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Department of Medicine, University of Auckland, Auckland, New Zealand
Address all correspondence and requests for reprints to: Dr. Andrew Grey, Department of Medicine, University of Auckland, Private Bag 92019, Auckland, New Zealand. E-mail a.grey{at}auckland.ac.nz
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionNote Added in RevisionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in RevisionReferences |
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LPA signals through a specific cell membrane-bound receptor, LPA1 (also known as edg-2 and vzg-1), which is a member of the G protein-coupled receptor superfamily (15, 16). The receptor couples to Gi/o, Gq, and G12/13 proteins, and previous reports have identified several signaling pathways activated by LPA, including inhibition of adenylyl cyclase, stimulation of phospholipases C and D, activation of phosphatidylinositol 3-kinases (PI-3-kinases) and mitogen-activated protein (MAP) kinases, and Rho-dependent phosphorylation of cytoskeletal proteins (1). It is generally thought that the p42/44 MAP kinases, which act as a convergence point for mitogenic signals originating from a variety of extracellular sources (17), mediate the proliferative effects of LPA (1), but direct evidence for their involvement in LPA-induced mitogenesis has not been reported.
There is currently considerable interest in understanding the biology of the osteoblast, the bone-forming cell of mesenchymal origin. Osteoblasts play a central role in bone remodeling in both physiological and pathophysiological conditions (18), and impaired osteoblast function may contribute to the pathogenesis of metabolic bone diseases characterized by low bone mass, such as osteoporosis (19). Thus, agents that increase osteoblast number and/or function have the potential to act as anabolic therapies in osteoporosis. In the current study we report that the LPA receptor LPA1/edg-2/vzg-1 is expressed in osteoblastic cells, that LPA is an osteoblast mitogen in vitro, and that LPA activates p42/44 MAP kinases via a signaling pathway that involves Gi proteins, cytosolic calcium, and protein kinase C (PKC). However, the mitogenic actions of LPA in osteoblastic cells, although in part dependent upon Gi proteins and PKC, do not require activation of p42/44 MAP kinases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in RevisionReferences |
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-Oleoyl lysophosphatidic acid,
EDTA, EGTA, thapsigargin, ionomycin, collagenase, leupeptin, pepstatin,
aprotinin, and sodium orthovanadate were purchased from
Sigma (St. Louis, MO).
[3H]Thymidine was obtained from Amersham Pharmacia Biotech (Little Chalfont, UK). RNase-free
deoxyribonuclease (DNase) was purchased from Promega Corp.
(Madison, WI), and proteinase K, deoxy (d)-NTPs, and AmpliTaq DNA
polymerase were obtained from Roche Molecular Biochemicals
(Mannheim, Germany). The antibodies to phosphorylated p42/44 MAP kinases and total p42/44 MAP kinases were purchased from New England Biolabs, Inc. (Beverly, MA), and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. Horseradish peroxidase-conjugated antimouse and antirabbit secondary antibodies and enhanced chemiluminescence (ECL) reagents were obtained from Amersham Pharmacia Biotech. PD-98059, U-0126, calphostin C, wortmannin, LY294002, H-89, and BAPTA-AM were purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). Pertussis toxin was obtained from List Biological Laboratories (Campbell, CA).
Preparation of RNA
Total cellular RNA was purified from primary rat osteoblastic
cells and UMR 106–01 cells by a modification of the single step
guanidinium thiocyanate-phenol-chloroform RNA extraction protocol
described by Chomczynski et al. (20). The
protocol was followed up to the step of isopropanol precipitation,
after which the RNA pellet was washed with ice-cold 70% ethanol,
air-dried, and dissolved in double distilled water. The RNA was then
reprecipitated using 0.3 M sodium acetate, pH
5.2, and 2.5 vol ethanol, incubated for 16 h at -20 C, pelleted
by centrifugation, lyophilized, and redissolved in water. RNA
concentration and purity were determined by spectrophotometry, and its
quality was determined by electrophoresis on a 1% agarose gel.
