Porphyromonas gingivalis-Stimulated TACE Activation for TGF-α Ectodomain Shedding and EGFR Transactivation in Salivary Gland Cells Requires Rac 1-Dependent p 38 MAPK Membrane Localization

Oral mucosal inflammatory responses to P. gingivalis and its key virulence factor, lipopolysaccharide (LPS), are characterized by a massive rise in proinflammatory cytokine production, up-regulation in mitogen-activated protein kinase (MAPK) cascade, and the induction in epidermal growth factor receptor (EGFR) activation. In this study, we report that stimulation of salivary gland acinar cells with P. gingivalis LPS leads to p38 MAPK-dependent release of soluble TGF-α ligand and the increase in EGFR phosphorylation. Further, we show that the LPS-induced TGF-α shedding and EGFR transactivation involve the activation of membrane-associated metalloprotease, TACE also known as ADAM17, through phosphorylation by p38 MAPK, and require Rac1 participation. Moreover, we demonstrate that blocking the Rac1 activation leads to the suppression in the membrane translocation of Rac1 as well as p38, thus indicating that the LPS-elicited p38 membrane recruitment for TACE phosphorylation requires colocalization with Rac1. Hence, our findings imply that Rac1 membrane translocation serves as an essential platform for the localization of p38 with TACE, TGF-α ectodomain shedding, and the EGFR activation.


Introduction
Lipopolysaccharide (LPS), a component of the outer membrane of P. gingivalis bacterium colonizing the oral cavity, is recognized as a potent endotoxin responsible for eliciting persistent mucosal inflammation that leads to periodontal lesions and progressive destruction of teeth-supporting tissue, including bone loss [1]- [4].The oral mucosal responses to P. gingivalis LPS are mediated through the interaction with Toll-like receptor-4 (TLR4), stimulation of which leads to the activation of transcriptional factors that exert control over a wide range of proinflammatory mediators [2] [5]- [7].The engagement of TLR4 by LPS, moreover, is known to elicit signaling events leading to the activation of mitogen-activated protein kinase (MAPK) cascade, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase [8]- [12].The activated MAPKs, in turn, exert their control over transcriptional factors activation through phosphorylation on the critical Ser/Thr residues [8]- [11].Moreover, the recent evidence indicates that LPS-induced proinflammatory signal propagation is associated with epidermal growth factor receptor (EGFR) activation [13]- [16].
The EGFR is a transmembrane glycosylated glycoprotein, consisting of an extracellular ligand binding domain, a single membrane-spanning region and a cytoplasmic tyrosine kinase domain, implicated in growth and repair processes of a variety of epithelial cells, including those of oral mucosa and salivary glands [17]- [19].Upon ligand binding to the extracellular domain, EGFR undergoes dimerization, and the intrinsic tyrosine kinase activation through phosphorylation of key tyrosine residues within the cytoplasmic region of the receptor [18] [20].Subsequently, these phosphorylated tyrosine residues serve as docking sites for a variety of the recruited signal transducer molecules [18].Studies indicate that in addition to its cognate epidermal growth factor (EGF) ligand, the EGFR activation occurs in response to transforming growth factor-α (TGF-α), heparin binding EGF-like growth factor (HB-EGF), epiregulin, amphiregulin, and epigen [18] [20].All these ligands are expressed as inactive membrane-anchored proteins that in response to a specific cellular stimulus undergo proteolytic cleavage, termed ectodomain shedding, to release the particular mature growth factor [18] [21].
The cleavage of EGFR ligands occurs with the involvement of membrane-anchored family of disintegrinmetalloproteases (ADAMs), which show preference with respect to stimulus response and exhibit various degree of substrate specificity [18].Among the metalloproteinases that respond to LPS stimulation, the primary role is assigned to TNF-α converting enzyme (TACE), also known as ADAM17 [12] [16] [22].Indeed, studies show that LPS-induced TACE activation leads to the enhancement in TGF-α shedding and EGFR transactivation [12] [16].Furthermore, EGFR transactivation by TACE-dependent TGF-α shedding has been linked to LPS-induced TLR4 signaling through p38 MAPK [12] [21], and there are strong indications for the involvement of Rac1 GTPase in the activation process [23] [24].
As oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis, are characterized by a massive rise in proinflammatory cytokine production, up-regulation in MAPK and Rac1 activation, and the induction in EGFR phosphorylation [2] [11] [19] [25], in this study, we examine the nature of factors associated with P. gingivalis LPS-induced amplification in salivary gland acinar cell EGFR transactivation.Our results demonstrate that stimulation of the acinar cells with the LPS leads to the enhancement in TACE-dependent shedding of TGF-α and the increase in EGFR activation.We also show that the LPS-induced TACE activation occurs through phosphorylation by p38 MAPK and requires Rac1 participation.

