Protein kinase C mediates induced secretion of vascular endothelial growth factor by human glioma cells

To understand how vascular endothelial growth factor (VEGF) production is activated in malignant glioma cells, we employed protein tyrosine kinase (PTK) and protein kinase C (PKC) inhibitors to evaluate the extent to which these protein kinases were involved in signal transduction leading to VEGF production. PTK inhibitors blocked glioma proliferation and epidermal growth factor (EGF)-induced VEGF secretion, while H-7, a PKC inhibitor, inhibited both EGF-induced and baseline VEGF secretion. Phorbol 12-myristate 13-acetate (PMA), a non-specific activator of PKC, induced VEGF secretion by glioma cells, which was enhanced by calcium ionophore A23187, but completely blocked after prolonged treatment of cells with 1 microM PMA, by presumably depleting PKC. All inhibitors (genistein, AG18, AG213, H-7, prolonged PMA treatment) which inhibited EGF-induced VEGF secretion in glioma cells also inhibited cell proliferation at similar concentrations. However, PKC inhibition only blocked 50% of the VEGF secretion induced by growth factors (EGF, platelet-derived growth factor-BB, or basic fibroblast growth factor). This reserve capacity could be ascribed to a PKC-independent effect, or to PKC isoenzymes not down-regulated by PMA. These findings extend our previous assertion that VEGF secretion is tightly coupled with proliferation by suggesting that activation of convergent growth factor signaling pathways will lead to increased glioma VEGF secretion. Understanding of signal transduction of growth factor-induced VEGF secretion should provide a rational basis for the development of novel strategies for therapy.     

Increases in intracellular calcium dephosphorylate histone H3 at serine 10 in human hepatoma cells: potential role of protein phosphatase 2A-protein kinase CbetaII complex

We present evidence that increases in intracellular calcium, induced by treatment with calcium ionophore A23187 or the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, dephosphorylated histone H3 at serine10 (histone H3-Ser10) in a dose-dependent manner in human hepatoma HepG2 cells. Inhibition of p42/44MAPK, pp90RSK, or p38MAPK did not affect the ability of A23187 to dephosphorylate histone H3-Ser10. This response is significantly blocked by okadaic acid, indicating a requirement for protein phosphatase 2A (PP2A). A23187 increased the activity of PP2A towards phosphorylated histone H3-Ser10. Furthermore, pretreatment with calphostin C, a selective protein kinase C (PKC) inhibitor, blocked A23187-dependent dephosphorylation of histone H3-Ser10, and coimmunoprecipitation analysis showed PP2A association with the PKCbetaII isoform. Unlike untreated cells, coimmunoprecipitated complex from A23187-treated cells showed greater dephosphorylation of histone H3-Ser10 in a PP2A-dependent manner. Inhibition of PP2A increased phosphorylation at Ser660 that determines calcium sensitivity and activity of PKCbetaII isoform, thus supporting a role for intracomplex regulation. Finally, chromatin immunoprecipitation assays following exposure to A23187 and okadaic acid revealed regulatory role of histone H3-Ser10 phosphorylation in selective gene induction. Altogether, our findings suggest a novel role for calcium in modulating histone H3-Ser10 phosphorylation level and led us to propose a model emphasizing PP2A activation, occurring downstream following perturbations in calcium homeostasis, as key event in dephosphorylating histone H3-Ser10 in mammalian cells.     

VEGF selectively induces Down syndrome critical region 1 gene expression in endothelial cells: a mechanism for feedback regulation of angiogenesis?

