Major house dust mite allergen, Der p I, activates phospholipase D in human peripheral blood mononuclear cells from allergic patients: involvement of protein kinase C

The major house-dust-mite allergen, Der p I, stimulates the phospholipase D (PLD) in peripheral blood mononuclear cells (PBMC) from allergic patients with maximal responses after 30 min exposure. At 30 min, Der p I stimulated PLD activity by 1.4-fold in mild, 1.6-fold in moderate and 2-fold in severe allergic patients over control values (p < 0.05). When the cells were pretreated for 24 h with phorbol myristate acetate to down-regulate protein kinase C (PKC), PLD stimulation by Der p I was largely abolished. These results indicate that in PBMC from allergic patients, Der p I can stimulate PLD activity, and that PKC activation is involved in this stimulation.     

Involvement of protein kinase C pathway in UVC-stimulated phospholipase D2 activity in Vero 76 cells

Phospholipase D (PLD) activity is known to be related to oxidant-induced cellular signaling and membrane disturbance. Previously, an induction of PLD activity in various cell lines by X-ray irradiation was observed. In this study, we examined the effect of UVC radiation on the PLD activity in Vero 76 cells. At a dose of 10 kJ/m2 of UVC irradiation, the PLD activity was stimulated approximately 10-fold over the basal activity. This UVC-induced PLD activity was found to be dependent on the presence of extracellular calcium and was inhibited by catalase as well as amifostine-an intracellular thiol antioxidant. Pretreatments with Ro32-0432-a selective inhibitor of protein kinase C (PKC)-and downregulation of PKC by preincubation of phorbol 12-myristate 13-acetate significantly inhibited the UVC-induced PLD activity. UVC-stimulated PLD activity was observed only in murine PLD2 (mPLD2)-transfected Vero 76 cells and not in human PLD1 (hPLD1)-transfected cells. Transient incorporation of PKC with mPLD2 and the phosphorylation of mPLD2 by a and b forms of PKC by UVC irradiation were observed. These results suggest that the UVC-stimulated PLD activity in Vero 76 cells is mediated through transient phosphorylation of PLD2 by the translocation of PKC to PLD2.     

Phospholipase D is involved in oxidative stress-induced migration of vascular smooth muscle cells via tyrosine phosphorylation and protein kinase C

Oxidative stress has been implicated in mediation of vascular disorders. In the presence of vanadate, H(2)O(2) induced tyrosine phosphorylation of PLD1, protein kinase C-alpha (PKC-alpha), and other unidentified proteins in rat vascular smooth muscle cells (VSMCs). Interestingly, PLD1 was found to be constitutively associated with PKC-alpha in VSMCs. Stimulation of the cells by H(2)O(2) and vanadate showed a concentration-dependent tyrosine phosphorylation of the proteins in PLD1 immunoprecipitates and activation of PLD. Pretreatment of the cells with the protein tyrosine kinase inhibitor, genistein resulted in a dose-dependent inhibition of H(2)O(2)-induced PLD activation. PKC inhibitor and down-regulation of PKC abolished H(2)O(2)-stimulated PLD activation. The cells stimulated by oxidative stress (H(2)O(2)) caused increased cell migration. This effect was prevented by the pretreatment of cells with tyrosine kinase inhibitors, PKC inhibitors, and 1-butanol, but not 3-butanol. Taken together, these results suggest that PLD might be involved in oxidative stress-induced migration of VSMCs, possibly via tyrosine phosphorylation and PKC activation.     

Activation of epidermal growth factor receptor is responsible for pervanadate-induced phospholipase D activation

Pervanadate, a complex of vanadate and H(2)O(2), has an insulin mimetic effect, and acts as an inhibitor of protein tyrosine phosphatase. Pervanadate-induced phospholipase D (PLD) activation is known to be dependent on the tyrosine phosphorylation of cellular proteins and protein kinase C (PKC) activation, and yet underlying molecular mechanisms are not clearly understood. Here, we investigated the signaling pathway of pervanadate-induced PLD activation in Rat2 fibroblasts. Pervanadate increased PLD activity in dose- and time- dependent manner. Protein tyrosine kinase inhibitor, genistein, blocked PLD activation. Interestingly, AG-1478, a specific inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR) blocked not only the PLD activation completely but also phosphorylation of p38 mitogen-activated protein kinase (MAPK). However, AG-1295, an inhibitor specific for the tyrosine kinase activity of pletlet drived growth factor receptor (PDGFR) did not show any effect on the PLD activation by pervanadate. We further found that pervanadate increased phosphorylation levels of p38, extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK). SB203580, a p38 MAPK inhibitor, blocked the PLD activation completely. However, the inhibitions of ERK by the treatment of PD98059 or of JNK by the overexpression of JNK interacting peptide JBD did not show any effect on pervanadate-induced PLD activation. Inhibition or down-regulation of PKC did not alter the pervanadate-induced PLD activation in Rat2 cells. Thus, these results suggest that pervanadate-induced PLD activation is coupled to the transactivation of EGFR by pervanadate resulting in the activation of p38 MAP kinase.     

