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Am J Physiol Lung Cell Mol Physiol 290: L291-L297, 2006. First published September 16, 2005; doi:10.1152/ajplung.00053.2005
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Involvement of protein kinase C in crystalline silica-induced activation of the MAP kinase and AP-1 pathway

Min Ding,1 Chuanshu Huang,2 Yongju Lu,1 Linda Bowman,1 Vince Castranova,1 and Val Vallyathan1

1Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia; and 2Nelson Institute of Environmental Medicine, New York University, School of Medicine, Tuxedo, New York

Submitted 28 January 2005 ; accepted in final form 9 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Crystalline silica has long been well established as a fibrogenic agent, and recent evidence has implicated it as a potential human carcinogen. However, the mechanisms of silica-induced disease development and progression are not well understood. Our previous studies demonstrated that crystalline silica is able to activate activator protein-1 (AP-1) through mitogen-activated protein kinase (MAPK) pathways. The present study investigates the possible involvement of protein kinase C (PKC) in silica-induced activation of the MAPK/AP-1 signal transduction pathway. Treatment of mouse epidermal cells (JB6 cell line) with freshly fractured silica stimulated translocation of PKC{alpha} and PKC{epsilon} from the cytosol to the membrane and activated AP-1 transcription activity. Pretreatment of cells with PKC inhibitors, including RO-32-0432, calphostin C, and bisindolylmaleimide I, inhibited silica-induced AP-1 activation and phosphorylation of ERKs and p38 kinase. These inhibitory effects by PKC inhibitors were dose dependent. Furthermore, overexpression of dominant negative mutant (DNM) of PKC{alpha} or PKC{epsilon} markedly blocked AP-1 activation as well as phosphorylation of ERKs and p38 kinase induced by freshly fractured silica. These results demonstrate that PKC{alpha} and PKC{epsilon} are essential in silica-induced AP-1 activation through the MAP kinase (ERKs and p38 kinases) pathway.

protein kinase C; mitogen-activated protein kinase; activator protein-1; silica

EPIDEMIOLOGICAL AND PATHOLOGICAL studies have established that occupational exposure to crystalline silica can lead to the development of either chronic or acute pulmonary disease (16, 20, 27). Increasing evidence from epidemiological and animal studies has also implicated crystalline silica as a potential carcinogen (3, 28, 32, 47). For example, inhalation of silica has been shown to cause lung tumors in rats (27–29, 33). Intrapleural administration of crystalline silica in rats leads to the induction of localized malignant histiocytic lymphomas. Epidemiological studies have also shown an increased lung cancer risk in workers occupationally exposed to silica (3, 26, 43). Based on evidence obtained from animal and epidemiological studies supported by limited molecular studies, the International Agency for Research on Cancer has classified crystalline silica and cristobalite in certain occupational settings as group I human carcinogens (28).

Although silica is a documented carcinogen, the molecular mechanisms involved in silica-induced carcinogenesis are largely unknown. Previous studies have shown that freshly fractured silica is capable of generating reactive oxygen species (ROS) upon reaction with aqueous media (42–44, 46). These reactive species were reported to be involved in the silica-induced lipid peroxidation and membrane damage that lead to the loss of membrane integrity and eventual pulmonary injury (45, 46). Silica-induced ROS generation can cause oxidative damage to DNA and mammalian cell transformation, which has been used as an in vitro analog of cancer induction (11, 44, 47). Recent studies have also shown that through ROS-mediated reactions, silica is able to activate nuclear transcription factor activator protein (AP)-1 (14, 15). Because AP-1 activation has been linked to cell transformation and cancer development in various in vitro and in vivo systems, it is reasonable to hypothesize that silica-induced AP-1 activation may play an important role in silica-induced carcinogenesis. To elucidate the mechanisms involved in silica-induced AP-1 activation, we investigated the mitogen-activated protein kinase (MAPK) family members, including extracellular signal-regulated protein kinases (ERKs) and p38 kinase in cell culture models and AP-1 reporter transgenic mice (14, 15). The present studies are designed to unravel the complex upstream regulators that mediate the activation of MAPK/AP-1 and to examine the possible involvement of PKCs in silica-induced signal transduction pathway.