The Down syndrome critical region 1 (DSCR1) gene (also known as MCIP1, Adapt78) encodes a regulatory protein that binds to calcineurin catalytic A subunit and acts as a regulator of the calcineurin-mediated signaling pathway. We show in this study that DSCR1 is greatly induced in endothelial cells in response to VEGF, TNF-alpha, and A23187 treatment, and that this up-regulation is inhibited by inhibitors of the calcineurin-NFAT (nuclear factor of activated T cells) signaling pathway as well as by PKC inhibition and a Ca(2+) chelator. We hypothesized that the up-regulation of DSCR1 gene expression in endothelial cells could act as an endogenous feedback inhibitor for angiogenesis by regulating the calcineurin-NFAT signaling pathway. Our transient transfection analyses confirm that the overexpression of DSCR1 abrogates the up-regulation of reporter gene expression driven by both the cyclooxygenase 2 and DSCR1 promoters in response to stimulators. Our results indicate that DSCR1 up-regulation may represent a potential molecular mechanism underlying the regulation of angiogenic genes activated by the calcineurin-NFAT signaling pathway in endothelial cells.     

Ergosterol elicits oxidative burst in tobacco cells via phospholipase A2 and protein kinase C signal pathway.

Ergosterol, a typical fungal sterol, induced in tobacco (Nicotiana tabacum L. cv. Xanthi) suspension cells the synthesis of reactive oxygen species and alkalization of the external medium that are dependent on the mobilization of calcium from internal stores. We used specific inhibitors to elucidate the signal pathway triggered by ergosterol compared with cryptogein, a proteinaceous elicitor of Phytophthora cryptogea. Herbimycin A and genistein, inhibitors of tyrosine protein kinases, had no effect on the oxidative burst and pH changes induced by both elicitors. Similarly, H-89, an inhibitor of protein kinase A, had no effect on the induction of these defense reactions. However, the response to both elicitors was completely blocked by NPC-15437, a specific inhibitor of animal protein kinase C (PKC). The responses induced by cryptogein but not those induced by ergosterol were inhibited by U73122 and neomycin, inhibitors of phospholipase C (PLC). On the other hand, the activity of phospholipase A2 (PLA2) measured using a fluorogenic substrate was stimulated by ergosterol and not by cholesterol and cryptogein. A specific inhibitor of PLA2, arachidonic acid trifluoromethyl ketone (AACOCF3), inhibited the pathway stimulated by ergosterol but not that induced by cryptogein. These results suggest that the cryptogein-induced signal pathway leading to the oxidative burst and DeltapH changes includes PLC and PKC, whereas this response induced by ergosterol includes PLA2 and PKC.     

Mechanism of isoproterenol-induced RGS2 up-regulation in astrocytes

Regulators of G protein signaling (RGSs) are inducibly expressed in response to various stimuli and the up-regulation of RGSs leads to significant decreases in GPCR responsiveness. Isoproterenol, an adrenergic receptor agonist, stimulated RGS2 mRNA in C6 rat astrocytoma cells. The up-regulation of RGS2 mRNA was abrogated by genistein, a protein tyrosine kinase inhibitor (PTK), and by broad-spectrum protein kinase C (PKC) inhibitors (staurosporine and GF109203X). alpha-Adrenergic antagonist (prazocin), beta-adrenergic antagonist (prazocin), and pertussis toxin only partially blocked the RGS2 up-regulation, suggesting that the RGS2 up-regulation is concomitantly mediated by Galphai, Galphas, and Galphaq. It is interesting to note that SB203580, a potent p38 mitogen-activated protein kinase (MAPK) inhibitor, completely inhibited the isoproterenol-mediated RGS2 expression. In addition, isoproterenol also markedly stimulated RGS2 mRNA in rat primary astrocytes, which were sensitive to SB203580 and staurosporine. Therefore, our data suggest that adrenergic receptor-mediated signaling (induced by isoproterenol) may be involved in the regulation of RGS2 expression in astrocytes via activating PTK, PKC, and p38 MAPK.     

