Gamma-interferon enhances expression of CD14/MyD88 and subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts

OBJECTIVES: CD14, toll-like receptor 4 (TLR4) and MyD88 have been shown to mediate responsiveness in host cells to lipopolysaccharide. We investigated here the regulatory effects of inflammatory cytokines on the expression of membrane CD14 (mCD14), TLR4 and MyD88, and on subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts. MATERIALS AND METHODS: Following treatment with either interleukin-1beta, tumor necrosis factor-alpha (TNF-alpha) or gamma-interferon (IFN-gamma), expression of mCD14/TLR4 and MyD88 was determined by flow cytometry and western blotting, respectively. After pretreatment with IFN-gamma, cells were pre-incubated with either anti-CD14 antibody MY4 or anti-TLR4 antibody HTA125 and subsequently treated with A. actinomycetemcomitans lipopolysaccharide. Then, phosphorylation of mitogen-activated protein (MAP) kinases and IkappaBalpha was examined by western blotting, and production of interleukin-6 and interleukin-8 was measured by their respective enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: IFN-gamma stimulated expression of mCD14, whereas -1beta and TNF-alpha did not. Expression of MyD88 but not TLR4 was also enhanced by IFN-gamma. The lipopolysaccharide activated MAP kinases, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, and IkappaBalpha and stimulated production of interleukin-6 and interleukin-8. The lipopolysaccharide-stimulated interleukin-6 and interleukin-8 production was markedly inhibited by MY4 or HTA125. Pretreatment with IFN-gamma augmented the following activation of MAP kinases and IkappaBalpha and production of interleukin-6 and interleukin-8 in response to the lipopolysaccharide. CONCLUSIONS: These results suggest that the augmentation by IFN-gamma of the responsiveness to A. actinomycetemcomitans lipopolysaccharide, such as activation of MAP kinases and IkappaBalpha and terminal cytokine production in human gingival fibroblasts, may be partially mediated by up-regulation of CD14 and MyD88 expression.     

Cigarette smoke condensate stimulates urokinase production through the generation of reactive oxygen species and activation of the mitogen activated protein kinase pathways in human gingival fibroblasts

Background and Objective:  Tobacco smoking is a significant risk factor for periodontal disease. It has been suggested that smoking may alter connective tissue remodeling in the periodontium. In the present study, we investigated whether cigarette smoke condensate modulates the production of the serine protease urokinase in human gingival fibroblasts.
Material and Methods:  Primary cultures of human gingival fibroblasts were stimulated with cigarette smoke condensate. Urokinase production was evaluated through casein zymography and western blotting. Plasmin activation was assessed by means of a radial diffusion assay. The roles of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and reactive oxygen species in cigarette smoke condensate-stimulated urokinase production were studied using distinct selective inhibitors (SP600125, PD98059, N -acetyl cysteine). Reactive oxygen species production was determined using a fluorometric assay. Activation of ERK and JNK pathways were evaluated using western blots.
Results:  In gingival fibroblasts, cigarette smoke condensate potently stimulated urokinase production and plasmin activation. Cigarette smoke condensate-stimulated urokinase production was dependent on the activity of ERK/JNK pathways and was inhibited by the reactive oxygen species scavenger, N -acetyl cysteine. Cigarette smoke condensate strongly stimulated ERK and JNK phosphorylation and the generation of reactive oxygen species.
Conclusion:  Cigarette smoke condensate stimulates urokinase production and plasmin activation in gingival fibroblasts. Moreover, cigarette smoke condensate-stimulated urokinase production depends on both the activation of ERK/JNK pathways and on the generation of intracellular reactive oxygen species. These results show that cigarette smoke may alter connective tissue remodeling by inducing production of the urokinase-type plasminogen activator through specific signaling pathways.     

