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Dental Pulp Fibroblasts Contain Target Cells for Lysophosphatidic Acid

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					         Journal of Dental Research
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Dental Pulp Fibroblasts Contain Target Cells for Lysophosphatidic Acid
            R. Gruber, B. Kandler, C. Jindra, G. Watzak and G. Watzek
                            J DENT RES 2004 83: 491
                       DOI: 10.1177/154405910408300611

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                        http://jdr.sagepub.com/content/83/6/491


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                                          International and American Associations for Dental Research
 RESEARCH REPORTS
 Biological

R. Gruber1*,2, B. Kandler1, C. Jindra1,
G. Watzak1,2, and G. Watzek1,2                                           Dental Pulp Fibroblasts
1Department of Oral Surgery, Vienna Medical University,
Waehringerstraße 25a, A-1090 Vienna, Austria; and
                                                                         Contain Target Cells for
2Ludwig Boltzmann Institute of Oral Implantology, Vienna,

Austria; *corresponding author, reinhard.gruber@akh-                     Lysophosphatidic Acid
wien.ac.at

J Dent Res 83(6):491-495, 2004




ABSTRACT                                                                  INTRODUCTION
Lysophosphatidic acid (LPA) is a locally produced
bioactive phospholipid which is involved in tissue
repair. The objective of this study was to
                                                                         Dental large repair is of highlyDental pulpfactors (Smith and Lesot, 2001;a
                                                                            by a
                                                                                 pulp           a         coordinated process, which is orchestrated
                                                                                      number locally produced
                                                                         Goldberg and Smith, 2004).                  fibroblasts (DPF), although
determine whether dental pulp tissue also responds                        heterogeneous cell population, can serve as an in vitro model to identify
to the phospholipid. Effects of LPA on                                    molecules which may act as paracrine mediators at sites of pulp repair
proliferation, differentiation, and mitogen-                              (Goldberg and Smith, 2004). Using this in vitro model, investigators have
activated protein kinase (MAPK) signaling of                              found vascular endothelial growth factor (Matsushita et al., 2000), platelet-
dental pulp fibroblasts (DPF) were examined in                            derived growth factor (Denholm et al., 1998), thrombin (Chang et al.,
vitro. We report that DPF express LPA receptors                           1999), basic fibroblast growth factor (Shiba et al., 1995), and bone
LPA1, LPA2, and LPA3 and respond to the ligand                            morphogenetic protein (Lianjia et al., 1993) to be mitogenic for DPF and to
with increased mitogenic activity. Involvement of                         modulate the expression of odontoblastic differentiation markers such as
extracellular signal-regulated kinase, p38 MAPK,                          alkaline phosphatase, dentin sialophosphoprotein, and osteocalcin
and c-Jun NH2-terminal kinase in LPA signaling                            (Papagerakis et al., 2002). The majority of the factors involved in dental
could be demonstrated by use of specific                                  pulp repair have been determined to be proteins. However, the question of
inhibitors and detection of the phosphorylation                           whether pulp tissue contains target cells for bioactive lipids has not been
status of the kinases. An increased mitogenic                             investigated.
activity paralleled a decreased number of alkaline-                            Lysophosphatidic acid (LPA; 1-acyl-sn-glycerol-3-phosphate) is a
phosphatase-positive cells and expression levels of                       membrane-derived phospholipid that exerts its functions in an autocrine-
dentin sialophosphoprotein and osteocalcin.                               paracrine mode of action (Goetzl and An, 1998; Mills and Moolenaar,
Together, these results suggest that dental pulp                          2003). LPA is a pleiotropic molecule, which affects cell proliferation,
fibroblasts can respond to LPA, a process that may                        migration, differentiation, survival, and the production of local factors by
play a role in pulp tissue repair.                                        the target cells (Goetzl and An, 1998; Mills and Moolenaar, 2003). In
                                                                          particular, LPA has been reported to be mitogenic for various cell types
KEY WORDS: lysophosphatidic acid, dental pulp                             such as osteoblasts (Caverzasio et al., 2000), endothelial cells (Panetti et
fibroblasts, mitogen-activated protein kinase,                            al., 1997), and smooth-muscle cells (Ediger and Toews, 2001) and to
proliferation, differentiation.                                           modulate osteogenic differentiation (Dziak et al., 2003). Platelets, which
                                                                          are activated at the sites of injury, are a main source of LPA, suggesting
                                                                          that LPA may play a role in tissue repair (Eichholtz et al., 1993; Pages et
                                                                          al., 2001). Moreover, topical application of LPA can enhance wound
                                                                          healing (Balazs et al., 2001). The effects of LPA are mediated via the G
                                                                          protein-coupled receptors LPA1, LPA2, and LPA3 that activate different
                                                                          intracellular signaling pathways (Kranenburg and Moolenaar, 2001).
                                                                          Extracellular-regulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal
                                                                          kinase (JNK) belong to the family of mitogen-activated protein kinases
                                                                          (Chang and Karin, 2001), which can be activated by LPA (Kranenburg and
                                                                          Moolenaar, 2001; Baudhuin et al., 2002; Sorensen et al., 2003; Xu et al.,
                                                                          2003). Antibodies that recognize their phosphorylation status and the
                                                                          kinase-specific inhibitors U0126, SB203580, and SP600125 are tools to
                                                                          determine whether ERK, p38 MAPK, or JNK, respectively, is involved in
                                                                          LPA signaling.
                                                                               Here we investigated whether dental pulp tissue contains potential target
Received June 4, 2003; Last revision April 6, 2004;                       cells for LPA by measuring proliferation, differentiation, and MAPK
Accepted April 19, 2004                                                   signaling in DPF.