The p38 mitogen-activated protein kinase is involved in stress-induced phospholipase D activation in vascular smooth muscle cells

Oxidative stress has been implicated in mediation of vascular disorders. Earlier study showed that the exposure of vascular smooth muscle cells (VSMC) to pervanadate (hydrogen peroxide plus orthovanadate) resulted in the accumulation of [3H]phosphatidylbutanol. In this study, the effect of pervanadate on the activation of p38 mitogen-activated protein kinase (p38 MAPK) was studied in the VSMC. Pervanadate treatment activated p38 MAPK in a dose-and time-dependent manner. Interestingly, specific inhibition of p38 MAPK with SB203580 attenuated pervanadate-induced PLD activation. This correlates with the finding that expression of dominant negative mutants of MKK3/6 inhibited the PLD activation. SB203580 pretreatment also inhibited other cellular stressors (i.e. high osmolarity and UV light)-induced PLD activation. The possible correlationship of p38 MAPK activation with PKC was examined since PKC is reported to be involved in the pervanadate-induced PLD activation. Calphostin C, a PKC inhibitor, suppressed pervanadate-induced p38 MAPK and PLD activation in a dose-dependent manner. These results suggest that PKC-p38 MAPK may represent an upstream pathway of PLD in the signal transduction of cellular stress.     

INHIBITION OF 80 kDa PROTEIN PHOSPHORYLATION BY SHORT-WAVELENGTH UV LIGHT IN NIH 3T3 CELLS

The exposure of NIH 3T3 fibroblast cells to 254 nm UV radiation resulted in a temporary depression of DNA synthesis and inhibition of 80 kDa protein phosphorylation. This inhibition of protein phosphorylation was correlated with decreased protein kinase C activity in the membrane fractions of UV-damaged cells. The inositol tnphosphate contents measured, by the competitive binding assay using bovine adrenal binding protein, showed 80% reduction in the fibroblasts treated with 15 J/m² of UV light. The intracellular diacylglycerol concentration was also markedly reduced in UV-damaged cells. The results suggest that UV light causes acute reductions of inositol triphosphate and diacylglycerol contents in cells along with decreases in membrane protein kinase C activity, which leads to the inhibition of phosphorylation of an acidic protein of 80 kDa.     

Differential effects of Fas cross-linking on phospholipase D activation and related lipid metabolism in Fas-resistant A20 cells

A20 murine lymphoma cells undergoing Fas-mediated apoptosis showed increase in the activity of phospholipase D (PLD), which is involved in proliferative or mitogenic cellular responses. Using A20 cell lines that were resistant to Fas-induced apoptosis, we investigated the differential effects of Fas cross-linking on PLD activity and sphingolipid metabolism. The basal PLD activities in all of the selected three Fas-resistant clones (#5, #8, and #11) were about 2~4 folds higher than that of wild type A20 cells. Among the PLD isoforms, PLD2 expression was increased in all of the selected Fas-resistant clones. The Fas downstream signaling events triggered by Fas cross-linking, including the activations of PLD, phosphatidylcholine-specific phospholipase C (PC-PLC), sphingomyelinase (SMase), the increase in diacylglycerol (DAG) and protein phosphorylation levels, and the translocation of protein kinase C to membrane were not changed in both of Fas-resistant clone #5 and #8. In contrast, Fas cross-linking stimulated the activity of PLD, PC-PLC, and SMase, translocation of PKC, and protein phosphorylation in Fas-resistant clone #11, similar to that of wild type cells. We also found that clone #11 had a different Fas sequence encoding Fas B which has been known to inhibit Fas-induced apoptosis. These findings suggest that increased PLD2 expression resulting in increased basal PLD activity and the blockade of Fas downstream signaling cascades may be involved to limit apoptosis induced by Fas cross-linking.     