Epidermal growth factor synergistically enhances interleukin-8 production in human gingival fibroblasts stimulated with interleukin-1beta

The chemokine interleukin-8 (IL-8) has been implicated in inflammatory diseases including periodontitis. In this study the effect of epidermal growth factor (EGF) on the production and regulation of interleukin-8 (IL-8) in human gingival fibroblasts challenged with interleukin-1beta (IL-1beta) was investigated. EGF, in comparison to the effect of IL-1beta, weakly increased the mRNA and protein expression of IL-8 in gingival fibroblasts. When the cells were treated simultaneously with EGF and IL-1beta, however, EGF synergistically enhanced the mRNA expression and production of IL-8. The stimulatory effect of EGF on IL-1beta-induced IL-8 production was completely abolished by the broad range tyrosine kinase inhibitor Herbimycin A, and considerably reduced by the receptor tyrosine kinase specific inhibitor PD 153035. Herbimycin A abolished IL-8 production induced by IL-1beta, whereas PD 153035 had no effect on the cytokine-induced IL-8 production. Furthermore, the p38 mitogen-activated protein (MAP) kinase inhibitor SB 203580 reduced IL-8 production induced by IL-1beta as well as by the combination of EGF and IL-1beta but had no effect on EGF-induced IL-8 production. In conclusion, the study demonstrates that EGF synergistically stimulates IL-8 production in the presence of IL-1beta and that tyrosine kinase(s) seem to be involved in the signalling pathway of IL-1beta and EGF. The synergistic interactions between EGF and IL-1beta on IL-8 production may play an essential role in the pathogenesis of the inflammatory disease periodontitis.     

Effects of cytokines on gingival fibroblasts in vitro are modulated by the extracellular matrix

Fibroblast function in gingival tissue is thought to be regulated by the local cellular environment – both the extracellular matrix and soluble factors. In an attempt to artificially re-create this situation fibroblasts have been cultured within 3-dimensional collagen gels in an environment more physiologically comparable to connective tissue. Using such a model we investigated the effects of the extracellular matrix on gingival fibroblast growth and synthetic activity and on the cellular responsiveness to 4 soluble factors – epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF- β ₁) and interleukin-1 β (IL-1 β ). Fibroblasts cultured within collagen gels showed similar growth rates, an increased production of collagen but reduced levels of hyaluronan synthesis in comparison to cells in monolayer culture. Cellular responsiveness to soluble mediators was also modulated by the collagen matrix, with a generalised reduction in response by cells embedded within the matrix. The stimulatory effects of EGF and PDGF on cell growth in monolayer over a 14-day period were only found during the initial stages of culture within gels. Similarly the stimulation of matrix production by cells induced by TGF- β ₁, on plastic was reduced or even negated when cells were cultured in collagen gels. On plastic IL-1 β significantly stimulated cell growth but had no effect on either collagen or hyaluronan production by fibroblasts. In gel cultures, this cytokine had no effect on cell proliferation, but significantly inhibited both collagen and hyaluronan synthesis. These findings further illustrate the usefulness of fibroblast-populated collagen gels as a model system for studying the modulatory effects of soluble factors and extracellular matrix molecules on fibroblast function in vitro .     

Possible involvement of extracellular signal-regulated kinases 1/2 in mitogenic response of periodontal ligament cells to enamel matrix derivative

The efficacy of enamel matrix derivative (EMD) as an adjunct to periodontal regenerative therapy has been demonstrated in recent clinical studies, however, little is known about its molecular mechanism (s). We examined the mitogenic response of cultured periodontal ligament (PDL) cells to EMD and characterized associated changes in proliferation-related intracellular signaling molecules, including mitogen-activated protein kinases (MAPK) and Akt kinases/protein kinase B (Akt/PKB) kinases. The DNA synthesis of PDL cells increased following treatment with EMD at concentrations higher than 1 microg ml(-1). This mitogenic response to EMD was associated with the selective activation of extracellular signal-regulated kinase (ERK) 1/2. No other MAPKs, or Akt/PKB kinases, responded to EMD stimulation. The EMD induction of DNA synthesis and activation of ERK 1/2 were diminished by pretreatment with suramin, an inhibitor of receptor tyrosine kinases (RTK). The signaling pathway induced by EMD from RTK to ERK 1/2 was similar to that activated by epidermal growth factor (EGF), although the specific binding of 125I-EGF to PDL cells was not affected by pretreatment or concomitant treatment with EMD. These findings suggest that EMD elicits its mitogenic signal through an EMD-specific RTK towards ERK 1/2.     

