Kinase-dependent Mechanism Research Articles

T-cell antigen receptor-mediated enhancement of the adenylate cyclase pathway depends on tyrosine protein kinases

We have examined the human T-cell line Jurkat the interaction between the activation through the T-cell receptor/CD3 complex and the adenylate cyclase pathway. OKT3, an anti-CD3 monoclonal antibody, did not activate by itself adenylate cyclase but produced a 3–7-fold increase of the cAMP accumulation induced by indirect (chloroadenosine, PGE 2) or direct (forskolin) agonists of adenylate cyclase. A more detailed study with forskolin showed that OKT3 enhanced the effect of low concentrations of the agonist without afffecting the maximal capacity of cAMP sysnthesis of the cells. The same concentrations of OKT3 produced both the enhancement of the adenylate cyclase pathway and the activation of phospholipase C. The enhancement of OKT3 of the adenylate cyclase pathway was inhibited by 0.5 μM staurosporine, a potent inhibitor of protein kinases, including tyrosine kinases and protein kinases C, whereas it was not inhibited by H7, a specific inhibitor of PKC. Staurosporine, at the same concentration, also inhibited the OKT3-induced activation of phospholipase C, a tyrosine kinase-dependent process. Taken together, these data that activation of T-cell through the T-cell receptor enhances the adenylate cyclase pathway by a tyrosine protein kinase-dependent mechanism.

Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism.

Recent biochemical studies have suggested that apoptotic cell death is the molecular mechanism underlying the degeneration of ovarian follicles during atresia. Using a sensitive autoradiographic method for the detection of DNA fragmentation, we studied apoptosis in ovarian granulosa cells or intact follicles placed in serum-free culture as model systems to elucidate the hormonal regulation of atresia. Immature rats (25 days old) were primed for 2 days with 10 IU equine CG to induce a homogeneous population of mature preovulatory follicles. Granulosa cells isolated from these follicles contained predominantly intact high mol wt DNA. However, a time-dependent, spontaneous onset of internucleosomal DNA fragmentation characteristic of apoptotic cell death occurred in granulosa cells during culture. Treatment of granulosa cells with epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), or basic fibroblast growth factor (bFGF) inhibited the spontaneous onset of apoptotic DNA cleavage found during culture by 40-60%. In contrast, insulin-like growth factor I, insulin, TGF beta and tumor necrosis factor-alpha were ineffective. Likewise, activation of the protein kinase A or C pathways with forskolin or phorbol 12-myristate 13-acetate, respectively, did not prevent the onset of DNA fragmentation, although inclusion of a tyrosine kinase inhibitor (genistein) completely blocked the ability of EGF, TGF alpha, and bFGF to suppress apoptosis in granulosa cells. Similar to cultured granulosa cells, a spontaneous onset of apoptosis was also observed to occur in isolated preovulatory follicles during culture. Furthermore, treatment of follicles with EGF or bFGF inhibited the spontaneous initiation of apoptosis, and the suppressive effects of these growth factors were also attenuated by co-treatment with genistein.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism

Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism

Functional expression of the macrophage colony-stimulating factor receptor in human THP-1 monocytic leukemia cells

Macrophage colony-stimulating factor (M-CSF) is required for the proliferation, differentiation, and activation of monocytes. High- affinity receptors for M-CSF are encoded by the c-fms proto-oncogene. In the present study, we show that c-fms transcripts are detectable in human THP-1 myeloid leukemia cells. Furthermore, radiolabeled 125I-M- CSF is rapidly internalized into THP-1 cells and then degraded intracellularly. The results also show that treatment of THP-1 cells with M-CSF is associated with the activation of protein kinase C (PKC) and the induction of tumor necrosis factor (TNF) gene expression. TNF transcript levels were low to undetectable in uninduced THP-1 cells, reached maximal levels by 1 hour of exposure to M-CSF, and returned to those of control cells by 24 hours. Transcriptional run-on analysis showed that a low level of TNF transcription is detectable in untreated THP-1 cells, and M-CSF treatment increased the rate of TNF transcription. Pretreatment of THP-1 cells with pertussis toxin inhibited the increase in PKC activity but not the induction of TNF transcripts by M-CSF. Moreover, exposure of THP-1 cells to inhibitors of protein kinase activity blocked the increase in TNF messenger RNA. These findings suggest that at least two M-CSF-mediated signaling pathways exist in THP-1 cells and that the induction of TNF may be regulated by a protein kinase-dependent mechanism distinct from PKC.

