Phorbol myristate acetate inhibits okadaic acid-induced apoptosis and downregulation of X-linked inhibitor of apoptosis in U937 cells

Okadaic acid is a specific inhibitor of serine/threonine protein phosphatase 1 (PP-1) and 2A (PP-2A). The phosphorylation and dephosphorylation at the serine/threonine residues on proteins play important roles in regulating gene expression, cell cycle progression, and apoptosis. In this study, phosphatase inhibitor okadaic acid induces apoptosis in U937 cells via a mechanism that appears to involve caspase 3 activation, but not modulation of Bcl-2, Bax, and Bcl-X(L) expression levels. Treatment with 20 or 40 nM okadaic acid for 24 h produced DNA fragmentation in U937 cells. This was associated with caspase 3 activation and PLC-gamma1 degradation. Okadaic acid-induced caspase 3 activation and PLC-gamma1 degradation and apoptosis were dose-dependent with a maximal effect at a concentration of 40 nM. Moreover, PMA (phorbol myristate acetate), PKC (protein kinase C) activator, protected U937 cells from okadaic acid-induced apoptosis, abrogated okadaic acid-induced caspase 3 activation, and specifically inhibited downregulation of XIAP (X-linked inhibitor of apoptosis) by okadaic acid. PMA cotreated U937 cells exhibited less cytochrome c release and sustained expression levels of the IAP (inhibitor of apoptosis) proteins during okadaic acid-induced apoptosis. In addition, these findings indicate that PMA inhibits okadaic acid-induced apoptosis by a mechanism that interferes with cytochrome c release and activity of caspase 3 that is involved in the execution of apoptosis.     

Involvement of p21 in the PKC-induced regulation of the G2/M cell cycle transition

Activation of protein kinase C (PKC) inhibits cell cycle progression at the G1/S and G2/M transitions. We found that phorbol 12-myristate 13-acetate (PMA) induced upregulation of p21, not only in MCF-7 cells arrested in the G1 phase as previously shown, but also in cells delayed in the G2 phase. This increase in p21 in cells accumulated in the G1 and G2/M phases of the cell cycle after PMA treatment was inhibited by the PKC inhibitor GF109203X. This indicates that PKC activity is required for PMA-induced p21 upregulation and cell cycle arrest in the G1 and G2/M phases of the cell cycle. To further assess the role of p21 in the PKC-induced G2/M cell cycle arrest independently of its G1 arrest, we used aphidicolin-synchronised MCF-7 cells. Our results show that, in parallel with the inhibition of cdc2 activity, PMA addition enhanced the associations between p21 and either cyclin B or cdc2. Furthermore, we found that after PMA treatment p21 was able to associate with the active Tyr-15 dephosphorylated form of cdc2, but this complex was devoid of kinase activity indicating that p21 may play a role in inhibition of cdc2 induced by PMA. Taken together, these observations provide evidence that p21 is involved in integrating the PKC signaling pathway to the cell cycle machinery at the G2/M cell cycle checkpoint.     

Phorbol ester-induced G1 phase arrest selectively mediated by protein kinase Cdelta-dependent induction of p21

Although protein kinase C (PKC) has been widely implicated in the positive and negative control of proliferation, the underlying cell cycle mechanisms regulated by individual PKC isozymes are only partially understood. In this report, we show that PKCdelta mediates phorbol ester-induced G1 arrest in lung adenocarcinoma cells and establish an essential role for this novel PKC in controlling the expression of the cell cycle inhibitor p21. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) in early G1 phase impaired progression of lung adenocarcinoma cells into S phase, an effect that was completely abolished by specific depletion of PKCdelta, but not PKCalpha. Although the PKC effect was unrelated to the inhibition of cyclin D1 expression, PKC activation significantly up-regulated p21 and down-regulated Rb hyperphosphorylation and cyclin A expression. Elevations in p21 mRNA and protein by PMA were mediated by PKCdelta but not PKCalpha. Studies using luciferase reporters also revealed an essential role for PKCdelta in the PMA-induced inhibition of Rb-dependent cyclin A promoter activity. Finally, we showed that the cell cycle inhibitory effect of PKCdelta is greatly attenuated by RNA interference-mediated knock-down of p21. Our results identify a novel link between PKCdelta and G1 arrest via p21 up-regulation and highlight the complexities in the downstream effectors of PKC isozymes in the context of cell cycle progression and proliferation.     