Tyrosine kinase regulates phospholipase D activation at a point downstream from protein kinase C in osteoblast-like cells

It has recently been shown that the activation of protein kinase C (PKC) induces protein tyrosine phosphorylation in osteoblast-like MC3T3-E1 cells. We previously reported that the activation of PKC stimulates phosphatidylcholine-hydrolyzing phospholipase D in these cells. In this study, we examined whether protein tyrosine kinase is involved in the PKC-induced activation of phospholipase D in MC3T3-E1 cells. Genistein, an inhibitor of protein tyrosine kinases, which by itself had little effect on choline formation, significantly suppressed the formation of choline induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC, in a dose-dependent manner. Tyrphostin, an inhibitor of protein tyrosine kinases chemically distinct from genistein, also dose-dependently suppressed the TPA-induced formation of choline. Sodium orthovandate, an inhibitor of protein tyrosine phosphatases, significantly enhanced the TPA-induced formation of choline in a dose-dependent manner. These results strongly suggest that protein tyrosine kinase regulates phospholipase D activity at a point downstream from PKC in osteoblast-like cells.     

Herpes simplex virus-1 up-regulates IL-15 gene expression in monocytic cells through the activation of protein tyrosine kinase and PKC zeta/lambda signaling pathways

IL-15 plays a seminal role in innate immunity through enhancing the cytotoxic function as well as cytokine production by NK and T cells. We have previously shown that exposure of PBMC as well as monocytic cells to different viruses results in immediate up-regulation of IL-15 gene expression and subsequent NK cell activation as an innate immune response of those cells to these viruses. However, no signaling pathway involved in this up-regulation has been identified. Here we show for the first time that HSV-1-induced up-regulation of IL-15 gene expression is independent of viral infectivity/replication. IL-15 gene is up-regulated by HSV-1 in human monocytes, but not in CD3+ T cells. HSV-1 induces the phosphorylation of protein tyrosine kinases (PTKs) and protein kinase C (PKC) for inducing IL-15 expression in monocytic cells. Inhibitors for PTKs reduced HSV-1-induced PTK activity, DNA binding activity of NF-kB as well as IL-15 gene expression. In contrast, an inhibitor for membrane-bound tyrosine kinases had no effect on these events. Experiments using PKC inhibitors revealed that phosphorylation of PKC zeta/lambda (PKC zeta/lambda), DNA binding activity of NF-kB and HSV-1-induced up-regulation of IL-15 were all decreased. Furthermore, we found that HSV-1-induced IL-15 up-regulation was also dependent on PTKs regulation of PKC phosphorylation. Thus, we conclude that IL-15 up-regulation in HSV-1-treated monocytic cells is dependent on the activity of both PTKs and PKC zeta/lambda.     

Sustained norepinephrine stimulation induces different regulation of expression in three alpha1-adrenoceptor subtypes.

The norepinephrine (NE)-induced regulation of alpha1-adrenoceptors (ARs) expression in human embryonic kidney (HEK) 293 cells stably expressing cloned alpha1-AR subtypes with similar receptor densities was investigated. In the presence of 10 microM propranolol, the treatment of cells with 10 microM NE for 4-72 h down-regulated alpha1A- and alpha1D-AR. but increased alpha1B-AR expression in a time-dependent manner. The down-regulation of alpha1A-AR reached maximum of 40.3 +/- 14.7 % at 48h. The down-regulation of alpha1D-AR reached maximum of 51.3 +/- 3.7% at 24h. With the stimulation of NE, alpha1B-AR density was increased maximally by 112.4 +/- 43.4% at 48h. The protein kinase C (PKC) inhibitor calphostin C or R0-31-8220 abolished the NE-induced down-regulation of alpha1A- and alpha1D-AR, but showed no effect on the up-regulation of alpha1B-AR. The PKC agonist PMA not only mimicked the NE-induced down-regulation of alpha1A- and alpha1D-AR, but also induced a down-regulation of alpha1B-AR. The endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) or thapsigargin, or the calcium chelator BAPTA/AM did not affect the down-regulation of alpha1A-AR, but inhibited the up-regulation of alpha1B-AR induced by NE. Calmodulin antagonist W-7. tyrosine kinase inhibitor genistein or tyrphostin A25 had no effect on NE-induced up-regulation of alpha1B-AR. The results suggest that three alpha1-AR subtypes are differently regulated by sustained NE stimulation with different signal transduction pathways.     