Salivary Gland Cell Incubation
The acinar cells of rat sublingual salivary gland were suspended in five volumes of ice-cold Dulbecco's modified (Gibco) Eagle's minimal essential medium (DMEM), supplemented with fungizone (50 µg/ml), penicillin (50 U/ml), streptomycin (50 µg/ml), and 10% fetal calf serum, and gently dispersed by trituration with a syringe and settled by centrifugation [25].The cells were then resuspended in the medium to a concentration of 2 × 10 7 cell/ml, and transferred in 1 ml aliquots to DMEM in culture dishes and incubated under 95% O 2 and 5% CO 2 at 37˚C for up to 4 h in the presence of 0 -100 ng/ml P. gingivalis LPS [25].P. gingivalis used for LPS preparation was cultured from clinical isolates obtained from ATCC No. 33277 [6].In the experiments evaluating the effect of p38 MAPK inhibitor, SB202190, ERK inhibitor, P98059, JNK inhibitor, SP600125, and TACE (TNF-α converting enzyme) inhibitor, TAPI-1 (Calbiochem), Rac1 inhibitor, NSC 23766, and EGFR inhibitor, AG1478 (Sigma), the cells were first preincubated for 30 min with the indicated dose of the agent or vehicle before the addition of the LPS.

M. TGF-α Shedding Assay
The quantification of TGF-α release by the acinar cells were carried out with TGF-α ELISA Kit (EMD Millipore).The cells were incubated with P. gingivalis LPS at 100 ng/ml for 4 h, and the supernatant was collected and concentrated.In the experiments on the effect of MAPK and TACE inhibitors, the cells prior to the addition of the LPS were first preincubated with the indicated dose of the agent or the vehicle.To prevent soluble TGF-α binding to EGFR, the cells before the addition of the LPS were also incubated with 2 µg/ml anti-EGFR neutralizing antibody.Following centrifugation at 5000 rpm for 10 min, the level of TGF-α in concentrated cell supernatants was measured by following the manufacturer's (EMD Millipore) instructions.

TACE Activity Assay
TACE activity measurement in the acinar cells of sublingual salivary gland was conducted with InnoZyme TACE Activity Kit (EMD Millipore).The cells from the control and experimental treatments were rinsed with 0.05 M phosphate buffer/saline, pH 7.4, settled by centrifugation, and suspended for 30 min at 4˚C in magnesium lysis buffer (MLB).Following sonication (3 × 10 sec pulses), the samples were centrifuged at 12,000 × g for 10 min and the resulting supernatant was subjected to protein determination using BCA protein assay kit (Pierce).The samples from various experimental treatments were added to the wells pre-coated with anti-TACE antibody, and the activity of captured TACE enzyme was measured using an internally quenched fluorescent peptide substrate after 10 min of incubation at room temperature.

Cell Membrane Preparation
To assess membrane translocation of Rac1 and p38 in response to P. gingivalis LPS, the sublingual salivary gland acinar cells from the control and experimental treatments were subjected to cell membrane preparation.The cells were homogenized for 10 s at 600 rpm in 3 volumes of 50 mM Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose, 25 mM magnesium acetate, 1 mM EDTA, 1 mM dithiothreitol, 10 mM aprotinin, 10 mM leupeptin, 10 mM chymostatin, and 1 mM PMSF [26].The lysate was centrifuged at 5000 × g for 15 min, the supernatant was diluted with two volumes of cold homogenization buffer and centrifuged at 10,000 × g for 20 min.The resulting supernatant was then subjected to centrifugation at 100,000 × g for 1 h at 4˚C, and the obtained membrane pellet was suspended in the extraction buffer, containing 20 mM HEPES, pH 7.9, 25% glycerol, 0.4 M NaCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, and 1 mM PMSF.After 30 min of incubation at 4˚C, the suspension was centrifuged at 15,000 × g for 15 min, and the supernatant containing solubilized membrane fraction was collected and stored at −70˚C until use.Protein content of the prepared membrane fraction was analyzed using BCA protein assay kit (Pierce).