Glycated human serum albumin enhances macrophage inflammatory protein-1beta mRNA expression through protein kinase C-delta and NADPH oxidase in macrophage-like differentiated U937 cells

BACKGROUND: In a previous report (Higai K et al., Biol Pharm Bull, 2007), glycated human serum albumin (Glc-HSA) was found to induce interleukin-8 (IL-8) mRNA expression in human monocyte-derived U937 cells through a reactive oxygen species (ROS)-dependent pathway; however, Glc-HSA signaling has not been elucidated in macrophages. METHODS: U937 cells were differentiated by treatment with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) for 2 days and the macrophage-like differentiated U937 (differentiated U937) cells were stimulated with Glc-HSA and glycolaldehyde dimer-modified HSA (GA-HSA) in the presence of various signaling inhibitors. Macrophage inflammatory protein-1beta (MIP-1beta) mRNA expression was determined by real-time PCR. Intracellular ROS generation was estimated by confocal laser microscopy. RESULTS: Glc-HSA and GA-HSA markedly enhanced MIP-1beta mRNA expression in differentiated U937 cells. Enhanced MIP-1beta mRNA expression was completely suppressed by the ROS scavenger N-acetyl-l-cysteine, the NADPH oxidase inhibitors diphenylene iodonium and apocynin, and the protein kinase C (PKC)-delta inhibitor rottlerin. Furthermore, ROS generation was suppressed completely by rottlerin but not by the PKC-gamma inhibitor Ro318425 or the PKC-alpha, -beta1 and -micro inhibitor Go6976. CONCLUSION: Glc-HSA and GA-HSA enhance MIP-1beta mRNA expression in differentiated U937 cells through PKC-delta-dependent activation of NADPH oxidase.     

Trp-Lys-Tyr-Met-Val-Met activates mitogen-activated protein kinase via a PI-3 kinase-mediated pathway independent of PKC

Trp-Lys-Tyr-Met-Val-Met (WKYMVM) is a novel potent peptide which can stimulate phosphoinositide hydrolysis in U937 as well as U266 and HL-60 cells (Baek et al., J. Biol. Chem. 271, 8170 (1996)). The peptide also induces superoxide generation in human neutrophils (Seo et al., J. Immunol. 158, 1896 (1997)). However, the signaling pathway down-stream of PLC set in motion by the peptide is not yet completely understood. We studied the signaling pathway of the peptide with the goal of elucidating the mechanism of the peptide's action. WKYMVM induced a rapid and transient activation of the ERKs in human histiocytic lymphoma cells, U937. The ERK1 activation peaked at 5 min and returned to the basal level after 30 min. The ERK1 stimulation by the peptide was partially inhibited by pretreatment of the cells with pertussis toxin (PTX), implicating G-protein involvement in the peptide's action. Pretreatment of staurosporine, protein kinase C (PKC) inhibitor, or PKC down-regulating PMA had no impact on the ERK1 activation by the peptide, indicating that the signaling pathway is independent of PKC activation. Pretreatment of the cells with neomycin and intracellular Ca2+ mobilizing reagents had also no effect on the ERK1 activation by the peptide. However, pretreatment with wortmannin or LY294002, the inhibitor of phosphatidylinositol 3 kinase (PI-3K), strongly inhibited peptide-stimulated ERK1 activation. Our results suggest that PI-3K may be an important participant in the ERK cascade induced by the peptide. Furthermore, the treatment of U937 cells with the peptide activated p74Raf-1, an upstream kinase of ERK. Taken together, our results suggest that the peptide activate ERK via a G-protein/PI-3K/Ras/Raf-1 mediated signaling pathway in U937 cells.     