PKC has emerged as a key enzyme, which is activated by transmembrane signals such as products of phospholipid hydrolysis, Ca2+, and ROS (5, 37, 40). Activation of PKC signal transduction proteins is one of the earliest events leading to cellular responses including secretion of cytokines. PKC has been shown to influence tumor cell differentiation, growth, motility, adhesion, invasion, tumor promotion, and metastasis (18, 31). PKC represents a family of more than 11 isoenzymes that are divided broadly into Ca2+-dependent and Ca2+-independent types. Differences in the subcellular localization and expression of these isoenzymes in various cell types enable the PKC isozymes to respond differently to a wide variety of cellular stimuli (5, 35, 40). The experimental tumor promoters, phorbol esters, have been widely studied for their ability to increase membrane translocation of PKC and its regulation (6, 18, 25). Because silica is both an oxidant-generating source and an inflammatory agent, it is proposed that this carcinogen might be able to activate PKC and modulate cellular responses. Therefore, the present study was focused on silica-induced PKC activation and its relationship to downstream regulators of MAPKs and AP-1 activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents. Eagle's MEM (EMEM) was obtained from Whittaker Biosciences (Walkersville, MD). FBS, gentamicin, and L-glutamine were purchased from Life Technologies (Gaithersburg, MD). Luciferase assay substrate was obtained from Promega (Madison, WI). PhosphoPlus MAPK antibody kits were purchased from New England BioLabs (Beverley, MA). Bisindolylmaleimide I, calphostin C, RO-32-0432, and rottlerin were purchased from Calbiochem (La Jolla, CA).

Preparation of freshly fractured silica. Crystalline silica was obtained from Pennsylvania State University, Generic Center, State College, PA. The detailed method for preparation of the freshly fractured silica was described elsewhere (14, 15). In brief, crystalline silica (0.2–10 mm in diameter) was ground for 30 min with a ball grinder equipped with agate mortar and balls. The ground silica was sieved through a 10-µm-mesh filter for 20 min before use. Purity was checked by X-ray diffraction spectrometry, and particle diameter was determined by morphometric analyses, which indicated that freshly fractured silica had a purity of 99.5% and a mean diameter of 3.7 µm.

Cell culture. The mouse JB6 cell line and its transfectants, including cells stably transfected with AP-1 luciferase reporter plasmid (14), a stable dominant negative mutant PKC{alpha} transfectant (DNM-PKC{alpha}), and a stable dominant negative mutant PKC{epsilon} transfectant (DNM-PKC{epsilon}), were established as reported earlier (21, 22). The cells were cultured in EMEM containing 5% FBS, 2 mM L-glutamine, and 50 µg/ml gentamicin.

Assay of AP-1 activity in vitro. A confluent monolayer of JB6 cells with AP-1 luciferase reporter plasmid was trypsinized, and 5 x 104 viable cells (suspended in 1 ml of EMEM supplemented with 5% FBS) were added to each well of a 24-well plate. Plates were incubated at 37°C in a humidified atmosphere of 5% CO2. Twelve hours later, cells were cultured in EMEM supplemented with 0.5% FBS for 12–24 h to minimize basal AP-1 activity and then exposed to silica in the same medium to monitor the effects on AP-1 induction. The cells were extracted with 200 µl of 1x lysis buffer provided in the luciferase assay kit by the manufacturer. Luciferase activity was measured with a model 3010 Monolight Luminometer (14). The results were expressed as relative AP-1 activity compared with untreated controls.

Protein kinase phosphorylation assay. Immunoblots for phosphorylation of ERKs and p38 kinase were carried out as described in the protocol of New England Biolabs, using phospho-specific antibodies against the phosphorylated sites of ERKs and p38 kinase. Nonphospho-specific antibodies against ERKs and p38 kinase proteins provided in each assay kit were used to normalize the phosphorylation assay using the same-transferred membrane blot.