RT-PCR
Before using RNA as a template for RT-PCR, it was treated with
DNase to remove contaminating traces of DNA. One hundred micrograms of
RNA were incubated for 30 min at 37 C with 4 U RNase-free DNase, 40 U
RNase inhibitor, 2 mM 1,4-dithiothreithol, 40
mM Tris-HCl (pH 7.9), 10 mM NaCl, 6
mM MgCl2, and 10 mM
CaCl2 in a final volume of 100 µl. The RNA
samples were then incubated for 20 min at 37 C with 50 µg/ml
proteinase K in a buffer containing 10 mM Tris (pH 8.0), 5
mM EDTA, and 0.5% SDS in a final volume of 500 µl.
Samples were extracted with equal volumes of water-saturated phenol and
then with equal volumes of chloroform and precipitated with 0.3
M sodium acetate (pH 5.2) and 2.5 vol ethanol.
All RT-PCR amplifications were carried out using aerosol barrier pipette tips. Complementary DNA (cDNA) was synthesized from 1 µg DNase-treated RNA in 1 x PCR buffer (10 mM Tris-HCl, pH 8.3, and 50 mM KCl) and 2 mM MgCl2 using 0.1 U random hexamer primer, 1 mM of each dNTP, 5 mM dithiothreithol, 40 U RNase inhibitor, and 200 U Moloney murine leukemia virus reverse transcriptase in a final volume of 20 µl. The reaction was incubated for 60 min at 37 C and then heat inactivated at 95 C for 5 min. The RT reaction was used directly for subsequent PCR amplification. PCR was carried out in a final volume of 50 µl containing 1 x PCR buffer, 2 mM MgCl2, 1 µM of each primer, 0.4 mM of each dNTP, and 1 U AmpliTaq DNA polymerase in an automatic DNA thermal cycler (Eppendorf, Hamburg, Germany). After an initial denaturation step (2 min at 94 C), 35 PCR cycles were performed. Denaturation was carried out at 94 C for 30 sec, annealing was performed at 60 C for 30 sec, and primer extension was carried out at 72 C for 30 sec for the first cycle, adding 5 sec/cycle after that. PCR products were purified from 1% agarose gels using the QIAquick gel extraction kit (QIAGEN, Valencia, CA), and their sequences were determined on an ABI 377 XL DNA sequencer (PE Biosystems, Foster City, CA).
The specific primers used were based upon the rat LPA1/vzg-1/edg-2 cDNA sequences 5'-CACAGCCATGAACGAACAACAG-3' (nucleotides 199–220) and 5'-CATAGTCCTCTGGCGAACATAG-3' (nucleotides 862–841). The expected amplification product was 663 bp. Controls for the RT-PCR reaction were samples processed in the absence of reverse transcriptase enzyme.
Northern blot analysis
Polyadenylated [poly(A)+] RNA was
prepared from 1 mg total RNA, using the PolyAT tract messenger RNA
(mRNA) Isolation System (Promega Corp., Madison, WI), and
was electrophoresed on a 1% agarose gel containing 1.2 M
formaldehyde, 3 µg/ml ethidium bromide, and 1 x MOPS buffer
[20 mM 3-[N-morpholino]propanesulfonic acid
(pH 7), 5 mM sodium acetate, and 1
mM EDTA]. RNA was transferred to a Hybond
N+ nylon membrane in 20 x SSC (3
M NaCl and 0.3 M sodium
citrate, pH 7), and immobilized on the membrane by UV cross-linking
(Stratalinker, Stratagene, La Jolla, CA). The probe used
for hybridization was extracted from an agarose gel containing
amplification products of LPA1 cDNA, labeled with
[32P]dCTP using the Random Primers DNA labeling
system, then denatured by adding NaOH to a final concentration of 0.2
M and incubating for 10 min at room temperature.
The RNA blot was prehybridized in 0.25 M
Na2HPO4 (pH 7.5), 7% SDS,
and 1 mM EDTA for 2 h at 65 C, transferred
to fresh hybridization buffer containing the denatured probe, and
hybridized for 20 h at 65 C. After the membrane was washed in
0.2 x SSC and 0.5% SDS for 10 min at room temperature and then
for 1 h at 65 C, it was analyzed by autoradiography. The film was
exposed for 16 h at -70 C.