UCN-01 (7-hydroxystauorsporine) blocks PMA-induced maturation and reciprocally promotes apoptosis in human myelomonocytic leukemia cells (U937)

Interactions between the protein kinase inhibitor UCN-01 and the PKC activator phorbol ester (PMA) have been examined in relation to differentiation and apoptosis in human myelomonocytic leukemia cells (U937). Coadministratation of 100 nM UCN-01 with a low concentration of PMA e.g., 2 nM, inhibited rather than promoted differentiation, reflected by reduced surface expression of the monocytic maturation marker CD11b and diminished cell adherence. Instead, administration of UCN-01 with PMA led to a marked increase in mitochondrial injury (e.g, cytochrome c release), activation of caspases-3 and -8, Bid cleavage, PARP degradation, and apoptosis, accompanied by a substantial reduction in viability and clonogenic survival. These phenomena were associated with multiple perturbations in cell cycle regulatory events, including abrogation of p21(CIP1) induction, p27(KIP1) cleavage, down-regulation of cyclin D1, dephosphorylation (activation) of p34cdc2, and degradation of underphosphorylated pRb. Potentiation of PMA-mediated apoptosis was partially mimicked by caffeine suggesting the involvement of Chk1 in the potentiation of apoptosis. Induction of cell death by UCN-01 and PMA was increased in cells stably expressing a p21(CIP1) mRNA antisense construct, suggesting that p21(CIP1) expression may protect cells from the lethal effects of this drug combination. Finally, ectopic expression of a Bcl-2 but not dominant-negative caspase-8 protected cells from UCN-01/PMA-mediated apoptosis, suggesting the lethal effects of this combination primarily involves the mitochondrial rather than the TNF-related extrinsic apoptotic pathway. Taken together, these findings suggest that UCN-01 disrupts a variety of cell cycle events in leukemic cells exposed to the maturation-inducing agent PMA, causing cells to engage an apoptotic rather than a differentiation-related program.     

Phorbol ester inhibits DNA damage-induced apoptosis in U937 cells through activation of protein kinase C

The effects of phorbol 12-myristate 13-acetate (PMA) on DNA damage-induced apoptosis were examined in promyelocytic leukemia cells, U937, in comparison with other differentiation-inducing agents to clarify the role of protein kinase C (PKC) vis-a-vis cellular differentiation in apoptosis. The apoptosis of U937 cells was observed at as early as 1-1.5 h following UV irradiation, with most cells being in apoptotic state at 3 h. Pretreatment with PMA for as short as 5 min was sufficient to inhibit apoptosis induced by UV irradiation, whereas apparent changes in cell cycle distributions and expression of differentiation markers by PMA were not observed until 12 h and 48 h, respectively. The inhibition of apoptosis by PMA was completely abolished by the pretreatment with calphostin C, a PKC inhibitor, and 4 alpha-phorbor 12,13-didecanoate, which is unable to activate PKC, did not protect U937 cells against apoptosis induced by UV irradiation. Other differentiation inducers, such as cyclic AMP and active vitamin D3, did not affect the UV-induced apoptosis of U937 cells. Taken together, it was suggested that PMA inhibits DNA damage-induced apoptosis through the activation of PKC rather than as a result of differentiation of U937 cells.     

