receptor in cord blood CD34(+)CD38(-) cells

OBJECTIVE: Opioid receptor expression and function traditionally have been studied in neuronal cells and recently in mature lymphoid cells; however, little is known about their possible functions in hematopoietic stem cells (CD34(+) cells). We studied the expression of the mu receptor on CD34(+) cells and assessed the signal transduction cascade it induces. MATERIALS AND METHODS: Mu-receptor expression on cord blood (CB) and peripheral blood (PB) CD34(+) cells was studied by microarrays, immunostaining, and fluorescence-activated cell sorting analysis. Signal transduction by the mu receptor was studied through Western blots and kinase assay of enkephalin-activated CB CD34(+) cells. Apoptotic, differentiation, and proliferation responses following mu-receptor activatioSn were studied by annexin V assay and inverted microscopy. RESULTS: A prominent difference in gene expression, in favor of CB compared to PB CD34(+) cells, was observed in the mu-receptor gene. This receptor was mainly expressed on the CB CD34(+)CD38(-) subpopulation. A MAP kinase signal transduction cascade was shown to be induced through activation of this receptor by enkephalin or morphine. CONCLUSIONS: We showed for the first time that the mu receptor is expressed on immature CB stem cells and that its activation by enkephalin or morphine induces a MAP kinase signal transduction cascade. Because the MAP kinase cascade is known to elicit proliferation and differentiation responses, these findings suggest a possible role of endogenous enkephalins in hematopoietic stem cell proliferation and differentiation and may lead to therapeutic applications of opiates in CB stem cell expansion and neuronal differentiation.     

Evidence that tumor necrosis factor alpha (TNF)-induced activation of neutrophil respiratory burst on biologic surfaces is mediated by the p55 TNF receptor

Polymorphonuclear leukocytes (PMN) residing on biologic surfaces respond with a vigorous respiratory burst when exposed to tumor necrosis factor alpha (TNF). PMN possess both the p55 and the p75 TNF receptors, but their role in the elicitation of the respiratory burst is not known. We addressed this problem by studying the effect of monoclonal antibodies (MoAbs) directed against the p55 TNF receptor (MoAb H398 and MoAb htr-9) and the p75 TNF receptor (MoAb utr-1) on TNF- induced production of O2- by PMN residing on fibronectin-coated surfaces. Neither the anti-p55 nor the anti-p75 MoAbs affected TNF- induced O2- production despite their known ability to competitively inhibit TNF binding to the corresponding receptor. Experiments with the antibodies alone showed that the anti-p55 MoAbs directly triggered PMN O2- production, whereas no response was elicited by the anti-p75 MoAb. PMN unresponsiveness to the anti-p75 MoAb could not be ascribed to low expression of p75 receptor, because binding of the anti-p75 MoAb utr-1 to PMN was, indeed, even higher than binding of the anti-p55 MoAb htr- 9. The agonistic activity of the anti-p55 MoAbs was comparable with that of TNF and was not or only minimally modified by the simultaneous presence of TNF. Triggering of the respiratory burst by TNF was completely prevented by Fab fragments of the anti-p55 MoAb H398. Moreover, the monovalent Fab fragments, which lacked any stimulatory effect on PMN O2- production, acquired strong agonistic activity on cross-linking with anti Fab antibodies, suggesting that the ability of the anti-p55 antibodies to stimulate PMN O2- production depends on their ability to cross-link the TNF receptors. The agonistic effect of the anti-p55 MoAbs was only observed with cells residing on fibronectin- coated surfaces and not with cells in suspension, and in terms of kinetics, dependence on beta 2 integrin-mediated adherence, microfilament integrity, and sensitivity to elevations of intracellular levels of cAMP, it was virtually indistinguishable from the agonistic effect of TNF. Taken together, these results suggest that the p55 receptor is responsible for TNF-induced triggering of the respiratory burst of PMN residing on biologic surfaces.     

