Degranulation in RBL-2H3 cells: regulation by calmodulin pathway

Involvement of the calmodulin pathway in Ca2+-induced degranulation was evaluated in RBL-2H3 mast cells. Pretreatment of RBL-2H3 cells with a calmodulin antagonist, W-13, blocked ionomycin-dependent release of beta-hexosaminidase into the supernatant, although W-13 treatment alone slightly but significantly increased the release. Ca2+/calmodulin activates various protein kinases and phosphatases including myosin-light chain kinase (MLCK), calmodulin-dependent protein kinases (CaMKs), and calcineurin. When RBL-2H3 cells were pretreated with a MLCK inhibitor, ML-7, or a CaMKs inhibitor, KN-93, the ionomycin-dependent release of beta-hexosaminidase into the supernatant was inhibited. In addition, pretreatment with calcineurin inhibitors, cyclosporin A and FR901725, resulted in blockage of the ionomycin-dependent release of beta-hexosaminidase into the supernatant. Our results indicate that Ca2+/calmodulin, activated calmodulin, is indispensable for Ca2+-induced degranulation, and that within the calmodulin pathways, at least MLCK, CaMKs and calcineurin positively regulate the release of granules initiated by increasing cytosolic Ca2+ concentrations in RBL-2H3 cells.     

Elevated levels of cyclooxygenase-2 in antigen-stimulated mast cells is associated with minimal activation of p38 mitogen-activated protein kinase

We have investigated possible factors that underlie changes in the production of eicosanoids after prolonged exposure of mast cells to Ag. Ag stimulation of cultured RBL-2H3 mast cells resulted in increased expression of cyclooxygenase (COX-2) protein and message. Other eicosanoid-related enzymes, namely COX-1, 5-lipoxygenase, and cytosolic phospholipase A(2) were not induced. Activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein (MAP) kinase preceded the induction of COX-2, whereas phosphatidylinositol 3' kinase and its substrate, Akt, were constitutively activated in RBL-2H3 cells. Studies with pharmacologic inhibitors indicated that of these kinases, only p38 MAP kinase regulated expression of COX-2. The induction of COX-2 was blocked by the p38 MAP kinase inhibitor SB202190, even when added 12-16 h after stimulation with Ag when p38 MAP kinase activity had returned to near basal, but still minimally elevated, levels. Interestingly, expression of COX-2 as well as cytosolic phospholipase A(2) and 5-lipoxygenase were markedly reduced by SB202190 in unstimulated cells. Collectively, the results imply that p38 MAP kinase regulates expression of eicosanoid-related enzymes, passively or actively, at very low levels of activity in RBL-2H3 cells. Also, comparison with published data suggest that different MAP kinases regulate induction of COX-2 in inflammatory cells of different and even similar phenotype and suggest caution in extrapolating results from one type of cell to another.     

Tyrosine phosphorylation of Vav stimulates IL-6 production in mast cells by a Rac/c-Jun N-terminal kinase-dependent pathway.

This study investigates whether the guanine nucleotide exchange activity of Vav is linked to cytokine production in mast cells. Overexpression of Vav in the RBL-2H3 mast cell line resulted in the constitutive tyrosine phosphorylation and activation of Vav. We analyzed the functional effect of Vav overexpression on cytokine production. IL-2 and IL-6 mRNA levels were dramatically increased in Vav-overexpressing cells and correlated with increased NF-AT activity. Little or no effect was observed on the mRNA levels of IL-3, IL-4, GM-CSF, TNF-alpha, and TGF-beta. FcepsilonRI engagement did not further enhance IL-2 and IL-6 mRNA levels and only slightly enhanced NF-AT activity, but dramatically increased the mRNA levels of other tested cytokines. To understand the signal transduction required, we focused primarily on IL-6 induction by measuring mitogen-activated protein kinase activity and analyzing the effects of mutant or dominant negative forms of Vav, Rac1, and c-Jun N-terminal kinase-1 (JNK1). Vav overexpression resulted in the constitutive activation of JNK1 with little or no effect on p38 mitogen-activated protein kinase and ERK2. This was dependent on Vav-mediated activation of Rac1 as a Dbl domain-mutated Vav, inactive Rac N17, and inactive JNK1 down-regulated the Vav-induced JNK1 or IL-6 responses. Vav expression, but not expression of domain-mutated Vav, increased IL-6 secretion from nonimmortalized bone marrow-derived mast cells upon FcepsilonRI engagement. We conclude that Vav phosphorylation contributes to IL-6 induction in mast cells.     