TIS21/BTG2/PC3 is expressed through PKC-delta pathway and inhibits binding of cyclin B1-Cdc2 and its activity, independent of p53 expression

Signal transduction pathway and a new function of TIS21/BTG2/PC3 were investigated in p53 null U937 cells; Expression of TIS21 by 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulation was mediated by PKC-delta activation, however, was strongly inhibited by cPKC isozymes. When U937 cells were treated with TPA+Go6976, but not TPA+Go6850, the level of TIS21 mRNA was maintained over that of TPA alone. When analyzed by FACS, TPA-induced G2/M arrest was significantly inhibited by Go6850, but not by Go6976, suggesting the involvement of TIS21 and nPKC isozymes. Indeed, PKC-delta was found to be a regulator of the G2/M arrest and TIS21 expression, confirmed by employing rottlerin and dnPKC-delta experiments. In vivo accumulation of TIS21 protein significantly induced cell death through caspase 3 activation, which was supported further by degradations of procaspase 3, full-length PKC-delta, pRB, and p21(WAF1) in TIS21DeltaC expresser. When the cells were synchronized by nocodazole, TIS21 overexpressers inhibited degradations of cyclin A and cyclin B1 in 3 h after release from the synchronization. Furthermore, TIS21 inhibited cyclin B1-Cdc2 binding and its kinase activity in vivo. In summary, TPA-induced TIS21 mRNA expression is mediated by PKC-delta, and TIS21 induces G2/M arrest and cell death by inhibiting cyclin B1-Cdc2 binding and the kinase activity through its binding to Cdc2.     

UCN-01 (7-Hydroxystauorsporine) Blocks PMA-Induced Maturation and Reciprocally Promotes Apoptosis in Human Myelomonocytic Leukemia Cells (U937)

Interactions between the protein kinase inhibitor UCN-01 and the PKC activator phorbol ester (PMA) have been examined in relation to differentiation and apoptosis in human myelomonocytic leukemia cells (U937). Coadministratation of 100 nM UCN-01 with a low concentration of PMA e.g., 2 nM, inhibited rather than promoted differentiation, reflected by reduced surface expression of the monocytic maturation marker CD11b and diminished cell adherence. Instead, administration of UCN-01 with PMA led to a marked increase in mitochondrial injury (e.g, cytochrome c release), activation of caspases-3 and -8, Bid cleavage, PARP degradation, and apoptosis, accompanied by a substantial reduction in viability and clonogenic survival. These phenomena were associated with multiple perturbations in cell cycle regulatory events, including abrogation of p21CIP1 induction, p27KIP1 cleavage, down-regulation of cyclin D1, dephosphorylation (activation) of p34cdc2, and degradation of underphosphorylated pRb. Potentiation of PMA-mediated apoptosis was partially mimicked by caffeine suggesting the involvement of Chk1 in the potentiation of apoptosis. Induction of cell death by UCN-01 and PMA was increased in cells stably expressing a p21CIP1 mRNA antisense construct, suggesting that p21CIP1 expression may protect cells from the lethal effects of this drug combination. Finally, ectopic expression of a Bcl-2 but not dominant-negative caspase-8 protected cells from UCN-01/PMA-mediated apoptosis, suggesting the lethal effects of this combination primarily involves the mitochondrial rather than the TNF-related extrinsic apoptotic pathway. Taken together, these findings suggest that UCN-01 disrupts a variety of cell cycle events in leukemic cells exposed to the maturation-inducing agent PMA, causing cells to engage an apoptotic rather than a differentiation-related program.

Key Words:

PMA, UCN-01, Differentiation, Apoptosis     

Regulation of platelet-derived growth factor A and B chain gene expression in bone marrow stromal cells