Effects of nicotine on apoptosis in human gingival fibroblasts

Aim

Cigarette smoke is a complex mixture of more than 4700 chemical compounds including free radicals and oxidants and it is a world widely known problem to health. Nicotine is the major compound of tobacco and known as the cause of gingivitis and periodontitis. It induces intracellular oxidative stress recognized as the important agent in the damage of biological molecules. The aim of this study is to clarify the cytotoxic pathway of nicotine in human gingival fibroblasts (HGFs).

Methods

Human gingival fibroblasts stimulated by nicotine were used as an in vitro model. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cell viability and reactive oxygen species (ROS) generation was assessed with 2,7-dichlorofluoroscein diacetate (DCF-DA). Morphological change was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay, stained with 4,6-diamidino-2-phenylindole (DAPI). To delineate the roles of extracellular signal-regulated kinase (ERK), P38 and c-Jun N-terminal kinase (JNK), Western blot and caspase-3 (CASP3) activity assay were performed.

Results

Exposure of the human gingival fibroblasts to nicotine reduced cell viability by time and dose dependent and increased the generation of ROS. It also showed morphological evidence of increased apoptosis, resulted in transient activation of JNK and ERK concomitant with activation of P38, and stimulated apoptosis as evidenced by CASP3 activation and Poly ADP ribose polymerase (PARP) cleavage.

Conclusion

These results suggest that nicotine induces apoptosis through the ROS generation and CASP3 dependent pathways in HGFs.     

Proliferation of dental pulp fibroblasts in response to thrombin involves mitogen-activated protein kinase signalling

AIM: To examine the involvement of mitogen-activated protein kinases (MAPK) signalling on thrombin-stimulated human dental pulp fibroblasts (DPF). METHODOLOGY: Dental pulp fibroblasts were isolated from dental pulp connective tissue of third molars and expanded in vitro. Expression of thrombin receptors was analysed by RT-PCR, and cell proliferation was measured by 3[H]-thymidine incorporation assay. Phosphorylation levels of MAPK were determined by Western blot analysis, and alkaline phosphatase activity was measured to serve as a marker for odontogenic differentiation. Statistical analysis was performed by Student's t-test. RESULTS: Dental pulp fibroblasts express the thrombin receptors protease-activated receptor-1 (PAR-1), PAR-3 and PAR-4. Measurement of 3[H]-thymidine incorporation revealed a dose-dependent increase of DNA synthesis in response to thrombin treatment. The thrombin-induced mitogenic activity was decreased by the extracellular signal-regulated protein kinase (ERK) signalling inhibitor PD98059 (P < 0.05), and by SB203580 (P < 0.05), a p38 MAPK inhibitor. Western blot analysis demonstrated increased phosphorylation of ERK in DPF following stimulation with thrombin, while p38 MAPK and c-Jun NH2-terminal kinase (JNK) were not activated. Alkaline phosphatase activity of DPF remained unchanged upon incubation with thrombin. CONCLUSIONS: These results suggest that signalling via MAPK mediates the mitogenic activity of thrombin on DPF and may thus play a role during the early stages of pulp repair.     

Signaling pathways regulating IL-1alpha-induced COX-2 expression

Interleukin-1alpha(IL-1alpha) stimulates the production of prostaglandin E(2) (PGE(2)) in odontogenic keratocyst fibroblasts. However, the signaling pathways remain obscure. In this study, we investigated IL-1alphasignaling pathways that regulate cyclooxygenase-2 (COX-2) expression in odontogenic keratocyst fibroblasts. IL-1alphaincreased the expression of COX-2 mRNA and protein, and PGE(2) secretion in the fibroblasts. IL-1alphaincreased the phosphorylation of extracellular signal-regulated protein kinase-1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). PD-98059, SB-203580, SP-600125, and PDTC-which are inhibitors of ERK1/2, p38, JNK, and nuclear factor-kappaB (NF-kappaB), respectively-attenuated the IL-1alpha-induced COX-2 mRNA expression and activated protein kinase C PGE(2) secretion. IL-1alpha(PKC), and PKC inhibitor staurosporine inhibited IL-1alpha-induced phosphorylation of ERK1/2, p38, and JNK, and decreased IL-1alpha-induced COX-2 mRNA expression. Thus, in odontogenic keratocyst fibroblasts, IL-1alphamay stimulate COX-2 expression both through the PKC-dependent activation of ERK1/2, p38, and JNK signaling pathways, and through the NF-kappaB cascade.     