Induction of apoptosis by vitamin D3 analogue EB1089 in NCI-H929 myeloma cells via activation of caspase 3 and p38 MAP kinase

EB1089, a novel 1,25-dihydroxyvitamin D3 analogue, has been known to have potent antiproliferative properties in a variety of malignant cells both in vitro and in vivo. In the present study, we analysed the effect of EB1089 on NCI-H929 human myeloma cells. EB1089 inhibited cell growth of NCI-H929 and efficiently induced the G1 phase arrest of the cell cycle in a dose-dependent manner. We could also detect apoptosis in NCI-H929 cells exposed to EB1089 (1 x 10-7 M for 72 h) using the sub-G1 group of the cell cycle by FACS and annexin V binding assays. Induction of apoptosis by EB1089 was associated with down-regulation of the Bcl-2 protein without change of the Bax protein. Regarding caspase activity, which plays a crucial role in apoptosis, EB1089-treated NCI-H929 cells revealed an increased activity of caspase 3 protease accompanied by degradation of the PARP protein in a dose- and time-dependent manner. In addition, EB1089 caused the down-regulation of p44 extracellular signal-related kinase (ERK) activity and up-regulation of the p38 kinase activity during apoptosis of NCI-H929 cells. These results suggest that EB1089 inhibits growth of NCI-H929 cells via G1 cell cycle arrest as well as apoptosis by activating p38 kinase and suppressing ERK activity.     

Retinoic acid stimulation of the sodium/iodide symporter in MCF-7 breast cancer cells is mediated by the insulin growth factor-I/phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase signaling pathways

CONTEXT: All-trans retinoic acid (tRA) induces differentiation in MCF-7 breast cancer cells, stimulates sodium/iodide symporter (NIS) gene expression, and inhibits cell proliferation. Radioiodine administration after systemic tRA treatment has been proposed as an approach to image and treat some differentiated breast cancer. OBJECTIVE: The objective of this work was to study the relative role of genomic and nongenomic pathways in tRA stimulation of NIS expression in MCF-7 cells. DESIGN: We inspected the human NIS gene locus for retinoic acid-responsive elements and tested them for function. The effects of signal transduction pathway inhibitors were also tested in tRA-treated MCF-7 cells and TSH-stimulated FRTL-5 rat thyroid cells, followed by iodide uptake assay, quantitative RT-PCR of NIS, and cell cycle phase analysis. RESULTS: Multiple retinoic acid response elements around the NIS locus were identified by sequence inspection, but none of them was a functional tRA-induced element in MCF-7 cells. Inhibitors of the IGF-I receptor, Janus kinase, and phosphatidylinositol 3-kinase (PI3K), significantly reduced NIS mRNA expression and iodide uptake in tRA-stimulated MCF-7 cells but not FRTL-5 cells. An inhibitor of p38 MAPK significantly reduced iodide uptake in both tRA-stimulated MCF-7 cells and TSH-stimulated FRTL-5 cells. IGF-I and PI3K inhibitors did not significantly reduce the basal NIS mRNA expression in MCF-7 cells. Despite the chronic inhibitory effects on cell proliferation, tRA did not reduce the S-phase distribution of MCF-7 cells during the period of NIS induction. CONCLUSION: The IGF-I receptor/PI3K pathway mediates tRA-stimulated NIS expression in MCF-7 but not FRTL-5 thyroid cells.     

Immune complexes formed following the binding of anti-platelet factor 4 (CXCL4) antibodies to CXCL4 stimulate human neutrophil activation and cell adhesion

We tested the possibility that immune complexes formed following platelet factor 4 (PF4/CXCL4) binding to anti-PF4 antibody can stimulate neutrophil activation, similar to previous reports with platelets. Monoclonal Abs against PF4 and IgG from a heparin-induced thrombocytopenia (HIT) patient were applied. We observed that although PF4 or anti-PF4 antibody alone did not alter neutrophil function, costimulation with both reagents resulted in approximately 3-fold increase in cell surface Mac-1 expression, enhanced cell adhesion via L-selectin and CD18 integrins, and degranulation of secondary and tertiary granules. The level of Mac-1 up-regulation peaked at an intermediate PF4 dose, suggesting that functional response varies with antigen-antibody stoichiometry. PF4 binding to neutrophils was blocked by chondroitinase ABC. Cell activation was inhibited by both chondroitinase ABC and anti-CD32/FcgammaRII blocking mAb, IV.3. Confocal microscopy demonstrated that immune complexes colocalize with CD32a. Studies with HIT IgG demonstrated that neutrophils could be activated in the absence of exogenous heparin. These data, together, show that leukocyte surface chondroitin sulfates promote neutrophil activation by enhancing immune-complex binding to CD32a. Studies with recombinant PF4 suggest a role for arginine 49 in stabilizing PF4-chondroitin binding. Neutrophils activated via this mechanism may contribute to thrombosis and inflammation in patients mounting an immune response to PF4-heparin.     