MBA-2, bone marrow-derived endothelial stromal cells, express platelet-derived growth factor (PDGF) A and B chain mRNAs and secrete PDGF activity that is induced by TGF-beta. Either chain of the PDGF molecule could modulate hematopoiesis and stromal cell growth. Intracellular pathways that regulate PDGF expression in the marrow microenvironment are unknown. In the present study, we examined the mechanisms that mediate PDGF A and B chain mRNA induction by TGF-beta and the role of protein kinase C (PKC) and cyclic AMP in PDGF regulation. TGF-beta was tested in parallel with PMA, an activator of phorbol ester-dependent PKC isoforms. Both PMA (10^?7M) and TGF-beta (2.5 ng/ml) increased PDGF A and B chain mRNA levels. The serine/threonine protein kinase inhibitor, H7, blocked PDGF A and B chain mRNA induction in response to TGF-beta. However, down-regulation of PKC by prolonged incubation with PMA failed to abolish TGF-beta induction of PDGF A and B chain mRNAs. These findings indicate that induction of PDGF A and B chain mRNAs can be mediated via phorbol ester-dependent PKC pathway. In contrast, H7-sensitive protein kinase(s) other than phorbol ester-sensitive protein kinase C mediate the effect of TGF-beta. Agents that increase cAMP were also tested for their effect on PDGF gene expression. TGF-beta-mediated induction of PDGF A and B chain mRNAs was markedly inhibited by cAMP. cAMP also blocked stimulation of PDGF A chain mRNA by PMA. The positive and negative signaling mechanisms involved in modulating PDGF in the microenvironment may be important for determining hematopoietic and stromal cell responses in vivo. © 1995 Wiley-Liss, Inc.     

Cooperation between PKC-alpha and PKC-epsilon in the regulation of JNK activation in human lung cancer cells

Phorbol esters can induce activation of two mitogen-activated protein kinase (MAPK) pathways, the extracellular signal-regulated kinase (ERK) pathway and the c-Jun N-terminal kinase (JNK) pathway. Unlike ERK activation, JNK activation by phorbol esters is somehow cell-specific. However, the mechanism(s) that contribute to the cell-specific JNK activation remain elusive. In this study, we found that phorbol 12-myristate 13-acetate (PMA) induced JNK activation only in non-small cell lung cancer (NSCLC) cells, but not in small cell lung cancer (SCLC) cells, whereas ERK activation was detected in both cell types. In NSCLC cells, PMA induced JNK activation in a time- and dose-dependent manner. JNK activation was attenuated by protein kinase C (PKC) down-regulation through prolonged pre-treatment with PMA and significantly inhibited by PKC inhibitors G?6976 and GF109203X. Subcellular localization studies demonstrated that PMA induced translocation of PKC-alpha, -betaII, and -epsilon isoforms, but not PKC-delta, from the cytosol to the membrane. Analysis of various PKC isoforms revealed that PKC-epsilon was exclusively absent in the SCLC cell lines tested. Ectopic expression of PKC-epsilon in SCLC cells restored PMA activation of JNK signaling only in the presence of PKC-alpha, suggesting that PKC-alpha and PKC-epsilon act cooperatively in regulating JNK activation in response to PMA. Furthermore, using dominant negative mutants and pharmacological inhibitors, we define that a putative Rac1/Cdc42/PKC-alpha pathway is convergent with the PKC-epsilon/MEK1/2 pathway in terms of the activation of JNK by PMA.     

Association of TGF-beta signaling in angiotensin II-induced PAI-1 mRNA upregulation in mesangial cells: role of PKC

This study aimed to identify the intracellular signaling pathway in angiotensin II (Ang II)-induced upregulation of plasminogen activator inhibitor type 1 (PAI-1) mRNA expression in cultured rat glomerular mesangial cells, and to examine the interaction between Ang II and TGF-beta signaling. Ang II-induced upregulation of PAI-1 mRNA expression was prevented by a protein kinase C (PKC) inhibitor, bisindorylmaleimide I. While phorbol 12-myristate 13-acetate (PMA) upregulated the PAI-1 mRNA expression, a calcium ionophore, ionomycin, had little effect. Mesangial cells pretreated with PMA for 24 h to downregulate PKC demonstrated attenuated response to Ang II. A protein tyrosine kinase inhibitor, genistein, completely blocked both Ang II- and PMA-induced PAI-1 mRNA expression. Transforming growth factor-beta1 (TGF-beta1) alone induced the expression, and in the presence of Ang II, TGF-beta1 superinduced PAI-1 mRNA expression to a higher extent. Both bisindorylmaleimide I and genistein suppressed the Ang II plus TGF-beta1-induced PAI-1 mRNA upregulation to the basal level, while downregulation of PKC attenuated the synergistic upregulation of PAI-1 mRNA expression to the level comparable to TGF-beta1 alone. These data suggest that, in rat mesangial cells, (1) PKC and protein tyrosine kinase(s) are involved in the Ang II signaling cascade, (2) protein tyrosine kinase(s) works downstream from PKC in the cascade, and (3) there is an interaction between the Ang II and TGF-beta signal pathways downstream from PKC. In in vivo settings, local activation of renin-angiotensin and TGF-beta systems in the glomeruli may synergistically augment PAI-1 expression, promote mesangial matrix accumulation and progression of glomerular injury.     