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                                                                                                                                                      491
                                                              International and American Associations for Dental Research
492                                                                          Gruber et al.                                                          J Dent Res 83(6) 2004

MATERIALS & METHODS
Isolation and Cultivation of Dental Pulp Fibroblasts
Third molars were collected from six adults (age 23 to 46 yrs) after
informed consent had been obtained according to our institutional
standards. Dental pulp connective tissue was separated from the
root and digested in 3 mg/mL collagenase type I and 4 mg/mL
dispase (Roche, Basel, Switzerland) for at least 3 hrs at 37°C
(Gronthos et al., 2002). Released cells were cultured in alpha-                              Figure 1. Expression profile of lysophosphatidic acid receptors LPA1,
modified Eagle's medium ( MEM) supplemented with 10% fetal                                   LPA2, and LPA3. LPA receptor expression profile in dental pulp
calf serum, antibiotics, and antimycotics (all from Gibco, Grand                             fibroblasts (DPF). RNA was extracted and subjected to RT-PCR analysis.
Island, NY, USA). DPF were kept in a humidified atmosphere at                                Amplification products were separated on 1.5% agarose gels, stained
                                                                                             with ethidium bromide, and photographed. Lanes #1-6 represent
37°C in 5% CO 2. Experiments were performed with cells not                                   amplification products of individual preparations of DPF.
exceeding 10 passages.
RT-PCR Analysis
Total RNA was extracted from 1 x 106 DPF with TRIzol reagent
(Gibco). Aliquots of 2 g total RNA were primed by random                                     min. Cells underwent lysis with SDS buffer containing
hexamers and converted into cDNA by means of a kit used                                      phosphatase and proteinase inhibitors. Cell preparations were
according to the instructions of the manufacturer (MBI Fermentas,                            heated for 5 min at 95°C and centrifuged at 10,000 g for 10 min.
St. Leon-Rot, Germany). RT-PCR analysis was performed in a                                   Cell extracts were separated by 10% SDS-PAGE and transferred
Perkin-Elmer GeneAmp PCR System 2400 with primer sets and                                    onto nitrocellulose membranes (Amersham Pharmacia Biotech,
amplification conditions as recently described (Panther et al.,                              Buckinghamshire, UK). Membranes were blocked in 5% bovine
2002; Papagerakis et al., 2002). PCR products were separated in                              serum albumin in TBS-T and incubated with a 1:1000 dilution of
1.5% agarose gels and photographed with a digital scanning                                   antibodies against phospho ERK1/2 (clone E-4, St. Cruz
system (Bio-Rad Laboratories, Hercules, CA, USA).                                            Biotechnology, Santa Cruz, CA, USA), ERK1 (clone K-23, St.
3[H]-thymidine    Incorporation                                                              Cruz), phospho p38 MAPK (clone #9211, Cell Signaling
                                                                                             Technologies, Beverly, MA, USA), p38 MAPK (clone C-20, St.
DPF were seeded at 5 x 104 cells/cm2 in 96-well plates (Packard,
                                                                                             Cruz), phospho JNK (clone #9251, Cell Signaling), and JNK
Meriden, CT, USA). The following day, DPF were stimulated with
                                                                                             antibodies (clone C-17, St. Cruz) overnight at 4°C. The first
0.1, 0.3, 1, 3, and 10 M LPA (Sigma, St. Louis, MO, USA) in
                                                                                             antibody was detected with the appropriate secondary antibody
serum-free medium, i.e., MEM supplemented with 2.5 g/mL
                                                                                             (Dako, Glostrup, Denmark) according to the ECL method
insulin-transferrin-selenium (Roche, Mannheim, Germany) and
                                                                                             (Amersham).
antibiotics. In indicated wells, U0126 (Cell Signaling Technology,
Beverly, MA, USA), SB203580 (Alexis Corporation, San Diego,                                  Statistical Analysis
CA, USA), and SP600125 (Calbiochem, San Diego, CA, USA), all                                 Statistical analysis was performed with data obtained from six
at 10 M, were added to the culture medium. 3[H]-thymidine                                    independent preparations of DPF. Single data points represent the
incorporation was performed as described (Gruber et al., 2003).                              mean of quadruplicates from 3[H]-thymidine incorporation assay
Ki-67 Immunohistochemistry                                                                   and of duplicates from the counting of alkaline-phosphatase-