Rapid activation of G2/M checkpoint after hypertonic stress in renal inner medullary epithelial (IME) cells is protective and requires p38 kinase

Cells in the kidney medulla are subject to variable and often extreme osmotic stress during concentration of the urine. Previous studies showed that renal inner medullary epithelial (IME) cells respond to hypertonicity by G(2) arrest. The purpose of the present study was to investigate the mechanisms involved in initiation and maintenance of G(2) arrest. Rapid initiation of G(2) arrest after UV radiation is mediated by p38 kinase. Here we find that p38 kinase is responsible for rapid initiation of the G(2) delay in IME cells after the hypertonic stress created by adding NaCl. High NaCl, but not high urea, rapidly initiates G(2) arrest. Inhibition of p38 kinase by SB202190 (10 microM) blocks the rapid initiation of this checkpoint both in an immortalized cell line (mIMCD3) and in second-passage IME cells from mouse renal inner medulla. p38 inhibition does not affect exit from G(2) arrest. The rapid initiation of G(2) arrest is followed by inhibition of cdc2 kinase, which is also prevented by SB202190. To assess the possible protective role of G(2) arrest, we measured DNA strand breaks as reflected by immunostaining against phospho-histone H2AX, which becomes phosphorylated on Ser-139 associated with DNA breaks. Abrogation of rapid G(2)/M checkpoint activation by SB202190 increases the histone H2AX phosphorylation in G(2)/M cells. We propose that the rapid initiation of G(2) delay by p38 kinase after hypertonicity protects the cells by decreasing the level of DNA breaks caused by aberrant mitosis entry.     

T-cell antigen CD28 interacts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-Met-Xaa-Met motif.

The T-cell antigen CD28 provides a costimulatory signal that is required for T-cell proliferation. T-cell receptor zeta/CD3 engagement without CD28 ligation leads to a state of nonresponsiveness/anergy, thereby implicating CD28 in the control of peripheral tolerance to foreign antigens or tumors. A key unresolved question has concerned the mechanism by which CD28 generates intracellular signals. Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase with Src-homology 2 (SH2) domain(s) that binds to the platelet-derived growth factor receptor (PDGF-R), an interaction that is essential for signaling by growth factor. In this study, we demonstrate that CD28 binds to PI 3-kinase by means of a Y(P)MXM motif within its cytoplasmic tail. CD28-associated PI 3-kinase was detected by lipid kinase and HPLC analysis as well as by reconstitution experiments with baculoviral-expressed p85 subunit of PI 3-kinase. CD28 bound directly to the p85 subunit without the need for the associated p110 subunit. Site-directed mutagenesis and peptide competition analysis using Y(P)-MXM-containing peptides showed that PI 3-kinase bound to a Y(P)MXM motif within the CD28 cytoplasmic tail (residues 191-194). Mutation of the Y191 within the motif resulted in a complete loss of binding, while mutation of M194 caused partial loss of binding. Binding analysis showed that the CD28 Y(P)-MXM motif bound to the p85 C- and N-terminal SH2 domains with an affinity comparable to that observed for PDGF-R and insulin receptor substrate 1. In terms of signaling, CD28 ligation induced a dramatic increase in the recruitment and association of PI 3-kinase with the receptor. CD28 is likely to use PI 3-kinase as the second signal leading to T-cell proliferation, an event with implications for anergy and peripheral T-cell tolerance.     

TGF-beta3 regulates the blood-testis barrier dynamics via the p38 mitogen activated protein (MAP) kinase pathway: an in vivo study

Recent studies using Sertoli cells cultured in vitro to permit tight junction (TJ) assembly have shown that TJ dynamics are regulated, at least in part, by TGF-beta3 via the p38 mitogen activated protein (MAP) kinase pathway. This in turn regulates the production of occludin, a TJ-integral membrane protein, by Sertoli cells. Yet it is not known if this pathways is used by Sertoli cells to regulate the blood-testis barrier (BTB) function in vivo. Using an in vivo model for studying BTB dynamics, we report herein the CdCl(2)-induced BTB damage in rats was associated with a significant reduction in testicular occludin along with a loss of immunoreactive occludin in the seminiferous epithelium at the site of the BTB. Also, this CdCl(2)-induced occludin loss from the BTB coincided with a surge in testicular TGF-beta3, as well as p-p38 MAP kinase (the phosphorylated/activated form of p38), but not p38 MAP kinase and neither extracellular signal-regulated kinase nor its phosphorylated form (ERK/p-ERK), consistent with results of in vitro studies. More important, intratesticular administration of SB202190, a specific p38 MAP kinase inhibitor, could block the CdCl(2)-induced occludin loss from the BTB. These results illustrate that BTB dynamics in vivo are regulated by the TGF-beta3/p38 MAP kinase pathway, which in turn determines the level of occludin at the site of Sertoli cells TJs.