Induction of nuclear factor-kappa B and the human immunodeficiency virus long terminal repeat by okadaic acid, a specific inhibitor of phosphatases 1 and 2A

We have used a specific phosphatase inhibitor, okadaic acid, to examine the role of two phosphatases, PP1 and PP2A, in the induction of NF-kappa B and the long terminal repeat of the human immunodeficiency virus type 1 (HIV-LTR). Treatment of Jurkat cells with okadaic acid induced NF-kappa B in nuclear extracts. The rate of induction by okadaic acid was delayed compared to the induction of NF-kappa B by phorbol myristate acetate (PMA). The induction of NF-kappa B by okadaic acid was enhanced by cycloheximide or phytohemagglutinin (PHA). In contrast to PMA, okadaic acid appeared to induce NF-kappa B independently of protein kinase C (PKC). That the NF-kappa B induced by okadaic acid was functional was demonstrated by the marked increase in CAT activity that occurred in Jurkat, BJA-B, and U251 cells that were transfected with HIV-LTR-CAT and treated with okadaic acid. The increase in CAT activity triggered by okadaic acid was dependent on the presence of the NF-kappa B sites in the long terminal repeat of HIV as assessed by deletion and mutation analysis. Similarly to its effect on the induction of NF-kappa B, PHA added together with okadaic acid resulted in a further increase in CAT activity. Somewhat surprisingly, the addition of PMA inhibited the increase in CAT activity in response to okadaic acid, which suggests that the activation of PKC may also induce inhibitory factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycated human serum albumin enhances macrophage inflammatory protein-1β mRNA expression through protein kinase C-δ and NADPH oxidase in macrophage-like differentiated U937 cells

Background:

In a previous report (Higai K et al., Biol Pharm Bull, 2007), glycated human serum albumin (Glc-HSA) was found to induce interleukin-8 (IL-8) mRNA expression in human monocyte-derived U937 cells through a reactive oxygen species (ROS)-dependent pathway; however, Glc-HSA signaling has not been elucidated in macrophages.

Methods:

U937 cells were differentiated by treatment with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) for 2 days and the macrophage-like differentiated U937 (differentiated U937) cells were stimulated with Glc-HSA and glycolaldehyde dimer-modified HSA (GA-HSA) in the presence of various signaling inhibitors. Macrophage inflammatory protein-1β (MIP-1β) mRNA expression was determined by real-time PCR. Intracellular ROS generation was estimated by confocal laser microscopy.

Results:

Glc-HSA and GA-HSA markedly enhanced MIP-1β mRNA expression in differentiated U937 cells. Enhanced MIP-1β mRNA expression was completely suppressed by the ROS scavenger N-acetyl-l-cysteine, the NADPH oxidase inhibitors diphenylene iodonium and apocynin, and the protein kinase C (PKC)-δ inhibitor rottlerin. Furthermore, ROS generation was suppressed completely by rottlerin but not by the PKC-γ inhibitor Ro318425 or the PKC-α, -β1 and -μ inhibitor Go6976.