PKC translocation assay. Cells (2 x 105) were seeded in a 10-cm dish. After reaching 85% confluence, the cells were then cultured in EMEM containing 0.1% FBS for 24 h. After incubation with silica (0–225 µg/cm2) for the desired time, the cells were washed once with ice-cold phosphate-buffered saline (Ca2+-free). Two hundred microliters of homogenization buffer A (20 mM Tris·HCl, pH 8.0, 10 mM EGTA, 2 mM EDTA, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 µl/ml aprotinin, and 10 µg/ml leupeptin) were added to each dish, and the cells were scraped into a 1.5-ml tube with a rubber policeman. The suspension was sonicated for 10 s at output 4 with a sonicator (Ultrasonics) and centrifuged at 100,000 g for 1 h at 4°C. The supernatant was collected as the cytosolic fraction. The pellet was resuspended in 200 µl of homogenization buffer B (1% Triton X-100 in homogenization buffer A) and sonicated for 10 s. The suspension was centrifuged at 15,000 g for 15 min at 4°C. The supernatant was collected as the membrane fraction. Protein concentration of each sample was determined, and 100 µl of 3x Laemmli sample buffer (187.5 mM Tris·HCl, pH 6.8, 6% SDS, 30% glycerol, 150 mM dithiothreitol, and 0.3% bromphenol blue) were added. Then the samples were subjected to Western blot analysis using antibodies specific for each subtype of PKC.

PKC activity assay. Total PKC activity was quantified using StressXpress PKC activity assay kit (Stressgen, Victoria, BC, Canada). Briefly, a confluent monolayer of JB6 cells (in 100-mm culture dish) treated with or without silica was harvested. The cells were washed and lysed in 1 ml of lysis buffer (20 mM MOPS, 50 mM {beta}-glycerolphosphate, 50 mM sodium fluoride, 1 mM sodium vanadate, 5 mM EGTA, 2 mM EDTA, 1% Nonidet P-40, 1 mM dithiothreitol, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin and aprotinin). The lysate was centrifuged at 13,000 rpm for 15 min, and the protein concentration was quantified by the Lowry method. PKC activity in the supernatant was determined as described in the protocol of the manufacturer.

Statistical analysis. Data presented are the means ± SE of n experiments as noted in the figure legends. Outcome variables were analyzed by analysis of variance and Student's t-tests. Significance was set at P ≤ 0.05 and is indicated with an asterisk.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Silica-induces PKC membrane translocation. Previous studies from our laboratory have shown that freshly fractured silica causes AP-1 activation in JB6 cells and that this activation is mediated through ROS and MAP kinases p38 and ERKs (14, 15). To study the role of PKC in the silica-induced AP-1 and MAPK signal pathway, the same cell system was used. It is known that the translocation of PKCs from the cytosol to the membrane is a critical step for the activation of this enzyme, and the PKC content in membranes or the ratio in membranes to cytosol reflects PKC activity (37, 38). To understand whether PKCs are involved in silica-induced signal transduction, we determined the membrane/cytosol distribution of the subtypes of PKC{alpha}, PKC{delta}, PKC{epsilon}, PKC{theta}, and PKC{zeta} in JB6 cells after treatment of cells with silica by Western blot analysis. The results indicate that silica significantly induced the activation or translocation of PKC{alpha} and PKC{epsilon} (Figs. 1 and 2) from cytosol fraction to the membrane. Silica-induced membrane translocation of PKC{epsilon} was transient, with maximal activation or translocation occurring within 30 min, and returned to basal level after 60 min (Fig. 2A). This transient translocation of PKC{epsilon} may be due to the degradation and/or selective modification of cysteine-rich regions of this isozyme (17, 19, 37, 38). In our studies we used 12-O-tetradecanoylphorbol-13-acetate (TPA) as a positive control for PKC{alpha} and PKC{epsilon} membrane translocation assay, which is reported to cause a significant activation of these PKC subtypes in JB6 cells (24, 25).



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  		Fig. 1. Translocation of PKC{alpha} from cytosol to membrane induced by silica. JB6 cells were treated with freshly fractured silica at various concentrations or time as indicated. The samples were fractured as described in MATERIALS AND METHODS and analyzed with 8% SDS-PAGE and Western blotting. The membrane blot was probed with PKC{alpha} antibodies. A: time course distribution of PKC{alpha} in the membrane or cytosol after exposure of cells with silica (75 µg/cm2). B: dose-dependent membrane translocation of PKC{alpha} after 30-min exposure. Data are representative of 3 independent experiments. TPA was used as a positive control.

 


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  		Fig. 2. Silica-induced translocation of PKC{epsilon} from cytosol to membrane. JB6 cells were treated with freshly fractured silica at various concentrations or time as indicated. The samples were fractured as described in MATERIALS AND METHODS and analyzed with 8% SDS-PAGE and Western blotting. The membranes were probed with PKC{epsilon} antibodies and visualized using ECF detection reagents. A: time course distribution of PKC{epsilon} in the membrane or cytosol after exposure of cells with silica (75 µg/cm2). B: dose-dependent membrane translocation of PKC{epsilon} after 30-min exposure. Data are representative of 3 independent experiments. TPA was used as a positive control.