Osteoblast cell culture and mitogenesis assay
Primary rat osteoblastic cells were prepared as previously
described (21). In brief, osteoblasts from collagenase
digests 3–4 of 20-day-old fetal rat calvariae were pooled,
centrifuged, washed in DMEM with 10% FCS, resuspended in DMEM/10%
FCS, and placed in 75-cm2 flasks. After 48
h, the medium was changed to MEM. Confluence was reached within 5–6
days, at which time the cells were subcultured. The osteoblast-like
character of these cells has been established by demonstration of
alkaline phosphatase staining in more than 95% of cells, osteocalcin
production, and a sensitive adenylyl cyclase response to PTH and
PGE2 (21).
Osteoblast proliferation studies (cell counts and thymidine incorporation) were performed in subconfluent cell populations. Twenty-four hours after subculturing, cells were changed to serum-free medium containing 0.1% BSA for an additional 24 h before the addition of the experimental compounds. Inhibitors were added 10–20 min before the LPA. If the inhibitor being studied was dissolved in dimethylsulfoxide, equivalent concentrations of dimethylsulfoxide were added to wells lacking the inhibitor. Cell numbers were analyzed 24 h after addition of the test compounds by detaching cells from the wells using trypsin/EDTA (0.05%/0.53 mM) for approximately 5 min at 37 C. Counting was performed in a hemocytometer chamber. [3H]Thymidine incorporation into growth-arrested cells was assessed by pulsing the cells with [3H]thymidine (0.5 µCi/well) 2 h before the end of the experimental incubation. Experiments were terminated at 24 h by washing the cells in MEM followed by the addition of 10% trichloroacetic acid. The precipitate was washed twice with ethanol/ether (3:1), and the wells were desiccated at room temperature. The residue was redissolved in 2 M KOH at 55 C for 30 min and neutralized with 1 M HCl, and an aliquot was counted for radioactivity. Each experiment was performed at least three times using experimental groups consisting of at least six wells. A similar method of cell culture, and assessment of DNA synthesis, was used for the osteoblastic cell line UMR 106–01.
Immunoblotting
Primary rat osteoblasts, prepared as described above, were
seeded in six-well tissue culture plates at an initial density of
5 x 104 cells/ml in MEM 5% FCS and grown
to 80–90% confluence. After serum starvation overnight, cells were
treated at room temperature with LPA at the indicated concentrations in
MEM/0.1% BSA. In experiments designed to determine the effects of
inhibitors of signal transduction on LPA-induced p42/44 MAP kinase
phosphorylation, the cells were pretreated with the inhibitor for 30
min before the addition of LPA. The exception was pertussis toxin,
which was added 18 h before LPA. After treatment for the indicated
period of time, the treatment medium was aspirated, the cells were
washed in ice-cold PBS and then scraped in ice-cold HNTG lysis buffer
[50 mM HEPES (pH 7.5), 150 mM NaCl, 1%
Triton, 10% glycerol, 1.5 mM MgCl2,
and 1 mM EDTA] containing a cocktail of protease and
phosphatase inhibitors (1 mM phenylmethylsulfonylfluoride,
1 µg/ml pepstatin, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1
mM sodium vanadate, and 500 mM NaF). The
lysates were briefly vortexed, clarified by centrifugation at 13,000
rpm at 4 C, then stored at -70 C until analyzed. The protein content
of the cell lysates was measured using the DC protein assay
(Bio-Rad Laboratories, Inc., Hercules, CA). Equal amounts
of whole cell lysate (30–50 µg) were subjected to 8% SDS-PAGE,
transferred to nitrocellulose membranes, and immunoblotted overnight at
4 C with an anti-phospho-p42/44 MAP kinase antibody (1:1000). As a
control for protein loading, the same filters were stripped and
reprobed with an antibody to total p42/44 MAP kinase (1:400).
Incubation with the horseradish peroxidase-conjugated secondary
antibody was performed for 1 h at room temperature, and the
membranes were analyzed by ECL. Quantitation of bands detected by ECL
was performed using scanning laser densitometry (Molecular Dynamics, Inc., Sunnyvale, CA). The immunoblots presented are
representative of at least three separate experiments in each case.