Conclusion:

Glc-HSA and GA-HSA enhance MIP-1β mRNA expression in differentiated U937 cells through PKC-δ-dependent activation of NADPH oxidase.     

p21ras and protein kinase C function in distinct and interdependent signaling pathways in C3H 10T1/2 fibroblasts.

Both p21ras and protein kinase C (PKC) are believed to function downstream of plasma membrane-associated tyrosine kinases in cellular signal transduction pathways. However, it has remained controversial whether they function in the same pathway and, if so, what their relative position and functional relationship in such a pathway are. We investigated the possibilities that p21ras and PKC function either upstream or downstream of each other in a common linear pathway or that they function independently in colinear signal pathways. Either decreased expression of endogenous normal ras in fibroblasts transfected with an inducible antisense ras construct or overexpression of a mutant ras gene reduced the capacity of the phorbol ester tetradecanoyl phorbol acetate to trigger expression of the tetradecanoyl phorbol acetate-responsive and ras-dependent reporter gene osteopontin (OPN). PKC depletion decreased basal OPN mRNA levels, and the overexpression of ras restored OPN expression to the level of non-PKC-depleted cells. We propose a model in which ras and PKC function in distinct and interdependent signaling pathways.     

Activation of distinct protein kinase C isozymes by phorbol esters: correlation with induction of interleukin 1 beta gene expression

Treatment of human promyelocytic leukemia cells U937 with phorbol 12-myristate 13-acetate (TPA) induces them to differentiate into monocytic cells [Harris, P., & Ralph, P. (1985) J. Leukocyte Biol. 37, 407-422]. Here we investigated the effects of TPA on interleukin 1 gene expression and the possible role of protein kinase C (PKC) in this process. Addition of TPA to serum-starved U937 cells induced the expression of the interleukin 1 beta (IL-1 beta) gene. This effect was apparent as early as 2 h and peaked at 24 h in the presence of 5 X 10(-8) M TPA. Higher concentrations of TPA, which partially or totally depleted protein kinase C levels in the cells (10(-9)-2 X 10(-5) M), had an inhibitory effect on IL-1 beta mRNA expression. Cell-permeable 1,2-dioctanoyl-sn-glycerol (diC8), a diacylglycerol that activates PKC in intact cells and cell-free systems, did not mimic the effect of TPA on the IL-1 beta mRNA induction. To determine the protein kinase C isozymes present in the control and TPA- (5 X 10(-8) M) treated U937 cells, we prepared antipeptide antibodies that specifically recognize the alpha, beta, and gamma isoforms of protein kinase C in rat brain cytosol and U937 cell extracts. In "control" U937 cells, 30% of PKC alpha was particulate, and PKC beta was cytosolic, while there was no detectable PKC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

The levels of leukemia inhibitory factor mRNA in a Schwann cell line are regulated by multiple second messenger pathways

Axotomy of sympathetic and sensory neurons leads to changes in their neuropeptide phenotypes. These changes are mediated in part by the induction of leukemia inhibitory factor (LIF) by nonneuronal cells. In the present study, we identified satellite/Schwann cells as a possible source of the injury-induced LIF. Using a Schwann cell line, SC-1 cells, we examined mechanisms of LIF induction. LIF mRNA levels increased rapidly when the cells were treated with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, phorbol 12-myristate 13-acetate (PMA), or A23187. Among these reagents, PMA was the most efficacious. Inhibition of protein kinase C (PKC) by GF-1 09203X significantly reduced the PMA-induced LIF mRNA levels. As PKC is known to activate the extracellular signal-regulated kinase (ERK) signaling pathway, the involvement of this pathway in the PMA-stimulated induction of LIF mRNA was examined. Phosphorylation of ERKs was increased following PMA treatment in SC-1 cells. Moreover, inhibition of ERK kinase activity by PD98059 dramatically reduced PMA-stimulated phosphorylation of ERKs and induction of LIF mRNA. These results indicate that LIF mRNA levels can be regulated by ERK activation via stimulation of PKC in Schwann cells.     

Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.