 
Silica stimulates PKC activity. To further confirm that silica induces PKC activation, PKC activity was performed. The results indicated that freshly fractured silica stimulated PKC activity in a dose- and time-dependent manner (Fig. 3). Time-dependent activation of PKC was observed at 10-min exposure at the concentration of 75 µg/cm2 of silica and continued to increase up to 90 min (Fig. 3B). These results were consistent with the PKC membrane translocation study.



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  		Fig. 3. Effect of silica on PKC activity. JB6 cells were treated with freshly fractured silica at various concentrations or time as indicated. The PKC activity was determined as described in MATERIALS AND METHODS. A: dose-dependent induction of PKC activity after 30-min exposure. B: time course stimulation of PKC activity after treatment of cells with 75 µg/cm2 of silica. Results are presented as relative PKC activity compared with untreated control cells are means of SE from 4 assay wells. *Significant increase from untreated cells (P < 0.05).

 
PKC activation is required for phosphorylation of MAPKs induced by silica. The above results indicate that silica-induced subtype-specific PKC activation and PKC may be involved in mediating silica-induced AP-1 signaling. Earlier studies from our laboratory have shown that both ERKs and p38 are important in regulating silica-induced AP-1 activation (14, 15). We postulated that silica might first cause activation of PKC, which could activate ERKs and p38 leading to AP-1 activation. To address this question, we used several PKC inhibitors to investigate their effects on silica-induced phosphorylation of ERKs and p38 kinase. Among these inhibitors, RO-32-0432 is a selective inhibitor for PKC{alpha}, whereas bisindolylmaleimide I and calphostin C are general inhibitors for most subtypes (4, 19, 30, 50). Our results indicate that RO-32-0432, at concentrations of 25 and 50 nM, inhibited activation of both ERKs and p38 activation induced by silica (Fig. 4A). A much stronger inhibition was observed at a concentration of 100 nM (Fig. 4A). It has been reported that at a concentration of 9–50 nM, RO-32-0432 functions as a specific PKC{alpha} inhibitor and a higher dose (100 nM) is required to inhibit PKC{epsilon} (4). The two general PKC inhibitors, calphostin C and bisindolylmaleimide I, also exhibited inhibitory effects on silica-induced activation of ERKs, as well as p38 (Fig. 4, B and C). These results suggest that those PKC family members tested in our studies are involved in silica-induced activation of ERKs and p38.



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  		Fig. 4. Effects of PKC inhibitors on silica-induced phosphorylation of ERKs and p38 kinase. JB6 cells were cultured in 6-well plates and then exposed to 75 µg/cm2 freshly fractured silica for 60 min in the absence or presence of various concentrations of RO-32-0432 (A), calphostin C (B), and bisindolylmaleimide I (BIM, C) as indicated. The cells were lysed, and phosphorylated ERKs and p38 kinase were assayed using a Phosphoplus MAPK kit from New England Biolabs. The nonphosphorylated proteins are used as an internal control for normalizing the protein loading on each lane. Data are representative of 2 experiments.

 
Because the PKC inhibitors are only relatively subtype specific, we used JB6 cells stably transfected with DNM-PKC{alpha} and DNM-PKC{epsilon} to further confirm the role of these two PKC isozymes in silica-induced signal transduction. These dominant negative mutants were constructed by site-directed mutagenesis of lysine residues in the ATP binding site (21, 22). We analyzed the effect of these dominant negative PKC mutants on the silica-induced activation of ERKs and p38 kinase. In JB6 control vector transfectant, silica induced a strong activation of both ERKs and p38 kinase (Fig. 5). However, silica-induced activation of ERKs and p38 was abolished by overexpression of the dominant negative mutants of PKC{alpha} and PKC{epsilon}. On the other hand, we observed that the basal levels of the phospho-ERKs in DNM-PKC{alpha} and DNM-PKC{epsilon} were even higher than those with silica-treated cells. This may have resulted due to the cells trying to overcome DNM effect by upregulating its basal ERK activity. These results suggested that these PKC isozymes might function as upstream regulators in silica-induced activation of the MAPK/AP-1 pathway.