                                                                                             positive cells. For Ki-67 staining, statistical analysis was
DPF were seeded at 5 x 10 4 cells/cm 2 in 96-well plates. The
                                                                                             performed with the mean of triplicate cultures from two random
following day, LPA was added to the cultures with and without
                                                                                             selected preparations. All experiments were performed at least
U0126, SB203580, and SP600125, all at 10 M, in serum-free
                                                                                             twice. Data were statistically analyzed by paired t test, with
medium. After 24 hrs, cells were fixed and stained for Ki67 as
                                                                                             significance assigned at the P < 0.05 level.
recently described (Gruber et al., 2003). The percentage of Ki67-
positive nuclei was determined.
                                                                                             RESULTS
Alkaline Phosphatase Activity
DPF were seeded at 5 x 104 cells/cm2 in 48-well plates. The next                             Expression Pattern of LPA Receptors in DPF
day, growth medium was replaced by serum-free medium alone or                                DPF from six donors expressed LPA1, LPA2, and LPA3
medium supplemented with LPA at 10 M, BMP-7 (R&D                                             receptors as determined by RT-PCR analysis. PCR products
systems, Minneapolis, MN, USA) at 300 ng/mL, and a                                           appeared at the predicted size, with LPA1 showing the
combination of both factors. DPF were cultured for another 72 hrs,                           strongest amplification signal. No amplification products were
fixed with 10% formalin, and incubated with a substrate containing                           obtained when total cellular RNA was subjected to PCR
4 mg of naphthol AS-TR phosphate in 0.15 mL of N,N -                                         amplification (Fig. 1).
dimethylformamide and 12 mg of fast blue BB salt (all from
                                                                                             LPA-induced Proliferation Requires MAPK Signaling
Sigma, St. Louis, MO, USA) in 15 mL of 100 mM Tris-HCl (pH
9.6). Positive cells were counted in two random selected                                     LPA caused a dose-dependent mitogenic response in DPF of
microscopic fields.                                                                          six independent preparations. At the highest investigated
                                                                                             concentration of 10 M LPA, a 7.3 + 2.3-fold increase of
Western Blot Analysis                                                                        3[H]thymidine incorporation over unstimulated control cultures

Subconfluent DPF were grown in serum-free medium for 24 hrs                                  was observed. Concentrations lower than 1 M LPA had no
followed by stimulation with LPA at 10 M for 5, 15, 45, and 135                              effect in this in vitro assay (P > 0.05; Fig. 2A). LPA-induced
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                                                            International and American Associations for Dental Research
J Dent Res 83(6) 2004                                         LPA and Dental Pulp Fibroblasts                                                                                493




                                                                                                                                           Figure 3. Effects of LPA on MAPK
                                                                                                                                           phosphorylation in DPF. Serum-starved
                                                                                                                                           DPF were exposed to LPA at a
                                                                                                                                           concentration of 10 M for 5, 15, 45,
                                                                                                                                           and 135 min. Cell lysates were
                                                                                                                                           separated on a 10% SDS-PAGE and
                                                                                                                                           blotted onto a nitrocellulose membrane.
                                                                                                                                           Phosphorylated (pERK, pp38 MAPK, and
                                                                                                                                           pJNK) and unphosphorylated MAPKs
                                                                                                                                           were detected by Western blot analysis.