Signal pathways involved in the production of MMP-1 and MMP-3 in human gingival fibroblasts

Periodontitis is associated with enhanced production of cytokines, prostaglandins and matrix metalloproteinases (MMPs). The aim of this study was to investigate the production and regulation of MMP-1 and MMP-3 in human gingival fibroblasts challenged with the cytokines interleukin-lbeta (IL-1beta), tumor necrosis factor alpha (TNFalpha) or epidermal growth factor (EGF). The results showed that gingival fibroblasts constitutively produce MMP-1 and MMP-3, and that the cytokines IL-1beta, TNFalpha and EGF increase both MMP-1 and MMP-3 production in gingival fibroblasts. The upregulation by the cytokines was apparent at 8 h of incubation and increased thereafter continuously during 48 h of incubation. The upregulation of MMPs, induced by IL-1beta or TNFalpha, was reduced by the cyxlooxygenase-2 (COX-2) inhibitor NS-398, the p38 MAP-kinase inhibitor SB 203580, and the tyrosine kinase inhibitor herbimycin A. In addition, MMP-1 and MMP-3 production, induced by IL-1beta, TNFalpha or EGF, was strongly reduced by the presence of the glucocorticoid dexamethasone. Our findings demonstrate that the cytokines IL-1beta, TNFalpha and EGF, respectively, enhance both MMP-1 and MMP-3 production in human gingival fibroblasts, and that the signal pathways COX-2, MAP-kinases and tyrosine kinases are partly involved in the production of MMPs.     

Enamel matrix derivative induces production of vascular endothelial cell growth factor in human gingival fibroblasts

Enamel matrix derivative (EMD) may enhance periodontal wound healing by inducing angiogenesis. We sought to investigate the effect and the mechanism of action of EMD on vascular endothelial growth factor (VEGF) production by human gingival fibroblasts. Cells were stimulated with EMD, transforming growth factor‐β1 (TGF‐β1), or fibroblast growth factor 2 (FGF‐2), with or without antibodies to TGF‐β1 or FGF‐2. The levels of VEGF in the culture media were measured using an ELISA. We examined the effects of SB203580 [a p38 mitogen‐activated protein kinase (MAPK) inhibitor], U0126 [an extracellular signal‐regulated kinase (ERK) inhibitor], SP600125 [a c‐Jun N‐terminal kinase (JNK) inhibitor], and LY294002 [a phosphatidylinositol 3‐kinase (PI3K)/Akt inhibitor] on EMD‐induced VEGF production. Enamel matrix derivative stimulated the production of VEGF in a dose‐ and time‐dependent manner. Treatment of human gingival fibroblasts with antibodies to TGF‐β1 or FGF‐2 significantly decreased EMD‐induced VEGF production, whereas the addition of exogenous TGF‐β1 and FGF‐2 stimulated VEGF production. Enamel matrix derivative‐induced VEGF production was significantly attenuated by SB203580, U0126, and LY294002. Our results suggest that EMD stimulates VEGF production partially via TGF‐β1 and FGF‐2 in human gingival fibroblasts and that EMD‐induced VEGF production is regulated by ERK, p38 MAPK, and PI3K/Akt pathways. Enamel matrix derivative‐induced production of VEGF by human gingival fibroblasts may be involved in the enhancement of periodontal wound healing by inducing angiogenesis.     

Down-regulation of epidermal growth factor receptor-dependent signaling by Porphyromonas gingivalis lipopolysaccharide in life-expanded human gingival fibroblasts