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  		Fig. 5. Effects of overexpression of dominant negative mutants (DNM) of PKC{alpha} or PKC{epsilon} on silica-induced activation of ERKs and p38. JB6 Cl 41 cells and Cl 41 stable transfectants with DNM-PKC{alpha} and DNM-PKC{epsilon} were cultured in 5% FBS/MEM in 6-well plates until 80% confluent and then exposed to 75 µg/cm2 freshly fractured silica for 60 min. The cells were lysed, and phosphorylated and nonphosphorylated ERKs and p38 kinase protein were assayed using a Phosphoplus MAPKs kit from New England Biolabs. The phosphorylated and nonphosphorylated proteins were analyzed by using the same membrane following a stripping procedure. Data are representative of 3 experiments. WT, wild type.

 
Effect of PKC inhibitors and dominant negative mutants of PKCs on silica-induced AP-1 activation. The above results demonstrate that PKCs play a pivotal role in silica-induced MAPK activation. To investigate the role of PKCs in mediating silica-induced signaling leading to the activation of AP-1, we used PKC inhibitors to study the relationship between PKCs and AP-1 activation. As shown in Fig. 6A, silica induced a dose-dependent activation of AP-1. This induction was significantly inhibited by all the PKC inhibitors tested here (Fig. 6B).



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  		Fig. 6. Effect of PKC inhibitors and DNM-PKC{alpha} and DNM-PKC{epsilon} on silica-induced activator protein (AP)-1 activation. JB6 cells with stable AP-1-luciferase reporter cells, JB6 Cl 41 vector control transfectant (CMV-neo), or stable transfectants with either DNM-PKC{alpha} or DNM-PKC{epsilon} were exposed to 75 µg/cm2 of freshly fractured silica in the presence or absence of PKC inhibitors for 24 h. Results, presented as relative AP-1 induction compared with untreated control cells, are means ± SE from 8 assay wells. *Significant increase from untreated cells (P < 0.05). **Significant decrease from JB6 WT cells treated with freshly fractured silica (P < 0.05). A: dose-dependent induction of AP-1 activation by silica; B: effect of PKC inhibitors on AP-1 activation induced by silica; C: effect of DNM-PKC{alpha} and DNM-PKC{epsilon} on silica-induced AP-1 activation.

 
To further investigate the role of PKCs in silica-induced activation of AP-1, JB6 stable transfectants DNM-PKC{alpha} and DNM-PKC{epsilon} were used. As shown in Fig. 6C, overexpression of either mutant blocked silica-induced AP-1 activation, with DNM-PKC{alpha} being more potent.

These results, together with the membrane translocation of the PKCs induced by silica, indicate that PKC{alpha} and PKC{epsilon} play important roles in mediating silica-induced AP-1 activity.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PKC plays a central role in the regulation of several important cellular events by mediating extracellular stimuli involved in cell proliferation, differentiation, apoptosis, and survival (5, 34). PKC family members consisting of multiple isozymes exhibit differences in subcellular localization, substrate specificity, and response to divergent stimuli and inhibitors (36). Different PKC isozymes have been implicated in divergent cellular responses of apoptosis, contraction, hypertrophy, permeability, proliferation, and secretion (12). PKC activation occurs in concert with the activation of phospholipase C and release of diacylglycerol, which are well-known players in cell signaling and enzymatic activation of PKC, respectively. In this respect, PKCs have emerged as key enzymes that are activated by transmembrane signals, such as products of phospholipid hydrolysis and Ca2+ (5, 35, 37, 40). Because PKC family members are involved in tumor promotion, progression, proapoptotic/antiapoptotic response, and drug resistance, these enzymes are currently investigated for the development of cancer chemo-prevention and therapy (1, 2, 13). Various chemicals and carcinogens, such as asbestos, arsenate, and UV, have been reported to induce PKC activation (6, 10, 21, 25, 48).