Phospholipase D is activated and phosphorylated by casein kinase-II in human U87 astroglioma cells

Elevated expression of protein casein kinase II (CKII) stimulated basal phospholipase D (PLD) activity as well as PMA-induced PLD activation in human U87 astroglioma cells. Moreover, CKII-selective inhibitor, emodin and apigenin suppressed PMA-induced PLD activation in a dose-dependent manner as well as basal PLD activity, suggesting the involvement of CKII in the activation of both PLD1 and PLD2. CKII was associated with PLD1 and PLD2 in co-transfection experiments. Furthermore, CKII induced serine/threonine phosphorylation of PLD2 in vivo, and the multiple regions of PLD2 were phosphorylated by CKII in vitro kinase assay using glutathione S-transferase-PLD2 fusion protein fragments. Elevated expression of CKII or PLD increased cell proliferation but pretreatment of cells with 1-butanol suppressed CKII-induced cell proliferation. These results suggest that CKII is involved in proliferation of U87 cells at least in part, through stimulation of PLD activity.     

Deoxyhypusine synthase is phosphorylated by protein kinase C in vivo as well as in vitro

Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. We earlier observed that yeast recombinant deoxyhypusine synthase was phosphorylated by protein kinase C (PKC) in vitro (Kang and Chung, 1999) and the phosphorylation rate was synergistically increased to a 3.5-fold following treatment with phosphatidylserine (P.Ser)/diacylglycerol (DAG)/ Ca(2+), suggesting a possible involvement of PKC. We have extended study on the phosphorylation of deoxyhypusine synthase in vivo in different cell lines in order to define its role on the regulation of eIF5A in the cell. Deoxyhypusine synthase was found to be phosphorylated by endogenous kinases in CHO, NIH3T3, and chicken embryonic cells. The highest degree of phosphorylation was found in CHO cells. Moreover, phosphorylation of deoxyhypusine synthase in intact CHO cells was revealed and the expression of phosphorylated deoxyhypusine synthase was significantly diminished by diacyl ethylene glycol (DAEG), a PKC inhibitor, and enhanced by phorbol 12-myristate 13-acetate (PMA) or Ca(2+)/DAG. Endogenous PKC in CHO cell and cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced by PMA or Ca(2+) plus DAG. Close association of PKC with deoxyhypusine synthase in the CHO cells was evident in the immune coprecipitation and was PMA-, and Ca(2+)/phospholipid dependent. These results suggest that phosphorylation of deoxyhypusine synthase was PKC-dependent cellular event and open a path for possible regulation in the interaction with eIF5A precursor for hypusine synthesis.     

Characterization of the interaction of hypericin with protein kinase C in U-87 MG human glioma cells

A fluorescence imaging technique was used to monitor intracellular localization of protein kinase C (PKC) in U-87 MG human glioma cells in the presence of hypericin (Hyp) and phorbol 12-myristate-13-acetate (PMA). It is shown that PKC localization, which reflects its activity, is influenced by Hyp and this influence is different from that observed for PMA which acts as PKC activator. Fluorescence binding experiments were used to determine the binding constants of Hyp to several isoforms of PKC. The obtained values of K(d)s (approximately 100 nM) suggest that Hyp binds with high affinity to PKC. Finally, molecular modeling was used to compare structural models of the interaction of C1B domain of PKC (PKC isoforms alpha, delta, gamma) with Hyp and our previously published model of the (C1B domain PKCgamma)/PMA complex. The influence of Hyp on PKC translocation in U-87 MG cells in comparison with PMA, colocalization fluorescence pattern of Hyp and PKC, the higher binding affinity of Hyp to PKC in comparison with known binding constants of phorbol esters, as well as the binding mode of Hyp and PMA to the C1B domain of PKC suggested by molecular modeling, support the idea that Hyp and PMA might competitively bind to the regulatory domain of PKC.     

An alternatively spliced form of Met receptor is tumorigenic

The Met tyrosine kinase receptor is a widely expressed molecule, which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). Previously, one of the authors identified an alternatively spliced form of Met (Met-SM) that lacked a single exon of a 47-amino-acid segment in the juxtamembrane domain. Here we report that Met-SM is a potent transforming gene in NIH3T3 mouse fibroblast cells. Met-SM-transfected NIH3T3 cells show stronger foci-forming activity than wild type- Met-transfected ones. In addition, Met-SM-transfected NIH3T3 cells form colonies in soft agar and are tumorigenic in athymic nu/nu mice. Furthermore, HGF/SF significantly increases the focus-forming activity of Met-SM comparing to wild type Met. The amount of protein and of tyrosine kinase activity of Met-SM accumulates to a high level following HGF/SF treatment. The accumulation of Met-SM correlated well with its delayed ubiquitination and increased stability. These results are consistent with the important role of the juxtamembrane domain in protein stability of Met receptor and suggest that the alternatively-spliced form may contribute to the development and progression of human cancer.     