Data Analysis
All experiments were carried out using duplicate sampling, and the results are expressed as means ± SD.Analy-sis of variance (ANOVA) and nonparametric Kruskal-Wallis tests were used to determine significance.Any difference detected was evaluated by means of post hoc Bonferroni test, and the significance level was set at P < 0.05.

Results
Taking into account recent evidence as to the role of LPS in pro-inflammatory stimulus propagation through the processes associated with EGFR transactivation [12] [15] [16], we investigated the factors involved in the release of EGFR ligand, TGF-α, by salivary gland acinar cells in response to LPS of periodontopathic bacterium, P. gingivalis.The results revealed that exposure of rat sublingual salivary gland cells to incubation with the LPS elicited a significant induction in MAPKs activation, as evidence by a marked increase in p38, ERK, and JNK phosphorylation (Figure 1).Moreover, we found that the effect of the LPS was also reflected in a massive induction in the release of soluble TGF-α ligand (Figure 2).Furthermore, following the literature indication as to the involvement of MAPK in EGFR transactivation [21], we examined the effect of EGFR kinase inhibitor, AG1478, on the LPS-induced MAPK phosphorylation.As shown in Figure 1, pretreatment of the acinar cells with AG1478 led to a substantial suppression of the LPS-induced ERK phosphorylation, while the extent of p38 and JNK phosphorylation remained essentially unaffected.Further, we found that the LPS-elicited induction in TGF-α release was susceptible to suppression by p38 inhibitor, SB202190, as well as the inhibitor of TACE,  TAPI-1, while the inhibitors of ERK (PD98059) and JNK (SP600125) had no effect (Figure 2).These results, while attesting to the role of EGFR transactivation in the mediation of LPS-induced ERK activation, strongly suggest that p38 along with TACE are involved in TGF-α ectodomain shedding induced by P. gingivalis LPS.
In further assessment of the pathways involved in P. gingivalis LPS-induced EGFR activation via TGF-α ligand shedding, we examined the acinar cell EGFR tyrosine phosphorylation in the presence of neutralizing antibodies to the EGF and TGF-α, as well as the inhibitors of p38 and TACE (Figure 3).Western blot analysis revealed that pretreatment of the cells with an antibody to TFG-α led to a profound inhibition in the LPS-induced EGFR phosphorylation, whereas preincubation with an EGF-neutralizing antibody produced less apparent effect on the LPS-induced EGFR phosphorylation.This indicates that TGF-α plays a major role in the acinar cell EGFR activation in response to P. gingivalis LPS.The phosphorylation of EGFR by the LPS, moreover, was susceptible to suppression by p38 inhibitor, SB202190, as well as the inhibitor of TACE, TAPI-1 (Figure 3), thus attesting to the involvement of p38 and TACE in the TGF-α ligand release.
Accordingly, to ascertain the nature of the relationship between p38 and TACE, we examined the impact of the LPS on TACE activity in the presence of p38 MAPK inhibition.The results revealed that the effect of the LPS was manifested by a significant increase in the acinar cell TACE activity, whereas preincubation with p38 inhibitor, SB202190, elicited a marked reduction in the LPS-elicited effect (Figure 4).Moreover, the LPSinduced TACE activity was also subject to suppression by Rac1 inhibitor, NSC23766, thus suggesting that TACE activation shows dependence on Rac1 GTPase.Hence, to assess the character of the cross talk between p38 MAPK and Rac1 leading to the LPS-induced TACE activation, we investigated the requirement and selectivity of the interactions by co-immunoprecipitation.The results revealed that while TACE did not co-precipitate with p38 in the absence of the LPS stimulation, the two proteins were found in complex in both p38 (Figure 5) and TACE (Figure 6) immunoprecipitates following the acinar cell incubation with the LPS.Moreover, we found that association between the p38 and TACE was subject to interference by p38 inhibitor, SB202190, which not only blocked the LPS-induced p38 phosphorylation (Figure 5), but also interfered with the LPSinduced phosphorylation of TACE on Thr 735 , which is the reflection of TACE activation (Figure 6).Furthermore, the LPS-induced association between p38 and TACE was blocked by the inhibitor of Rac1, NSC23766, which interfered with the interaction between phosphorylated p38 and the TACE (Figure 5), as well as TACE phosphorylation (Figure 6).Together, these data suggest that the LPS-induced process of TACE activation by p38 MAPK requires Rac1 GTPase participation.
In further assessment of the role of Rac1 in P. gingivalis LPS-induced TACE activation in the acinar cells, we examined the requirement and selectivity of the membrane translocation of p38 MAPK and Rac1.The results of Western blot analysis of the whole cell lysates as well as the cell membrane fraction, using anti-p38 and anti-Rac1 antibody, revealed that incubation with the LPS resulted in recruitment of both p38 and Rac1 to the   The cells, preincubated with 20 µM SB202190 (SB), or 50 µM NSC23766 (NSC), were treated with the LPS at100 ng/ml and incubated for 1 h.Cell lysates were immunoprecipitated (IP) with anti-p38 antibody and immunoblotted (WB) with anti-p38 and anti-TACE antibody (a).The p38 immunoblots were also reblotted with anti-phospho-p38 antibody (pp38), and the relative densities of proteins (b) are expressed as fold of p38 control.The data represent the means ± SD of four separate experiments.* P < 0.05 compared with that of control.** P < 0.05 compared with that of LPS.membrane fraction (Figure 7).Further, we found that blocking p38 activation with SB202190, while affecting the membrane localization of p38 had no effect on the LPS-induced membrane recruitment of Rac1.On the other hand, blocking the Rac1 activation with NSC23766 led to a marked suppression in the membrane translocation of Rac1 as well as p38.Hence, we surmised that the recruitment of p38 for TACE activation by P. gingivalis LPS is dependent upon the membrane colocalization with Rac1.