Complete inhibition of anisomycin and UV radiation but not cytokine induced JNK and p38 activation by an aryl-substituted dihydropyrrolopyrazole quinoline and mixed lineage kinase 7 small interfering RNA

Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC50 of 70 nM for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nM or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1beta or tumor necrosis factor-alpha-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stress-activated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.     

Regulation of human beta-defensin-2 in gingival epithelial cells: the involvement of mitogen-activated protein kinase pathways, but not the NF-kappaB transcription factor family.

Stratified epithelia of the oral cavity are continually exposed to bacterial challenge that is initially resisted by neutrophils and epithelial factors, including antimicrobial peptides of the beta-defensin family. Previous work has shown that multiple signaling pathways are involved in human beta-defensin (hBD)-2 mRNA regulation in human gingival epithelial cells stimulated with a periodontal bacterium, Fusobacterium nucleatum, and other stimulants. The goal of this study was to further characterize these pathways. The role of NF-kappaB in hBD-2 regulation was investigated initially due to its importance in inflammation and infection. Nuclear translocation of p65 and NF-kappaB activation was seen in human gingival epithelial cells stimulated with F. nucleatum cell wall extract, indicating possible involvement of NF-kappaB in hBD-2 regulation. However, hBD-2 induction by F. nucleatum was not blocked by pretreatment with two NF-kappaB inhibitors, pyrrolidine dithiocarbamate and the proteasome inhibitor, MG132. To investigate alternative modes of hBD-2 regulation, we explored involvement of mitogen-activated protein kinase pathways. F. nucleatum activated p38 and c-Jun NH(2)-terminal kinase (JNK) pathways, whereas it had little effect on p44/42. Furthermore, inhibition of p38 and JNK partially blocked hBD-2 mRNA induction by F. nucleatum, and the combination of two inhibitors completely blocked expression. Our results suggest that NF-kappaB is neither essential nor sufficient for hBD-2 induction, and that hBD-2 regulation by F. nucleatum is via p38 and JNK, while phorbol ester induces hBD-2 via the p44/42 extracellular signal-regulated kinase pathway. Studies of hBD-2 regulation provide insight into how its expression may be enhanced to control infection locally within the mucosa and thereby reduce microbial invasion into the underlying tissue.     

In situ analysis of the changes in expression of ovarian inhibin subunit mRNAs during follicle recruitment after ovulation in pigs

In situ hybridization was used on frozen tissue sections with digoxigenin-labelled antisense riboprobes to inhibin/activin alpha and beta(A) subunits to determine whether inhibin/activin subunit mRNA expression was associated with development of growing, steroidogenically active follicles during follicle recruitment after ovulation. Cell proliferation-associated nuclear antigen Ki-67 protein and cytochrome P450 aromatase expression in granulosa cells were determined immunohistochemically and used as markers for granulosa cell proliferation and steroidogenesis, respectively, on days 3, 5 and 7 after the onset of oestrus. The amounts of inhibin/activin alpha and beta(A) subunit mRNA and P450 aromatase protein were greater (102, 93, and 238%, respectively; P < 0.05) in medium than in small non-atretic follicles and were positively correlated with Ki-67 and with each other. Inhibin/activin alpha and beta(A) mRNA, P450 aromatase, and Ki-67 in granulosa cells were reduced by 66-83% (P < 0.001) in atretic follicles compared with non-atretic follicles. In addition, inhibin/activin alpha and beta(A) mRNA and P450 aromatase in small (1-2 mm) non-atretic follicles decreased (P < 0.05) between day 3 and day 7 independently of morphological or biochemical signs of atresia. The pattern of inhibin/activin subunit mRNA expression supports the notion that activin and inhibin have roles in growth and steroidogenesis in follicle recruitment during the early luteal phase of the oestrous cycle.     

An activin/furin regulatory loop modulates the processing and secretion of inhibin alpha- and betaB-subunit dimers in pituitary gonadotrope cells

Of all ligands of the transforming growth factor beta superfamily, inhibins and activins are a physiologically relevant pair that are functional antagonists of each other. Activin stimulates whereas inhibin blocks follicle-stimulating hormone biosynthesis and secretion from pituitary gonadotrope cells, and together, inhibin and activin control the pituitary gonadal axis essential for normal reproductive function. Sharing a similar beta-subunit, the secretion of inhibin heterodimers (alpha/beta) or activin homodimers (beta/beta) as mature bioactive ligands depends, in part, on the proteolytic processing of precursor proteins. A short loop regulatory pathway controlling precursor processing and dimer secretion was discovered. Activin stimulates endogenous inhibin alpha- and betaB-subunit mRNA, protein, and proteolytic processing. Simultaneously, activin stimulated the proconvertase furin through a Smad2/3-dependent process. The data provide a mechanism where the regulation of furin and inhibin subunits cooperates in an important positive short feedback loop. This regulatory loop augments the secretion of bioactive mature activin B, as well as inhibin B dimers, necessary for local follicle-stimulating hormone beta regulation.     