Background and Objective: Human gingival fibroblasts exhibit proliferative responses following epidermal growth factor exposure, which are thought to enhance periodontal regeneration in the absence of bacterial products such as lipopolysacharide. However, lipopolysaccharide challenge activates human gingival fibroblasts to release several inflammatory mediators that contribute to the immune response associated with periodontitis and attenuate wound repair. We tested the hypothesis that Porphyromonas gingivalis lipopolysaccharide‐activated signaling pathways down‐regulate epidermal growth factor receptor‐dependent events. Material and Methods: To study lipopolysaccharide/epidermal growth factor interactions in human gingival fibroblasts, we introduced the catalytic subunit of human telomerase into human gingival fibroblasts, thereby generating a more long‐lived cellular model. These cells were characterized and evaluated for lipopolysaccharide/epidermal growth factor responsiveness and regulation of epidermal growth factor‐dependent pathways. Results: Comparison of human telomerase‐transduced gingival fibroblasts with human gingival fibroblasts revealed that both cell lines exhibit a spindle‐like morphology and express similar levels of epidermal growth factor receptor, CD14 and Toll‐like receptors 2 and 4. Importantly, human telomerase‐transduced gingival fibroblasts proliferation rates are increased 5–9 fold over human gingival fibroblasts and exhibit a longer life span in culture. In addition, human telomerase‐transduced gingival fibroblasts and human gingival fibroblasts exhibit comparable profiles of mitogen‐activated protein kinase kinase (extracellular signal‐regulated kinase 1/2) activation upon epidermal growth factor or P. gingivalis lipopolysaccharide administration. Interestingly, treatment with P. gingivalis lipopolysaccharide leads to a down‐regulation of epidermal growth factor‐dependent extracellular signal‐regulated kinase 1/2, p38 and cyclic‐AMP response element binding protein phosphorylation in both cell types. Conclusion: These studies demonstrate that human telomerase‐transduced gingival fibroblasts exhibit an extended life span and recapitulate human gingival fibroblasts biology. Moreover, this system has allowed for the first demonstration of lipopolysaccharide down‐regulation of epidermal growth factor activated pathways in human gingival fibroblasts and should facilitate the analysis of signaling events relevant to the pathogenesis and treatment of periodontitis.     

Enamel matrix derivative (EMDOGAIN®) rapidly stimulates phosphorylation of the MAP kinase family and nuclear accumulation of smad2 in both oral epithelial and fibroblastic human cells

In our previous study, we demonstrated that porcine enamel matrix derivative (EMD) induces p21WAF1/cip1 within 8 hours and subsequently arrests the cell cycle of human oral epithelial cells in G1 phase. In contrast, EMD markedly stimulates the proliferation of gingival fibroblasts without inducing p21WAF1/cip1. To investigate the mechanism of how EMD produces these differential effects, we have focused on the initial response of these two cell types to EMD. In epithelial cell cultures, EMD stimulated cytoskeletal actin polymerization within 30 min and promoted cell adhesion in our experimental system. EMD failed to stimulate either intracellular Ca2+ mobilization or cAMP production in either cell type. In both epithelial and fibroblastic cells, EMD (25-100 microgram/ml) rapidly produced dose-dependent phosphorylation of the mitogen-activated protein kinase (MAPK) family: extracellular signal response kinase (ERK), p38-MAPK (p38-K), and c-Jun-terminal kinase/stress-activated protein kinase (JNK). However, neither inhibitors of MEK (ERK kinase) nor p38-K could block EMD's anti-proliferative action on epithelial cells. On the other hand, EMD rapidly stimulated translocation of smad2 into the nucleus in both cell types. Spurred by this finding, we assayed for TGF-beta1, a ligand for one receptor associated with smad2 activation, and detected significant levels in EMD preparations. The sum of these pharmacological findings indicates that EMD contains at least one bioactive factor, which is most probably TGF-beta1 (or TGF-beta-like substances). In conjunction with the similarities in the differential growth-modulating actions between EMD and what is known for TGF-beta, we suggest that TGF-beta might act as the principal growth regulating agent of oral fibroblastic and epithelial cell types in EMD despite being present in only low levels.     

p38 MAPK在LPS诱导牙龈成纤维细胞表达uPA中作用的研究

目的：研究p38 MAPK在LPS诱导牙龈成纤维细胞表达uPA中的作用。方法：采用Western blotting观察LPS对牙龈成纤维细胞内p38 MAPK活性的影响；蛋白激酶活性实验SB203580地p38 MAPK活性的抑制作用；Northern blotting观察SB203580对LPS诱导uPA表达的影响。结果：LPS能够迅速地激活牙龈成纤维细胞内p38 MAPK的活性；SB203580能够有效地抑制牙龈成纤维细胞内的p38 MAPK的活性；经SB203580处理后，LPS对uPA的诱导作用受到显著的抑制。结论：LPS通过p38 MAPK信号转导途径诱导牙龈成纤维细胞表达uPA。     