Discussion
Oral mucosal responses to periodontopathic bacterium, P. gingivalis and its key endotoxin, LPS, are characterized by a massive rise in proinflammatory cytokine production, disturbances in cyclooxygenase (COX) and nitric oxide synthase (NOS) isozyme systems, and up-regulation in MAPK activation [2] [3] [6] [27].Moreover, there are strong indications that LPS-induced proinflammatory signal propagation is also associated with EGFR transactivation [12]- [15].Indeed, studies show that LPS-induced EGFR transactivation is an essential part of the inflammatory and repair process, and involves TACE-dependent release of inflammatory regulators, including EGFR ligand, TGF-α [12] [16].As recent literature evidence has linked the course of TACE induction to LPSelicited MAPK phosphorylation and Rac GTPase activation [12] [21], in the present study we investigated the nature of factors involved in P. gingivalis LPS-induced EGFR transactivation in salivary gland acinar cells.
Our data revealed that the exposure of the acinar cells to incubation with P. gingivalis LPS elicited a significant increase in ERK, JNK, and p38 MAPK phosphorylation, as well as produced a massive increase in the soluble TGF-α ligand release.However, while the LPS-induced ERK phosphorylation was prone to suppression by EGFR kinase inhibitor, AG1478, the extent of p38 and JNK phosphorylation remained essentially unaffected.Further, the LPS-induced increase in soluble TGF-α release was susceptible to inhibition by p38 inhibitor, The cells, preincubated with 20 µM SB202190 (SB), or 50 µM NSC23766 (NSC), were treated with the LPS at 100 ng/ml and incubated for 1 h.Cell lysates were immunoprecipitated (IP) with anti-TACE antibody and immunoblotted (WB) with anti-TACE and anti-p38 antibody (a).The TACE immunoblots were also reblotted with anti-phospho-TACE antibody (pTACE), and the relative densities of proteins (b) are expressed as fold of TACE control.The data represent the means ± SD of four separate experiments.* P < 0.05 compared with that of control.** P < 0.05 compared with that of LPS.SB202190, as well as the inhibitor of TACE, TAPI-1, whereas the inhibitors of ERK and JNK had no effect on the extent of TGF-α release.Moreover, examination of the acinar cell EGFR phosphorylation in the presence of neutralizing antibodies to the TGF-α and EGF revealed that P. gingivalis LPS-induced acinar cell EGFR transactivation occurs mainly in response to up-regulation in TGF-α ligand release.Indeed, the literature data indicate that TGF-α also plays the major role in LPS-induced EGFR phosphorylation in biliary epithelial cells [12] [16], and that metalloprotease, TACE, mediates ectodomian shedding of inflammatory cytokines and TGF-α family ligands [12] [16] [21] [28].Moreover, our finding that P. gingivalis LPS-induced EGFR phosphorylation was susceptible to suppression by p38 inhibitor, SB202190 as well as the inhibitor of TACE, TAPI-1, attests to the involvement of p38 MAPK and TACE in the TGF-α ligand shedding.
Therefore, considering the evidence as to the involvement of Rac GTPase in p38 activation [23] [24] [29], we ascertained further the nature of the relationship between p38 MAPK and TACE in mediation of TGF-α release in response to P. gingivalis in the presence of p38 and Rac Inhibition.We observed a profound inhibitory effect on of the LPS-induced TACE activity not only in the presence of p38 inhibitor, SB202190, but also with the inhibitor of Rac1, NSC23766, thus pointing to the existence of crosstalk between Rac1 and p38 in TACE activation.This assertion is further supported by the results of co-immunoprecipitation analysis, in which the lysates of the acinar cells were immunoprecipitated with anti-p38 or TACE antibody, and subjected to probing with antiphospho-p38 and anti-TACE, or anti-p38 and anti-phospho-TACE antibody.The results revealed that the association between the p38 and TACE was subject to interference by p38 inhibitor, SB202190, as well as the inhibitor of Rac1, NSC23766.Moreover, the inhibitor of p38, SB203190, not only blocked the p38 phosphorylation,  but also interfered with the LPS-elicited TACE phosphorylation on Thr 735 , which is considered the target of p38induced TACE activation [9] [21].On the other hand, the inhibition of Rac1 with NSC23766, was found to interfere with the interaction between phosphorylated p38 and the TACE, as well as TACE phosphorylation.The fact that the activation of TACE by the LPS was susceptible to suppression by Rac1 inhibition led us to conclude that Rac1-GTP may be required for p38 localization with the membrane-anchored TACE for its activation.Indeed, studies show that in the absence of specific localization partners, the activated p38 preferentially accumulates in the cytosol [9].
Hence, taking into account that Rac proteins undergo regulatory control by GTP binding and membrane translocation for activation and hydrolysis to GDP for inactivation [30] [31], we evaluated the influence of P. gingivalis LPS on membrane recruitment of Rac1 and p38 translocation.Western blot analysis revealed that the effect of the LPS was reflected in the elevation in membrane recruitment of both p38 and Rac1, whereas inhibition of the p38 activation with SB202190 while affecting p38 translocation had no effect on the LPS-induced membrane translocation of Rac1.However, blocking the Rac1 activation with NSC23766 produced a marked suppression in the membrane translocation of Rac1 as well as p38.Therefore, we surmised that up-regulation by P. gingivalis LPS in Rac1 membrane translocation plays a major role in recruitment of the activated p38 to the cytosolic aspect of the membrane for TACE activation through phosphorylation of its cytoplasmic domain on Thr 735 .This assertion is supported by the findings demonstrating that membrane proximity of Rac1 is also essential for the recruitment and the enhancement in PLCγ2 activation [25] [32].
Although the detailed functional paradigm of the role of Rac1 in p38 membrane recruitment and TACE acti- vation requires further defining, our findings are consistent with the with the studies suggesting that Rac GTPases function as sophisticated modulators of a remarkably complex and diverse range of cellular processes, including regulation of p38 expression, PLCγ2 membrane localization, and metalloproteinase activation [9] [25] [28] [30]- [34].