Impaired FcepsilonRI-dependent gene expression and defective eicosanoid and cytokine production as a consequence of Fyn deficiency in mast cells

Fyn kinase is a key contributor in coupling FcepsilonRI to mast cell degranulation. A limited macroarray analysis of FcepsilonRI-induced gene expression suggested potential defects in lipid metabolism, eicosanoid and glutathione metabolism, and cytokine production. Biochemical analysis of these responses revealed that Fyn-deficient mast cells failed to secrete the inflammatory eicosanoid products leukotrienes B4 and C4, the cytokines IL-6 and TNF, and chemokines CCL2 (MCP-1) and CCL4 (MIP-1beta). FcepsilonRI-induced generation of arachidonic acid and normal induction of cytokine mRNA were defective. Defects in JNK and p38 MAPK activation were observed, whereas ERK1/2 and cytosolic phospholipase A2 (S505) phosphorylation was normal. Pharmacological studies revealed that JNK activity was associated with generation of arachidonic acid. FcepsilonRI-mediated activation of IkappaB kinase beta and IkappaBalpha phosphorylation and degradation was defective resulting in a marked decrease of the nuclear NF-kappaB DNA binding activity that drives IL-6 and TNF production in mast cells. However, not all cytokine were affected, as IL-13 production and secretion was enhanced. These studies reveal a major positive role for Fyn kinase in multiple mast cell inflammatory responses and demonstrate a selective negative regulatory role for certain cytokines.     

Activin stimulation of inhibin secretion and messenger RNA levels in cultured granulosa cells

Recent reports suggest that activin (the dimer of inhibin beta subunits with FSH-releasing activity) has specific receptors on ovarian granulosa cells. The present study examined the effects of purified porcine activin on inhibin secretion and mRNA levels in granulosa cells obtained from immature, estrogen-treated rats. Cells were cultured for 48 h in culture media, or media containing FSH (10 ng/ml) and/or activin (30 ng/ml). Western blot analyses performed with affinity-purified antisera to inhibin alpha- and beta A-subunits revealed that treatment with either FSH or activin increased the secretion of inhibin alpha beta dimer (Mr 30,000), with a further increase after cotreatment. These results were confirmed by an inhibin alpha-subunit RIA, which revealed 7-, 14-, and 71-fold increases in the secretion of immunoreactive inhibin-alpha by activin, FSH, and activin plus FSH, respectively. TGF beta, a structural homolog of activin, also stimulated inhibin release, whereas follistatin was ineffective. Total RNA from cultured cells was hybridized with 32P-labeled inhibin alpha-subunit cRNA or beta-actin cDNA probes, and inhibin-alpha message levels were normalized with beta-actin mRNA levels. Northern blot analysis revealed that treatment with FSH and activin increased hybridization of a 1.5 kilobase (kb) message, corresponding to the inhibin alpha-subunit mRNA. Slot blot analyses indicated a 6- and 8-fold stimulation of inhibin alpha-subunit mRNA levels by FSH and activin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization and secretion of inhibins in the equine fetal ovaries

To clarify the source of inhibins in equine female fetuses, concentrations of immunoreactive (ir-) inhibin, inhibin pro-alphaC, and inhibin A in both fetal and maternal circulation and in fetal ovaries were measured. In addition, the localization of inhibin alpha and inhibin/activin beta(A), and beta(B) subunits and the expression of inhibin alpha(A) and inhibin/activin beta(A) subunit mRNA in fetal ovaries were investigated using immunohistochemistry and in situ hybridization. Concentrations of circulating ir-inhibin, inhibin pro-alphaC, and inhibin A were remarkably more elevated in the fetal than in the maternal circulation between Days 100 and 250 of gestation. Fetal ovaries contained large amounts of ir-inhibin, inhibin pro-alphaC, and inhibin A. In contrast, these inhibin forms were undetectable in both the maternal ovaries and placenta. The inhibin alpha and inhibin/activin beta(A) and beta(B) subunit proteins were localized to enlarged interstitial cells of the equine fetal ovary. Expression of inhibin alpha and inhibin/activin beta(A) subunit mRNAs were also observed in the interstitial cells. We conclude that the main source of large amounts of inhibins in fetal circulation is interstitial cells of fetal ovary and is not of maternal origin. Furthermore, these inhibins may play some important physiological roles in the development of gonads in the equine fetus.     