Effect of phenytoin on intracellular 45Ca2+ accumulation in gingival fibroblasts in vitro

Effect of 5,5 diphenylhydantoin (phenytoin; PHT) alone or in combination with epidermal growth factor (EGF) on the intracellular accumulation of the radioisotope 45Ca2+ (4 min labelling period) was determined in gingival fibroblasts. EGF as well as PHT increased the intracellular accumulation of the radioisotope in normal gingival fibroblasts by approximately 2 and 1.6-fold, respectively. In contrast, in fibroblasts derived from the phenytoin-induced gingival overgrowth, neither EGF nor PHT stimulated intracellular accumulation of 45Ca2+. When normal gingival fibroblasts were treated in vitro with EGF in combination with PHT, the EGF-induced increase in intracellular accumulation of the radioisotope 45Ca2+ was abolished. The rate of efflux of the radioisotope 45Ca2+ in prelabelled normal gingival fibroblasts was decreased by PHT treatment in vitro to a level already present in fibroblasts derived from PHT-induced gingival overgrowth. This study indicates that PHT influences the cellular calcium metabolism in fibroblasts which may contribute to the pathogenesis of gingival overgrowth.     

Regulation of extracellular signal-regulated protein kinase signaling in human osteosarcoma cells stimulated with nicotine

BACKGROUND: Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. Currently, there is limited information on the regulation of mitogen-activated protein kinases (MAPK) expression in smoking-associated periodontal disease. OBJECTIVES: The aim of the present study was to investigate the effects of nicotine on the expression of MAPKs in human osteosarcoma cell line U2OS cells. Furthermore, various pharmacological agents were added to search the possible regulation mechanisms on nicotine-induced MAPKs expression. METHODS: Cytotoxicity and western blot assays were used to investigate the effects of U2OS cells exposed to nicotine. In addition, various pharmacological agents [NS-398, dexamethasome, 2-oxothiazolidine-4-carboxylic acid (OTZ), herbimycin A, and curcumin] were added to test how they modulated the effects of nicotine-induced MAPKs expression. RESULTS: Concentrations of nicotine higher than 5 mm demonstrated cytotoxicity to U2OS cells (p<0.05). A nicotine concentration of 5 mm was found to induce extracellular signal-regulated kinase (ERK) phosphorylation in a time-dependent manner (p<0.05). In addition, amounts of ERK protein were unaffected by nicotine during the same time interval. By contrast, nicotine has no effect on either c-Jun N-terminal kinase (JNK) or p38, respectively. In addition, NS-398, dexamethasone, OTZ, herbimycin A, and curcumin were found to inhibit the nicotine-induced ERK expression (p<0.05). CONCLUSIONS: The activation of ERK expression by nicotine suggests a potential role for nicotine in the pathogenesis of cigarette smoking-associated periodontal disease. In addition, nicotine-induced ERK expression was down-regulated by NS-398, dexamethasone, OTZ, herbimycin A, and curcumin.     

Dental pulp fibroblasts contain target cells for lysophosphatidic Acid

Lysophosphatidic acid (LPA) is a locally produced bioactive phospholipid which is involved in tissue repair. The objective of this study was to determine whether dental pulp tissue also responds to the phospholipid. Effects of LPA on proliferation, differentiation, and mitogen-activated protein kinase (MAPK) signaling of dental pulp fibroblasts (DPF) were examined in vitro. We report that DPF express LPA receptors LPA1, LPA2, and LPA3 and respond to the ligand with increased mitogenic activity. Involvement of extracellular signal-regulated kinase, p38 MAPK, and c-Jun NH(2)-terminal kinase in LPA signaling could be demonstrated by use of specific inhibitors and detection of the phosphorylation status of the kinases. An increased mitogenic activity paralleled a decreased number of alkaline-phosphatase-positive cells and expression levels of dentin sialophosphoprotein and osteocalcin. Together, these results suggest that dental pulp fibroblasts can respond to LPA, a process that may play a role in pulp tissue repair.