Phorbol and calcium decreased atriopeptin response in a human renal cell line.

We investigated regulation of atrial natriuretic factor (ANF)-stimulated cellular cGMP accumulation (ANF-s-cGMP) in an ANF-responsive human renal cell line, SK-NEP-1. Dose-response data indicated that the EC50 for ANF(99-126) was 1.1 x 10(-9) M. Brain natriuretic peptide (10(-6) M) increased cGMP to a level indistinguishable from that of ANF (10(-6) M). [Met-(O)]ANF was only half as potent as ANF, and atriopeptin I (10(-6) M) did not increase cGMP over basal levels. Preincubation of SK-NEP-1 cells with ANF, but not atriopeptin I (API), for two hours or longer, caused a concentration-dependent down-regulation of ANF-s-cGMP. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, and A23187 and its 4-bromo derivative, calcium ionophores, inhibited ANF-s-cGMP in a dose-dependent manner. A23187 inhibition was calcium dependent and promoted net cGMP degradation. Thirty-six hour preincubation with PMA, a procedure used to down-regulate PKC, abolished acute PMA inhibition of ANF-s-cGMP without having an effect on ANF-s-cGMP or on 4-bromo-A23187 inhibition thereof. These data indicate that PKC activation specifically inhibited ANF-s-cGMP but that PKC was not required for ANF-s-cGMP in SK-NEP-1 cells. Thus structurally related ANF peptides, protein kinase C (PKC) activators, calcium ionophores are potential modulators of ANF-s-cGMP in cells from this human renal cell line.     

Angiotensin type 1 receptor is linked to inhibition of nitric oxide production in pulmonary endothelial cells

We previously demonstrated that angiotensin II (Ang II) stimulates an increase in nitric oxide synthase (NOS) mRNA levels, eNOS protein expression and NO production via the type 2 (AT2) receptor, whereas signaling via the type 1 (AT1) receptor negatively regulates NO production in bovine pulmonary artery endothelial cells (BPAECs). In the present study, we investigated the components of the AT1 receptor-linked signaling pathway(s) that are involved in the downregulation of eNOS protein expression in BPAECs. Treatment of BPAECs with either AT1 receptor antagonists or an anti-AT1 receptor antibody induced eNOS protein expression. Furthermore, intracellular delivery of GP-Antagonist-2A, an inhibitor of Galphaq proteins, and treatment of BPAECs with U73122, a phosphatidylinositol-phospholipase C (PLC)-specific inhibitor, enhanced eNOS protein expression. Treatment of BPAECs with the cell-permeable calcium chelator, BAPTA/AM, increased eNOS protein expression at 8 h, while increasing intracellular calcium with either thapsigargin or A23187 prevented Ang II-induced eNOS protein expression. Phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, completely prevented Ang II-stimulated eNOS protein expression at 8 h, whereas depletion of PKC by long-term treatment with PMA, induced eNOS protein expression. Treatment of BPAECs with a PKCalpha-specific inhibitor or transfection of BPAECs with an anti-PKCalpha neutralizing antibody stimulated eNOS protein expression. Conversely, rottlerin, a PKCdelta specific isoform inhibitor had no effect on basal or Ang II-stimulated eNOS protein expression. Moreover, treatment of BPAECs with U73122, BAPTA/AM and PKCalpha-specific inhibitors increased NO production at 8 h. In conclusion, Ang II downregulates eNOS protein expression via an AT1 receptor-linked pathway involving Galphaq/PLC/calcium/PKCalpha signaling pathway in BPAECs.     