In this report, we present data demonstrating that silica is capable of activating of PKC{alpha} and PKC{epsilon} and that the silica-induced activation of these isozymes is important in the activation of MAPKs and AP-1 signaling. Previous studies from our laboratory have shown that freshly fractured silica contains more reactive species with a potential to induce AP-1 and MAPK signaling in a dose-dependent manner (14, 15). Recent studies have suggested that stimulation of AP-1 may be a common mechanism for diverse types of tumor promoters (23, 25). Therefore in the present study, we investigated the possible involvement of PKCs in silica-induced activation of MAPK/AP-1 pathway. JB6 cells were chosen for these studies since this cell line was previously used for silica-induced activation of AP-1 studies in our laboratory and they have been shown to respond to a greater degree than rat lung epithelial cells (14, 15). The results show that silica is able to induce translocation of PKC{alpha} and PKC{epsilon} from the cytosol to the membrane and stimulate PKC activity. With regard to the mechanism of silica-induced PKC activation, it may be noted that the structural aspects of PKC, the cysteine residues present within the regulatory and catalytic domains, make them very sensitive targets for ROS (19, 34). PKCs can be activated and inactivated as an effective on/off signaling for several cellular mechanisms involved in signal transduction regulating cell proliferation or growth promotion. It has been demonstrated that freshly fractured silica is capable of generating ROS upon reaction with cultured cells (15). ROS may cause oxidative modification of both regulatory and catalytic domains of PKCs leading to their activation (19). Prolonged oxidant exposure is linked to tumor promotion and antioxidants appear to prevent this process.

Our earlier studies (14, 15) have also shown that silica is able to activate MAPKs through ROS-mediated reactions. Among these MAPK family members, ERKs and p38 play important roles in silica-induced AP-1 activation. To investigate the role of PKCs in silica-induced MAPKs and AP-1 activation, several established PKC inhibitors were used. The results indicate that inhibition of various PKC isozymes resulted in the decrease of silica-induced phosphorylation of ERKs and p38. Furthermore, all the PKC inhibitors tested in this study, including calphostin C, bisindolylmaleimide I, and RO-32-0432, inhibited silica-induced AP-1 activation although their potencies were different. Thus it appears that PKCs are upstream regulators for silica-induced MAPK/AP-1 cellular signaling. In the present study, we also used dominant negative mutants of PKC{alpha} and PKC{epsilon} to further investigate the role of PKCs in silica-induced ERKs and p38 kinase activation. The results show that silica-induced activation of these two MAPK members was significantly decreased in these mutant transfectants. These studies provide additional support to show that PKC isozymes PKC{alpha} and PKC{epsilon} are involved in silica-induced activation of ERKs and p38 kinase, which in turn lead to the activation of AP-1.

Crystalline silica and crystobalite are classified as group I carcinogens to humans in certain occupational settings (28), but animal experimental support and molecular mechanisms involved in silica-induced carcinogenesis are poorly understood (43). Earlier studies from our laboratories have focused on silica-induced cellular injury and the involvement of oxidative damage in injury (7–9, 41–49). In our recent studies, we have focused on the increasing recognition that prolonged oxidant exposure and the effects of oxidative stress are involved in signal transduction and other cascades of cellular events promoting the activation of oncogenes leading to silica-induced carcinogenesis (14, 15). In this respect, PKCs, MAPKs, AP-1, as well as NF-{kappa}B, are key regulators involved in immediate cellular response to oxidative stress associated with cellular proliferation and carcinogenesis. Thus paradoxically the connection of oxidative stress-induced changes in second messengers and activation of transcription factors leading to gene regulation and expression through involvement of PKCs, MAPKs, and AP-1 pathway play a central role in carcinogenesis.

Our present studies together with those reported earlier (14, 15) suggest a model for the elucidation of events involved in cell proliferation and carcinogenesis by freshly fractured silica. Upon reacting with the cells, silica causes the generation of ROS and translocation of PKCs from the cytosol to the membrane, leading to activation of AP-1 through MAPK signal transduction pathways. It is possible that activation of AP-1 is a crucial step that initiates cell proliferation and progression through the cell cycle. The biopersistence of silica may result in continued oxidant generation and sustained redox signaling stimulating growth, cell proliferation, genetic changes leading to the eventual fixation of genetic changes, and cancer development. The activation of AP-1 and transcription factors associated with crystalline silica exposure may contribute to cell phenotype leading to neoplastic transformation.

In conclusion, the results obtained from the present study demonstrate that silica is able to cause translocation of PKC{alpha} and PKC{epsilon} from the cytosol to the membrane in JB6 cells. Prolonged oxidant exposure to PKC is linked to carcinogenesis. Therefore, the activation of PKCs induced by silica and subsequent activation of ERKs and p38 kinase, as well as AP-1, may be involved in silica-induced lung disease.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Ding, PPRB, NIOSH, 1095 Willowdale Rd., Morgantown, WV 26505 (e-mail: mid5{at}cdc.gov)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
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