Molecular assembly of mitogen-activated protein kinase module in ras-transformed NIH3T3 cell line

The ras, is a G-like protein that controls the mitogen-activated protein kinase (MAPK) pathway involved in control and differentiation of cell growth. MAPK is a key component of its signaling pathway and the aberrant activation may play an important role in the transformation process. To better understand roles of ras in the activation of MAPKs, we have established ras transformed NIH3T3 fibroblast cell line, and analyzed the MAPK module. The ras transformed cells formed numerous spikes at the edges of cells and showed loss of contact inhibition. The levels of ERK1/2 MAPKs as revealed by Western blot analysis were not significantly different between ras transformed and non-transformed cells. However, phosphorylation of ERK MAPKs and the level of MEK were significantly increased although the heavily expressed level of Raf-1, an upstream component of MAPK pathway was unchanged in ras transformed NIH3T3 cells. The sedimentation profile of the MAPK module kinases in a glycerol gradient showed the presence of a rather homogeneous species of multimeric forms of ERK1/2 and MEK as indicated by the narrow distribution peak areas. The broad sedimentation profile of the Raf-1 in a glycerol gradient may suggest possible heterologous protein complexes but the identification of interacting molecules still remains to be identified in order to understand the organization of the MAPK signal transduction pathway.     

Inhibitory effects of enalaprilat on rat cardiac fibroblast proliferation via ROS/P38MAPK/TGF-β1 signaling pathway

Enalaprilat (Ena.), an angiotensin II (Ang II) converting enzyme inhibitor (ACEI), can produce some therapeutic effects on hypertension, ventricular hypertrophy and myocardial remodeling in clinic, but its precise mechanism, especially its signaling pathways remain elusive. In this study, cardiac fibroblasts (CFb) was isolated by the trypsin digestion method; a BrdU proliferation assay was adopted to determine cell proliferation; an immunofluorescence assay was used to measure intracellular reactive oxygen species (ROS); immunocytochemistry staining and Western blotting assay were used to detect phosphorylated p38 mitogen activated protein kinase (p-p38MAPK) and transforming growth factor-β(1) (TGF-β(1)) protein expression, respectively. The results showed that Ang II (10(-7) M) stimulated the cardiac fibroblast proliferation which was inhibited by NAC (an antioxidant), SB203580 (a p38MAPK inhibitor) or enalaprilat; Ang II caused an burst of intracellular ROS level within thirty minutes, an increase in p-p38MAPK (3.6-fold of that in the control group), as well as an elevation of TGF-β(1) meantime; NAC, an antioxidant, and enalaprilat treatment attenuated cardiac fibroblast proliferation induced by Ang II and decreased ROS and p-p38MAPK protein levels in rat cardiac fibroblast; SB203580 lowered TGF-β(1) protein expression in rats' CFb in a dose-dependent manner. It could be concluded that enalaprilat can inhibit the cardiac fibroblast proliferation induced by Ang II via blocking ROS/P38MAPK/TGF-β(1) signaling pathways and the study provides a theoretical proof for the application of ACEIs in treating myocardial fibrosis and discovering the primary mechanism through which ACEIs inhibit CFb proliferation.     

LONG-WAVE ULTRAVIOLET RADIATION INDUCES PROTEIN KINASE C IN NORMAL HUMAN KERATINOCYTES

Abstract Skin tumor promotion by phorbol ester is believed to be mediated by the phospholipid-dependent ser/ thr kinase, protein kinase C (PKC). Long-wave ultraviolet radiation (320-400 nm, UVA), which has also been shown to promote skin tumors, induces elevated levels of PKC in murine fibroblasts, suggesting that UVA may promote the development of basal and squamous cell skin cancers by a mechanism involving PKC. To examine UVA effects on PKC in a model relevant to skin, we maintained normal human epidermal keratinocytes (NHEK) in serum-free medium and exposed the cultured cells to various doses of UVA or to the phorbol ester, 12- O -tetradecanoylphorbol-13-acetate (TPA). Fifty minutes after exposure to UVA (5-20 J/cm²), PKC activity was elevated up to three-fold in NHEK cytosolic fractions, and membrane-associated PKC activity was elevated up to two-fold by UVA. The TPA treatment induced a 10-fold increase in membrane-associate PKC activity only. Immunoblot analysis suggested that a UVA-induced increase in PKC protein occurred. Both UVA and TPA reduced the cell number by 50-75% in the first 24-48 h; however, irradiated cultures began to recover at 72 h post-UVA due to an increased proliferative rate beginning after 48 h. Treatment with TPA induced a high level of differentiation as measured by cornified envelope formation. Ultraviolet A irradiation exposure was not followed by increased differentiation. These findings suggest that acute UVA exposure elevates PKC activity in human keratinocytes and may act through PKC to promote actinic skin cancer. The molecular mechanism is like to differ from that of the phorbol esters, however.     