Involvement of protein kinase C in translocation of desmoplakins from cytosol to plasma membrane during desmosome formation in human squamous cell carcinoma cells grown in low to normal calcium concentration

The intracellular signal transduction mechanism leading to desmosome formation in low-calcium-grown keratinocytes after addition of calcium to the medium was studied by immunofluorescence using antibodies to desmoplakins I and II (cytoplasmic desmosomal proteins) and by electron microscopy before and after addition of calcium; protein kinase C (PKC) activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBu), and 1,2-dioctanoylglycerol (DOG); calcium ionophore A23187; selective PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine; and a Ca2+/calmodulin-dependent kinase inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). In previous studies using a low-calcium-grown human epidermal squamous cell carcinoma, we have shown that an increase in extracellular Ca2+ caused a four-fold increase in PKC activity and addition of TPA (10 ng/ml) induced a transient increase in membrane-bound PKC activity in association with cell-cell contact formation. The present study showed that TPA (10 ng/ml). PDBu (10 ng/ml), and DOG (1 mg/ml) induced a rapid cell-cell contact and redistribution of desmoplakins from cytoplasm to the plasma membrane with desmosome formation within 60-120 min, which was similar, although less marked, to the effect of increased Ca2+. The TPA-induced desmosome formation was inhibited by selective PKC inhibitors, H-7 (20 microM) or staurosporine (100 nM). On the other hand, calcium ionophore A23187 induced only a temporary increase in the number of desmoplakin-containing fluorescent spots in the cytoplasm and a temporary cell-cell attachment without desmosome formation. The calcium-induced desmosome formation was partially inhibited by 20-100 microM H-7 or 100 nM staurosporine; however, it was not inhibited by W-7 at a concentration of 25 microM, at which this agent selectively inhibits calmodulin-dependent protein kinase. These results suggest that PKC activation plays an important role in desmoplakin translocation from the cytoplasm to the plasma membrane as one of the processes of calcium-induced desmosome formation.     

Ouabain-induced enhancement of rat mast cells response. Modulation by protein phosphorylation and intracellular pH

The digitalic glicoside ouabain induces potentiation of rat mast cell histamine release in response to several stimuli, which is mediated by Na+/Ca2+ exchanger. In this work, we studied the effect of ouabain on cytosolic calcium, intracellular pH and histamine release with Ca2+ ionophore A23187 in conditions designed to maximize ouabain-induced potentiation of rat mast cells response. The effect of protein kinase C (PKC), cAMP and phosphatase inhibition was also tested. Ouabain induced an enhancement in histamine release, cytosolic calcium and intracellular pH. The adenylate cyclase activator forskolin reduced the effect of ouabain on histamine release and intracellular pH, but enhanced the effect on cytosolic calcium. PKC activator PMA enhanced the effect of ouabain on histamine release and cytosolic calcium, without affecting intracellular pH. A PKC inhibitor, GF-109203X, reduced ouabain-induced enhancement of histamine release and intracellular pH, but increased the enhancement on cytosolic calcium. Finally, inhibition of protein phosphatases 1 and 2A with okadaic acid, increased the effect of ouabain on histamine release and intracellular pH, but reduced cytosolic calcium in presence of ouabain. This result suggest that ouabain-induced potentiation of rat mast cell histamine release with A23187 is modulated by kinases, and this modulation may be carried out by changes in intracellular alkalinization. However, the mechanism underlying cellular alkalinization remains to be elucidated.     

Regulation of CD93 cell surface expression by protein kinase C isoenzymes

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.     

Multifactorial regulation of prostaglandin synthesis in preovulatory goldfish ovarian follicles.

Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (PG) production. The protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 micrograms/ml), and 1,2-dioctanoylglycerol (10 and 50 micrograms/ml) stimulated PGE production; the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23187 (0.25-4.0 microM) stimulated PGE production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23187. The direct activator of phospholipase A2, melittin (0.1-1.0 microM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 microM), a putative inhibitor of phospholipase A2, blocked basal and PMA + A23187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), forskolin (10 microM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23187-stimulated PGE production. Melittin-stimulated production of PGE was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian follicles.     