Statistical analyses
All data were analyzed using either SAS statistical software,
version 6.12 (SAS Institute, Inc., Cary, NC), or Prism
(GraphPad Software, Inc., San Diego, CA). Data from at
least three separate experiments in each treatment condition were
pooled and analyzed by two-way ANOVA to determine the effects of LPA on
osteoblast cell number and DNA synthesis in the presence and absence of
the indicated inhibitor. All data shown are the mean ±
SEM unless otherwise stated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in RevisionReferences |
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, lanes 1 and 3) or UMR 106–01 cells
(lanes 2 and 4) was subjected to RT-PCR as described in Materials
and Methods. As shown in Fig. 1A, lanes 1 and 2 (RT-), no
amplification products were detected in the absence of reverse
transcriptase. However, amplification products of the predicted size of
the LPA1 fragment (663 bp) were detected in both
primary rat osteoblasts (Fig. 1A, lane 3) and UMR 106–01 cells (Fig. 1A, lane 4) in the presence of reverse transcriptase (RT+). Sequencing
of the amplification products extracted from the gel confirmed them to
be identical to the known sequence of rat
LPA1/vzg-1/edg-2 cDNA (data
not shown). Northern analysis of poly(A)+ RNA
from UMR 106–01 cells demonstrated the presence of an
LPA1 transcript of about 2.2 kb (Fig. 1B), a size
consistent with previous reports (22, 23). These data
demonstrate that osteoblastic cells express LPA receptor mRNA.
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, 24 h of treatment with LPA
induced a dose-dependent increase in DNA synthesis. The stimulation of
DNA synthesis was evident at concentrations of LPA as low as 100
nM and peaked at a concentration of 50
µM, which induced a 3.3-fold increase in this
measure of cell proliferation. The proliferative effect (mean ±
SEM fold stimulation) induced by 50
µM LPA (3.25 ± 0.18) was almost 80% of
that induced by 5% FCS (3.98 ± 0.36; Fig. 2, A and B). The
EC50 for LPA-induced DNA synthesis in primary rat
osteoblast cultures was 1.1 µM. LPA also
increased primary rat osteoblast cell numbers. Thus, 1 and 10
µM LPA induced mean increases of 40% and 45%,
respectively, in cell number after 24 h of treatment (Fig. 2C).
LPA also stimulated DNA synthesis in the UMR 106–01 osteoblastic cell
line (Fig. 2B). Although the magnitude of the proliferative effect
induced by LPA in UMR 106–01 cells was smaller than that observed in
the primary cell cultures, it was proportionally similar to that of 5%
FCS in the respective cell types (Fig. 2B). Treatment of primary rat
osteoblastic cells with 10 µM LPA did not alter
the proportion of nonosteoblastic cells, which remained below 5% as
assessed by alkaline phosphatase staining (data not shown).
Collectively, these data demonstrate that LPA is an osteoblast
mitogen.
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(left panel), 10
µM LPA stimulated rapid phosphorylation of
p42/44 MAP kinases in primary rat osteoblastic cells. This effect
peaked at 10–30 min and was sustained for at least 4 h.
LPA-induced p42/44 MAP kinase phosphorylation in primary rat
osteoblasts was dose dependent and observed at a concentration as low
as 0.1 µM (Fig. 3B). LPA also induced
phosphorylation of p42/44 MAP kinases in UMR 106–01 cells (Fig. 3A, right panel).
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, left panel, compare lanes 3 and 4;
right panel, compare lanes 2 and 4). Each of the MEK
inhibitors also inhibited basal p42/44 MAP kinase activity (Fig. 3C, left panel, compare lanes 1 and 2; right panel,
compare lanes 1 and 3).
LPA-induced phosphorylation of p42/44 MAP kinases in osteoblastic
cells involves Gi proteins, calcium and PKC
Having established that LPA potently induced p42/44 MAP kinase
activation in osteoblastic cells, we next examined which proximal
signaling events were involved in this process. As shown in Fig. 4A, pretreatment of osteoblastic cells
with pertussis toxin, which ADP-ribosylates and inactivates
Gi proteins, completely abrogated the
phosphorylation of p42/44 MAP kinases induced by LPA (compare lanes 3
and 4). In contrast, the PKC inhibitor calphostin C caused only a
partial inhibition of LPA-induced p42/44 MAP kinase phosphorylation. In
five separate experiments, calphostin C inhibited LPA-induced p42/44
MAP kinase phosphorylation by 65 ± 14% (mean ±
SEM; Table 1). Figure 4B
shows the effect of inhibiting calcium signaling on LPA-induced p42/44
MAP kinase phosphorylation in osteoblastic cells. Pretreatment of cells
for 30 min with the calcium-chelating agent EGTA (5 mM),
which abrogates both rapid and sustained agonist-induced cytosolic
calcium transients (24), strongly inhibited LPA-induced
p42/44 MAP kinase phosphorylation (compare lanes 2 and 6, Fig. 4B).