Functional Expression of the Macrophage Colony-Stimulating Factor Receptor in Human THP-1 Monocytic Leukemia Cells

Macrophage colony-stimulating factor (M-CSF) is required for the proliferation, differentiation, and activation of monocytes. High-affinity receptors for M-CSF are encoded by the c-fms proto-oncogene. In the present study, we show that c-fms transcripts are detectable in human THP-1 myeloid leukemia cells. Furthermore, radiolabeled 125I-M-CSF is rapidly internalized into THP-1 cells and then degraded intracellularly. The results also show that treatment of THP-1 cells with M-CSF is associated with the activation of protein kinase C (PKC) and the induction of tumor necrosis factor (TNF) gene expression. TNF transcript levels were low to undetectable in uninduced THP-1 cells, reached maximal levels by 1 hour of exposure to M-CSF, and returned to those of control cells by 24 hours. Transcriptional run-on analysis showed that a low level of TNF transcription is detectable in untreated THP-1 cells, and M-CSF treatment increased the rate of TNF transcription. Pretreatment of THP-1 cells with pertussis toxin inhibited the increase in PKC activity but not the induction of TNF transcripts by M-CSF. Moreover, exposure of THP-1 cells to inhibitors of protein kinase activity blocked the increase in TNF messenger RNA. These findings suggest that at least two M-CSF-mediated signaling pathways exist in THP-1 cells and that the induction of TNF may be regulated by a protein kinase-dependent mechanism distinct from PKC.

A Novel Mechanism for the Induction of Aromatase in Ovarian Cells in Vitro: Role of Transforming Growth Factor Alpha-induced Protein Tyrosine Kinase

In the developing follicle, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are the primary stimulators of steroidogenesis in granulosa and theca cells. The steroidogenic actions of both these gonadotropins are largely if not exclusively mediated through cAMP. Previous studies have shown that EGF and TGF alpha do not affect basal estrogen production but attenuate FSH-stimulated estrogen production in vitro in rat granulosa cells. Here we present evidence that TGF alpha stimulates basal estrogen production in prepubertal porcine ovarian granulosa and theca cells in culture under defined conditions. In granulosa cells, TGF alpha is more potent than FSH in stimulating estrogen production. LH does not stimulate estrogen production in prepubertal porcine theca cells but rather attenuates that stimulated by TGF alpha. Treatment of follicular cells with genistein (inhibitor of protein tyrosine kinase) attenuates TGF alpha-induced estrogen biosynthesis suggesting that the action of TGF alpha is mediated through protein tyrosine kinase. These studies provide evidence for an alternative cAMP-independent and TGF alpha-induced tyrosine kinase-dependent mechanism for the induction of ovarian aromatase.

Arachidonic acid mobilization and phosphoinositide turnover by the terminal complement complex, C5b-9, in rat oligodendrocyte x C6 glioma cell hybrids.

Previously, we have shown that rat oligodendrocytes release phospholipid and generate arachidonic acid (AA) and leukotriene B4 in response to sublytic C5b-9 formation. In the present study, we investigated the biochemical pathways by which C5b-9 generates AA from clone ROC-1, a fusion product of rat oligodendrocytes and C6 glioma. Cells were incubated for 24 h in the presence of [3H]AA or [3H]myoinositol. They were then sensitized with antibody against hybrid cell stroma and treated for 1 h with C9-depleted human serum (C9D-HS) or C9D-HS reconstituted with C9. Alternatively, cells were treated with C8,C9D-HS or C8,C9D-HS reconstituted with C8 or C8 plus C9 for 1 h. Qualitative and quantitative analysis of the released [3H]AA and [3H]myoinositol radiolabeled products were performed by thin layer chromatography/autoradiography and anion exchange chromatography, respectively. The major [3H]AA radiolabeled products after C5b-9 stimulation comigrated with intact phospholipid and AA standards, and the major [3H]myoinositol radiolabeled product was inositol-1-phosphate. Treatment of cells with phospholipase A2 inhibitors, mepacrine and bromophenacyl bromide, abolished AA release by C5b-9. In the absence of extracellular Ca2+, C5b-9 also failed to induce the release of AA. Interestingly, 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinases, inhibited AA release by C5b-9, whereas AA release stimulated by the calcium ionophore A23187 was not blocked by H-7. The results suggest that AA generation by C5b-9 from the ROC-1 clone involves activation of Ca2+-dependent phospholipase A2 which is regulated by protein kinase-dependent mechanisms.

Regulation of Phospholipase D in HL-60 Granulocytes: Activation by Phorbol Esters, Diglyceride, and Calcium Ionophore Via Protein Kinase C-independent Mechanisms