Protein kinase C dependent and independent activation of phospholipase A2 under calcium ionophore (A23187) exposure in rabbit pulmonary arterial smooth muscle cells

Exposure of rabbit pulmonary arterial smooth muscle cells to the calcium ionophore A23187, dose-dependently stimulates arachidonic acid (AA) release and phospholipase A2 (PLA2) activity. The protein kinase C (PKC) inhibitor, sphingosine does not prevents AA release and PLA2 activity caused by low doses of A23187. In contrast, sphingosine markedly prevents AA release and PLA2 activity caused by higher doses of A23187. PKC activity profile indicates that treatment of the cells with low doses of A23187 does not cause significant alteration of PKC translocation from cytosol to membrane whereas higher concentrations of the ionophore dose-dependently enhance PKC translocation from cytosol to membrane in the smooth muscle cells.     

Role of protein kinase activation in the induction of B cell adhesion by MHC class II ligands.

Engagement of MHC class II (Ia) molecules on B cells induces tyrosine phosphorylation, phosphoinositide turnover, elevation of intracellular calcium concentrations, and a rise in cAMP levels. However, a role for these biochemical signals in mediating functional responses induced by Ia ligands remains largely undefined. In this study, we utilized the induction of B cell adhesion by Ia ligands to demonstrate a role for signals transduced via Ia molecules in the generation of a functional response. Ia ligands that induced B cell aggregation induced tyrosine phosphorylation, whereas Ia ligands that did not induce B cell aggregation failed to induce any detectable tyrosine phosphorylation. Ia-induced B cell aggregation and tyrosine phosphorylation were inhibited by genistein and by herbimycin A, inhibitors of tyrosine kinases (PTK). Sphingosine and calphostin C, inhibitors of protein kinase C (PKC), also inhibited Ia-induced adhesion whereas HA1004, an inhibitor of cyclic nucleotide-dependent kinases, did not. Ia ligands induced both LFA-1-dependent and LFA-1-independent B cell adhesion. These two pathways of cell adhesion differed in their requirement for activation signals. PKC activation was sufficient for LFA-1-dependent adhesion, whereas LFA-1-independent adhesion required independent phosphorylation events mediated by PKC and by PTK. These results provide functional relevance for biochemical signals transduced via Ia molecules by demonstrating that Ia-induced B cell adhesion is mediated by the activation of PKC and by one or more PTK.     

Activation of a suicide process of thymocytes through DNA fragmentation by calcium ionophores and phorbol esters

Calcium ionophore, A23187, is known to be a comitogen, but it activates a suicide process characterized by DNA fragmentation at linker regions in mouse immature thymocytes. It did not induce DNA fragmentation in T lymphocytes prepared from lymph node and spleen cells. Induction of DNA fragmentation by A23187 depends on protein phosphorylation and synthesis of mRNA and protein, because an inhibitor of protein kinase, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7), actinomycin D, and cycloheximide, respectively, inhibits the DNA fragmentation and cell death. Studies adding the inhibitors at various times show that protein phosphorylation and mRNA synthesis occur within a few hours after incubation with A23187 followed by the protein synthesis responsible for inducing DNA fragmentation. Phorbol esters, 12-O-tetradecanoyl 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBD), which are capable of activating protein kinase C, also induced similar DNA fragmentation in immature thymocytes, followed by cell death. PBD committed the suicide process after 6 h of incubation, because the DNA fragmentation above the control level was not induced when PDB was removed from the medium before 6 h of incubation. A23187 or a phorbol ester alone induced DNA fragmentation followed by cell death, whereas the addition of TPA at low concentration inhibited the DNA fragmentation induced by A23187 accompanied with an increase in DNA synthesis. The result suggests that TPA switched a suicide process induced by A23187 to an opposite process: stimulation of DNA synthesis. Physiologic factors and mechanisms which regulate cell proliferation and death in the thymus are not known at present, but the signals by protein kinases and calcium ions may regulate both cell proliferation and death, independently, synergistically or antagonistically.