Conclusion
The data presented in this report reveal that stimulation of salivary gland acinar cells with P. gingivalis LPS leads to p38 MAPK-dependent increase in TGF-α ligand shedding and EGFR transactivation.We further show that the LPS-induced TGF-α ectodomain shedding and TGFR transactivation involve the activation of membrane-anchored TACE through phosphorylation by p38 that requires Rac1 participation (Figure 8).Moreover, we demonstrate that the LPS-elicited Rac1 membrane translocation serves as an essential platform for the localization of p38 with the TACE, its activation through phosphorylation on Thr 735 , and subsequent increase in TGF-α ectodomain shedding for EGFR transactivation.

Figure 1 .
Figure 1.Effect of EGFR tyrosine kinase inhibitor, AG1478 (AG), on P. gingivalis LPS-induced changes in sublingual salivary gland acinar cell MAPK phosphorylation.The cells were preincubated with 0 or 5 µM of AG and incubated for 30 min with the LPS at 100 ng/ml.Cell lysates were analyzed by Western blotting for total and phosphorylated MAPKs (a), and the relative densities of phosphorylated MAPK proteins (b) are expressed as fold of control.Actin blot shows equal lane load.The data represent the mean ± SD of four experiments.* P < 0.05 compared with that of control.** P < 0.05 compared with that of LPS.