                                                                                                                                           LPA Decreased Expression of
Figure 2. Effects of LPA on 3[H]-thymidine incorporation of DPF. (A) Dose-response curve of the                                            Differentiation Markers in DPF
indicated concentrations of LPA on 3[H]-thymidine incorporation of DPF. The Fig. shows the mean and                        LPA at 10 M decreased the average
standard deviation of results from 6 independent preparations, each performed in quadruplicate. *P <
0.05 and **P < 0.01 vs. unstimulated cells. (B) Effects of inhibitors of MAPK signaling U0126,
                                                                                                                           number of cells staining positive for
SB203580, and SP600125 on 3[H]-thymidine incorporation by DPF of 6 individual preparations.                                alkaline phosphatase by 58.6 + 33.4%
Results are shown as mean and standard deviation. *P < 0.05 and **P < 0.01 vs. LPA alone. (C)                              when compared with untreated
Staining pattern of the nuclear antigen Ki67 in DPF incubated for 24 hrs with and without LPA at 10                        controls (P < 0.05; Figs. 4A, 4B, 4C).
  M. (D) Relative number of Ki67-positive cells in the indicated cultures. Data are means and standard                     LPA also decreased the number of
deviation of results from triplicates of one donor. *P < 0.05.
                                                                                                                           alkaline-phosphatase-positive cells in
                                                                                                                           the presence of BMP-7 at 300 ng/mL
                                                                                                                           by 37.8 + 12.5% (P < 0.01; Figs. 4A,
                                                                                                                           4B). RT-PCR analysis showed that
3[H]-thymidine    incorporation was decreased by inhibitors of                                 LPA treatment resulted in lower mRNA levels for dentin
ERK, p38 MAPK, and JNK signaling: U0126 decreased the                                          sialophosphoprotein and osteocalcin in DPF (Fig. 4D).
mitogenic activity by 39 + 6% (P < 0.05), SB203580 by 51 +
5% (P < 0.01), and SP600125 by 47 + 12% (P < 0.01) (Fig.
2B). Immunohistochemical evaluation of the proliferating cell                                  DISCUSSION
nuclear antigen Ki67 showed that the relative number of Ki67-                                  LPA is a locally produced bioactive lipid released from
positive cells increased from 49 + 6% in medium controls to 73                                 activated platelets during the early stages of wound healing and
+ 7% in cultures treated with LPA at 10 M (P < 0.05; Fig.                                      inflammation. It is therefore reasonable to suggest that LPA
2C,D).                                                                                         can also be released at sites of injured dental pulp tissue, where
                                                                                               it may play a role during the subsequent healing process. We
LPA Increased Phosphorylation of Different MAPK                                                have shown here that fibroblasts isolated from dental pulps
Signaling Pathways in DPF                                                                      express LPA receptors, indicating the presence of
Western blot analysis showed that DPF constitutively express                                   corresponding target cells in dental pulp tissue. The functional
unphosphorylated ERK, p38 MAPK, and JNK. Incubation of                                         responses of LPA receptors to their ligands have been
DPF with LPA at 10 M increased the phosphorylation status                                      demonstrated by the increased mitogenic activity. LPA at
of all MAPKs investigated, with pERK and pJNK remaining                                        concentrations mitogenic for DPF are likely to occur at sites of
elevated during the observation period. Phosphorylation of                                     inflammation and wound healing (Eichholtz et al., 1993). It is
p38 showed a maximum after 15 min and declined thereafter                                      possible that, also in dental pulp tissue, locally produced LPA
(Fig. 3).                                                                                      can reach levels that are sufficient to cause a cellular response.
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                                                              International and American Associations for Dental Research
494                                                                            Gruber et al.                                                          J Dent Res 83(6) 2004