Ligand-independent activation of the androgen receptor by insulin-like growth factor-i and the role of the MAPK pathway in skeletal muscle cells

In this study, the roles of the p38 MAPK, ERK1/2 and JNK signaling pathway in IGF-I-induced AR induction and activation were examined. C2C12 cells were treated with IGF-I in the absence or presence of various inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), and JNK (SP600125). Inhibition of the MAPK pathway with SB203580, PD98059, or SP600125 significantly decreased IGF-I-induced AR phosphorylation and total AR protein expression. IGF-I-induced nuclear fraction of total AR and phosphorylated AR were significantly inhibited by SB203580, PD98059, or SP600125. Furthermore, IGF-I-induced AR mRNA and skeletal α-actin mRNA were blocked by those inhibitors in dose-dependent manner. Confocal images showed that IGF-I-induced AR nuclear translocation from cytosol was significantly blocked by SB203580, PD98059, or SP600125, suggesting that the MAPK pathway regulates IGF-I-induced AR nuclear localization in skeletal muscle cells. The present results suggest that the MAPK pathways are required for the ligand-independent activation of AR by IGF-I in C2C12 skeletal muscle cells.     

N-linked oligosaccharides direct the differential assembly and secretion of inhibin alpha- and betaA-subunit dimers

The biosynthetic pathway governing inhibin heterodimer (alpha/beta) and activin homodimer (beta/beta) assembly and secretion from ovarian granulosa cells is not fully understood. Here, we examined the role of inhibin subunit glycosylation in the assembly and secretion of mature inhibin A and activin A. Inhibition of subunit glycosylation by tunicamycin treatment of alpha- and beta(A)-expressing CHO cell lines reduced inhibin but not activin secretion. Dimeric inhibin A is preferentially secreted from parental isogenic wild-type (wt) cell lines (alpha(wt)beta(wt)). Mutation of a single glycosylation site at asparagine 268 (alpha(Delta268)beta(wt)) reduces inhibin secretion by 78% and permits beta/beta assembly and secretion. Conversely, gain of a glycosylation (GOG) site in the analogous region of the beta(A)-subunit (alpha(wt)beta(GOG327)) enhances inhibin A secretion. The present study demonstrates that N-linked glycan sites direct heterodimer vs. homodimer assembly, and prevention of glycosylation abrogates inhibin secretion. These data support a definitive role for site-specific N-glycosylation in governing inhibin/activin dimer assembly and secretion.     

Positive regulation of c-Jun N-terminal kinase and TNF-alpha production but not histamine release by SHP-1 in RBL-2H3 mast cells

The SH2-containing protein tyrosine phosphatase1 (SHP-1) is important for signaling from immune receptors. To investigate the role of SHP-1 in mast cells we overexpressed the wild-type and the phosphatase-inactive forms of SHP-1 in rat basophilic leukemia 2H3 (RBL-2H3) mast cell line. The phosphatase-inactive SHP-1 (C453S or D419A) retains its ability to bind tyrosine phosphorylated substrates and thereby competes with the endogenous wild-type enzyme. Overexpression of wild-type SHP-1 decreased the FcepsilonRI aggregation-induced tyrosine phosphorylation of the beta and gamma subunits of the receptor whereas the dominant negative SHP-1 enhanced phosphorylation. There were also similar changes in the tyrosine phosphorylation of Syk. However, receptor-induced histamine release in the cells expressing either wild-type or dominant negative SHP-1 was similar to that in the parental control cells. In contrast, compared with the parental RBL-2H3 cells, FcepsilonRI-induced c-Jun N-terminal kinase phosphorylation and the level of TNF-alpha mRNA was increased in the cells overexpressing wild-type SHP-1 whereas the dominant negative SHP-1 had the opposite effect. The substrate-trapping mutant SHP1/D419A identified pp25 and pp30 as two major potential substrates of SHP-1 in RBL-2H3 cells. Therefore, SHP-1 may play a role in allergy and inflammation by regulating mast cell cytokine production.