Figure 3 .
Figure 3.Effect of p38 and TACE inhibitors on P. gingivalis LPS-induced EGFR activation in sublingual salivary gland acinar cells.The cells, preincubated with 20 µM of p38 inhibitor, SB202190 (SB), or 10 µM TACE inhibitor, TAPI-1 (TAPI), or 4 µg/ml neutralizing antibody against TGF-α (TGFab), or 4 µg/ml neutralizing antibody against EGF (EGFab), were treated with the LPS at 100 ng/ml and incubated 10 min.Cell lysates were analyzed by Western blotting for total and phosphorylated EGFR (a).The relative density of pEGFR protein is expressed as fold of control (b), and the total EGFR was used as loading control.The data represent the means ± SD of four separate experiments.* P < 0.05 compared with that of control.** P < 0.05 compared with that of LPS.

Figure 4 .
Figure 4. Effect of p38 and Rac1 inhibitors on P. gingivalis LPS-induced changes in sublingual salivary gland acinar cell expression of TACE activity.The cells, preincubated with 20 µM p38 inhibitor, SB202190 (SB) or 50 µM Rac1 inhibitor, NSC23766 (NSC), were treated with the LPS at 100 ng/ml and incubated for 10 min.Values represent the means ± SD of four experiments.* P < 0.05 compared with that of control.** P < 0.05 compared with that of LPS.

Figure 5 .
Figure 5. Impact of p38 and Rac1 inhibition on the changes induced by P. gingivalis LPS in sublingual salivary gland acinar cell p38 phosphorylation and its association with TACE.The cells, preincubated with 20 µM SB202190 (SB), or 50 µM NSC23766 (NSC), were treated with the LPS at100 ng/ml and incubated for 1 h.Cell lysates were immunoprecipitated (IP) with anti-p38 antibody and immunoblotted (WB) with anti-p38 and anti-TACE antibody (a).The p38 immunoblots were also reblotted with anti-phospho-p38 antibody (pp38), and the relative densities of proteins (b) are expressed as fold of p38 control.The data represent the means ± SD of four separate experiments.* P < 0.05 compared with that of control.** P < 0.05 compared with that of LPS.

Figure 6 .
Figure 6.Effect of p38 and Rac1 inhibition on the changes induced by P. gingivalis LPS in sublingual salivary gland acinar cell TACE phosphorylation and its association with p38.The cells, preincubated with 20 µM SB202190 (SB), or 50 µM NSC23766 (NSC), were treated with the LPS at 100 ng/ml and incubated for 1 h.Cell lysates were immunoprecipitated (IP) with anti-TACE antibody and immunoblotted (WB) with anti-TACE and anti-p38 antibody (a).The TACE immunoblots were also reblotted with anti-phospho-TACE antibody (pTACE), and the relative densities of proteins (b) are expressed as fold of TACE control.The data represent the means ± SD of four separate experiments.* P < 0.05 compared with that of control.** P < 0.05 compared with that of LPS.

Figure 7 .
Figure 7. Impact of Rac and p38 inhibitors on the changes induced by P. gingivalis LPS in membrane recruitment of Rac1 and p38.The sublingual salivary gland acinar cells were treated with the LPS at 100 ng/ml in the presence of Rac1 inhibitor, NSC-23766 (NSC) at 50 µM, or p38 inhibitor, SB202190 (SB) at 50 µM, and incubated for 2 h.The lysates of whole cells (T) and the corresponding membrane (M) fractions were analyzed for p38 and Rac1 with specific antibodies (a).The relative densities of proteins are expressed as fold of control (b), and the total (T) p38 and Rac1 were used as loading control.The data represent the means ± SD of four separate experiments.* P < 0.05 compared with that of control.** P < 0.05 compared with that of LPS.

Figure 8 .
Figure 8. Schematic representation of the proposed signaling mechanism leading to P. gingivalis LPS-induced EGFR transactivation by TGF-α.Ligation by the LPS of salivary gland acinar cell TLR4 triggers up-regulation in p38 MAPK phosphorylation, as well as prompts the activation and translocation of Rac1-GTP to the vicinity of membrane-anchored latent TACE.This, in turn, leads to the recruitment and localization of p38 to the complex, and TACE activation through phosphorylation.The subsequent cleavage by the TACE of pro-TGF-α results in a soluble TGF-α ligand that binds to and activates EGFR, thus causing the increased signaling to ERK/MAPK.