MODULATION OF 8-METHOXYPSORALEN-DNA PHOTOADDUCT FORMATION BY CELL DIFFERENTIATION, MITOGENIC STIMULATION AND PHORBOL ESTER EXPOSURE IN MURINE T LYMPHOCYTES

Abstract The effects of cell differentiation and mitogen and phorbol ester stimulation on the formation of 8-methoxypsoralen (8-MOP)-DNA photoadducts in murine T lymphocytes were examined using ³H-8-MOP. While there were no significant differences in 8-MOP photoadduct formation among BALB/c thymocytes, splenocytes, splenic T cells and MRL/1pr lymph node cells, BALB/c bone marrow cells showed fewer photoadducts than did the lymphocytes. This suggested that proliferating progenitor cells may be resistant to 8-MOP photoadduct formation. Incubation of purified splenic T cells with lectin mitogens for 2 h or with phorbol 12-myristate 13-acetate (PMA) for 2–43 h resulted in reduction of 8-MOP photoadduct formation in the DNA, whereas 64 h cultivation with these agents augmented the photoadduct formation. The reduction of photoadduct formation induced by phytohemagglutinin was restored by the further addition of a protein kinase C (PKC) inhibitor, H-7, to the culture. Thus, it is assumed that the reduction of adduct formation evoked by mitogens and PMA is mediated in part by the activation of PKC in the cells. On the other hand, the augmentation of the adduct formation induced by the longer-period cultures with mitogens and PMA appeared to be caused by down-regulation of PKC. The present study showed that the stimulatory signals in which PKC is presumably involved affect the ability of cells to form 8-MOP-DNA photoadducts.     

Rottlerin enhances IL-1β-induced COX-2 expression through sustained p38 MAPK activation in MDA-MB-231 human breast cancer cells

Cyclooxygenase-2 (COX-2) is an important enzyme in inflammation. In this study, we investigated the underlying molecular mechanism of the synergistic effect of rottlerin on interleukin1β (IL-1β)-induced COX-2 expression in MDA-MB-231 human breast cancer cell line. Treatment with rottlerin enhanced IL-1β-induced COX-2 expression at both the protein and mRNA levels. Combined treatment with rottlerin and IL-1β significantly induced COX-2 expression, at least in part, through the enhancement of COX-2 mRNA stability. In addition, rottlerin and IL-1β treatment drove sustained activation of p38 Mitogen-activated protein kinase (MAPK), which is involved in induced COX-2 expression. Also, a pharmacological inhibitor of p38 MAPK (SB 203580) and transient transfection with inactive p38 MAPK inhibited rottlerin and IL-1β-induced COX-2 upregulation. However, suppression of protein kinase C δ (PKC δ) expression by siRNA or overexpression of dominant-negative PKC δ (DN-PKC-δ) did not abrogate the rottlerin plus IL-1β-induced COX-2 expression. Furthermore, rottlerin also enhanced tumor necrosis factor-α (TNF-α), phorbol myristate acetate (PMA), and lipopolysaccharide (LPS)-induced COX-2 expression. Taken together, our results suggest that rottlerin causes IL-1β-induced COX-2 upregulation through sustained p38 MAPK activation in MDA-MB-231 human breast cancer cells.     

Phospholipase D inhibitor enhances radiosensitivity of breast cancer cells

Radiation and drug resistance remain the major challenges and causes of mortality in the treatment of locally advanced, recurrent and metastatic breast cancer. Dysregulation of phospholipase D (PLD) has been found in several human cancers and is associated with resistance to anticancer drugs. In the present study, we evaluated the effects of PLD inhibition on cell survival, cell death and DNA damage after exposure to ionizing radiation (IR). Combined IR treatment and PLD inhibition led to an increase in the radiation-induced apoptosis of MDA-MB-231 metastatic breast cancer cells. The selective inhibition of PLD1 and PLD2 led to a significant decrease in the IR-induced colony formation of breast cancer cells. Moreover, PLD inhibition suppressed the radiation-induced activation of extracellular signal-regulated kinase and enhanced the radiation-stimulated phosphorylation of the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Furthermore, PLD inhibition, in combination with radiation, was very effective at inducing DNA damage, when compared with radiation alone. Taken together, these results suggest that PLD may be a useful target molecule for the enhancement of the radiotherapy effect.