                                                                                                             differentiation (Manolagas, 2000).
                                                                                                             In agreement with these findings,
                                                                                                             LPA reduced mRNA levels of
                                                                                                             dentin sialophosphoprotein and
                                                                                                             osteocalcin, which are character-
                                                                                                             istically expressed by odontoblast-
                                                                                                             like cells (Papagerakis et al.,
                                                                                                             2002). Analysis of the data led us
                                                                                                             to speculate that LPA can maintain
                                                                                                             odontoblast-like cells in an
                                                                                                             undifferentiated state. The
                                                                                                             mechanism may include the
                                                                                                             transcellular activation of the
                                                                                                             transcription factor peroxisome
                                                                                                             proliferator-activated receptor
                                                                                                             gamma (PPAR) 2 by LPA
                                                                                                             (McIntyre et al., 2003). PPAR 2
                                                                                                             is a "master gene" that suppresses
                                                                                                             osteogenic differentiation of
                                                                                                             mesenchymal progenitor cells,
                                                                                                             whereas it is responsible for their
                                                                                                             differentiation into the adipogenic
                                                                                                             lineage (Manolagas, 2000). It
                                                                                                             remains to be determined whether
                                                                                                             DPF staining positive for alkaline
                                                                                                             phosphatase activity are derived
                                                                                                             from stem cells capable of forming
                                                                                                             ectopic dentin upon transplantation
                                                                                                             into immunodeficient mice
 Figure 4. Effects of LPA on alkaline phosphatase activity and expression of differentiation markers. (A)
 DPF were stimulated for 72 hrs with bone morphogenetic protein-7 (BMP-7) as indicated under serum-          (Gronthos et al., 2002). Moreover,
 free conditions and stained for alkaline phosphatase activity where positive cells appear blue (AP+). (B)   the question of which of the LPA
 Amount of alkaline phosphatase activity per microscopic field, normalized to unstimulated controls. Data    receptors are responsible for the
 are given as means and standard deviation from results of two random selected preparations, each            observed effects requires further
 performed in triplicate. *P < 0.05 vs. unstimulated control; **P < 0.01 between marked groups. (C)
 Representative picture of a microscopic field that shows alkaline-phosphatase-positive cells given in blue. investigation.
 (D) RT-PCR analysis of mRNA isolated from DPF incubated with LPA for 72 hrs.                                     Analysis of our data indicates
                                                                                                             that LPA is a potent mitogen for
                                                                                                             DPF under in vitro conditions.
                                                                                                             The mitogenic activity of LPA
                                                                                                             requires signaling via ERK, p38
The proliferative response of cells to LPA requires activation of             MAPK, and JNK. Moreover, LPA decreased the
intracellular signaling pathways such as MAPKs, connecting                    differentiation of progenitor cells into odontoblast-like cells,
receptor activation in the cell membrane with gene expression                 also in the presence of BMP-7. From analysis of the data
in the nucleus (Chang and Karin, 2001). Binding of LPA to its                 reported here, we suggest that dental pulps contain a
receptors has been reported to increase MAPK signaling in                     proportion of cells that can respond to LPA, a mechanism that
various cell types, such as smooth-muscle cells, astrocytes, and              may play a role in tissue repair.
ovarian cancer cells (Baudhuin et al., 2002; Sorensen et al.,
2003; Xu et al., 2003). Our experiments show that all MAPKs                   ACKNOWLEDGMENTS
investigated—i.e., ERK, p38 MAPK, and JNK—were
                                                                              The authors thank M. Pensch for skillful technical assistance,
phosphorylated in response to LPA. Moreover, kinase-specific
                                                                              and M.B. Fischer for helpful discussion. The authors also thank
inhibitors decreased LPA-induced proliferation. Our findings
                                                                              M. Kunschak for improving the fidelity of English grammar
suggest that the three MAPKs are involved in the mitogenic
                                                                              and syntax of the manuscript. This work was supported by the
process. These observations are in agreement with those from
                                                                              Austrian Nationalbank grant No. 9269.
other reports showing that LPA can induce mitogenic activity
via ERK and p38 MAPK signaling (Sorensen et al., 2003; Xu
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                                                              International and American Associations for Dental Research
J Dent Res 83(6) 2004                                         LPA and Dental Pulp Fibroblasts                                                                    495

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