It has recently been demonstrated that the chemotactic peptide N-formyl-Met-Leu-Phe activates phospholipase D (PLD) in dimethyl sulfoxide-differentiated HL-60 granulocytes to produce phosphatidic acid (PA) and, in the presence of ethanol, phosphatidylethanol (PEt) (Pai, J.-K., Siegel, M. I., Egan, R. W., and Billah, M. M. (1988) J. Biol. Chem. 263, 12472-12477). We now report that biologically active phorbol esters, a cell-permeable diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), and calcium ionophore A23187 are also potent inducers of PLD in these HL-60 granulocytes. HL-60 granulocytes have been selectively labeled in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P by incubating the cells with alkyl-[32P]lyso-phosphatidylcholine (PC). When these labeled cells are treated with phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate, OAG, or A23187, alkyl-[32P]PA is formed. Because cellular ATP has not been labeled with 32P, the formation of alkyl-[32P]PA conclusively demonstrates PLD activation by these agents. In the presence of 0.5% ethanol, phorbol esters, OAG, and A23187 also induce formation of alkyl-[32P]PEt, demonstrating that the activated PLD catalyzes transphosphatidylation between the phosphatidyl moiety of the alkyl-[32P]PC and ethanol. Formation of alkyl-[32P]PA and alkyl-[32P]PEt in response to these various agents occurs in a time- and dose-dependent manner and exhibits differential Ca2+ requirements. Based on experiments with both [3H]alkyl-PC and alkyl-[32P]PC, it is concluded that alkyl-PA and alkyl-PEt formed in response to PMA, OAG, or A23187 are derived exclusively from PLD action on alkyl-PC. Furthermore, subthreshold concentrations of PMA (0.5-2.0 nM) or OAG (1.0-25 microM) combined with subthreshold levels of A23187 (15-60 nM) induce the formation of alkyl-[32P]PA and alkyl-[32P]PEt, suggesting that receptor-mediated activation of PLD might involve cooperative interactions between Ca2+ and diglyceride. Although PLD is activated by agents that also activate protein kinase C, the protein kinase C inhibitor, K252a, inhibits PMA-induced protein phosphorylation but causes only partial inhibition of PLD activation. We conclude that phorbol esters, OAG, and A23187 activate PLD in HL-60 granulocytes via protein kinase-independent as well as protein kinase-dependent mechanisms.

Cuprammonium membranes stimulate interleukin 1 release and arachidonic acid metabolism in monocytes in the absence of complement

Contact of blood with foreign surfaces, specifically dialysis membranes, causes cell activation which has widely been assumed to be mediated by complement (C). To explore the possibility of C-independent activation, we examined different cell types: PMN, human monocytes and the cell lines U937 or HL60, washed human platelets and rat glomerular epithelial cell (primary) cultures (GEC), under serum-free conditions and after addition of anti-C3 F(ab)2, respectively. The monitored biological effects were release of PGE2, TXB2 or interleukin 1 and generation of O2- radicals. To further explore the mechanisms involved, phospholipid metabolism was studied by measuring IP3 and DG (14C-arachidonic or oleic acid prelabeled U937 and HL60 cells); changes of cytosolic Ca++ (Quin2 technique) were also determined. The results show that in absence of C, brief (2 min) contact with cuprammonium (CU) stimulated: (a) PGE2 release in U937 and human monocytes or GEC; (b) TXB2 release in washed platelets; (c) slow interleukin 1 release by monocytes; and (d) generation of O2- radicals in PMN. Artifacts due to endotoxin were excluded by appropriate polymyxin control experiments and by comparison of effects with those of bacterial LPS. Potential synthesis of C3 by U937 was excluded by addition of anti-C3 F(ab)2. C-independent cell activation was accompanied by increase of DG, but not IP3 (suggesting involvement of protein C kinase dependent mechanisms) and by increased cytosolic Ca++. To further explore the initial signal involved, incubations were carried out with covalently modified CU members (DEAE cellulose) and in the presence of mM concentrations of monosaccharides. Cationic modification of CU membranes reduced C-independent cell activation.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of steroids on sexual imprinting in male zebra finches

Tumour necrosis factor-alpha (TNFα) is up-regulated by cross-linking of major histocompatibility complex (MHC) class II [human leucocyte antigen (HLA)-DR] antigens on monocytes. This is done by a bacterial superantigen or anti-HLA-DR monoclonal antibody (MAb). We have previously shown that HLA-DR cross-linking results in inhibition of haematopoiesis and apoptosis. TNFα acts as a negative regulator of haematopoiesis. Here we investigated whether HLA-DR-mediated inhibition of haematopoiesis involved TNFα and TNFα-dependent secondary signals. Anti-HLA-DR MAb H81.9 up-regulated TNFα, as well as transforming growth factor β, interleukin (IL)-1β and IL-6 in human marrow cells at the ribonucleic acid (RNA) and protein level. The effect on TNFα was investigated further. Up-regulation was blocked by herbimycin A, consistent with a tyrosine kinase-dependent mechanism. Up-regulation was also blunted by the soluble TNF-receptor fusion protein TNFR:Fc, suggesting an autocrine amplification loop. Following TNFα up-regulation, there was increased expression of Fas (CD95) and Fas-ligand (Fas-L). Up-regulation of Fas and Fas-L was blocked by TNFR:Fc. Furthermore, MAb H81.9-induced apoptosis was prevented by anti-TNFα MAb and by the soluble Fas receptor, Fas-Ig, providing further evidence that the TNF effect was mediated via Fas. At the transcriptional level, cross-linking of HLA-DR by MAb H81.9 affects nuclear localization of NKκB, which is involved in the transcription of TNFα. NFκB activity is modified by changes in cellular redox potential, and we have shown that H81.9 affects redox potential as determined by the generation of nitric oxide. These data show that HLA-DR-initiated signals are able to trigger a cascade of negative regulators of haematopoiesis. This model provides an opportunity to dissect signalling pathways that may be involved in the development of spontaneous marrow failure, and to devise interventions aimed at protecting haematopoiesis.