However, neither loading osteoblastic cells with the intracellular
calcium-chelating agent BAPTA-AM
[1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
tetrakis (acetoxymethyl ester)] nor depleting intracellular
calcium stores by pretreatment with thapsigargin affected the ability
of LPA to phosphorylate p42/44 MAP kinases (compare lanes 2 and 4, Fig. 4B, and lanes 2 and 5, Fig. 4C, respectively). Taken together, these
data suggest that a calcium signal is required for LPA-induced p42/44
MAP kinase phosphorylation in osteoblastic cells, and that the
important component of the calcium signal is influx of extracellular
calcium. However, although a calcium signal is required for LPA-induced
p42/44 MAP kinase phosphorylation, it is not sufficient for the effect.
Thus, neither the calcium ionophore ionomycin nor the endoplasmic
reticulum Ca2+-ATPase inhibitor thapsigargin was
able to stimulate p42/44 MAP kinase phosphorylation (Fig. 4C, compare
lane 1 with lanes 3 and 4). Taken together, these data demonstrate that
Gi proteins, PKC, and influx of extracellular
calcium are involved in mediating the effect of LPA to induce
phosphorylation of p42/44 MAP kinases in osteoblastic cells.
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, A and B, respectively, neither
of the specific PI-3-kinase inhibitors, wortmannin (100 nM)
and LY294002 (50 µM), blocked LPA-induced phosphorylation
of p42/44 MAP kinases (compare lanes 3 and 4 in the top
panel of Fig. 5A and lanes 2 and 3 in the top panel of
Fig. 5B). Pretreatment of osteoblastic cells with the protein kinase A
inhibitor H-89 (10 µM) also failed to inhibit
LPA-induced p42/44 MAP kinase phosphorylation (Fig. 5C).
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(left panel), pertussis toxin
partially inhibited LPA-induced DNA synthesis. The 57% inhibition of
LPA-induced DNA synthesis by pertussis toxin at a concentration of 10
ng/ml was similar to that observed (71%) at a much higher
concentration (200 ng/ml), suggesting that Gi
proteins mediate only part of the proliferative effect of LPA in
osteoblasts. The PKC inhibitor, calphostin C, also significantly
inhibited LPA-induced DNA synthesis (Fig. 6A, right panel).
At a concentration of 100 nM, calphostin C
reduced LPA-stimulated [3H]thymidine
incorporation by an average of 42%. Pretreatment of osteoblastic cells
with a combination of pertussis toxin and calphostin C did not alter
the inhibitory effect produced by either inhibitor alone (data not
shown).
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, concentrations of PD-98059 up to 50 µM and of U-0126
up to 10 µM did not inhibit the proliferative effect of
LPA. The failure of PD-98059 and U-0126 to attenuate LPA-induced DNA
synthesis was not due to biological inactivity of either MEK inhibitor,
because at similar concentrations each completely blocked the
phosphorylation of p42/44 MAP kinases induced by LPA (Fig.
3C).
As PI-3-kinases have been implicated in mitogenic signaling induced by
LPA in fibroblasts (28), we also tested the effect of the
specific PI-3-kinase inhibitor LY294002 on LPA-induced DNA synthesis in
osteoblastic cells. As shown in Fig. 6C, LY294002 had no effect on the
proliferative response induced by LPA in these cells. Taken together,
these data suggest that the mitogenic effect of LPA in osteoblastic
cells is mediated in part by Gi proteins and PKC,
but does not require activation of either p42/44 MAP kinases or
PI-3-kinases.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in RevisionReferences |
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To our knowledge, the current study is the first to describe the biological activity of LPA and/or the presence of an LPA receptor in bone cells. Our data identify the osteoblast, the cell primarily responsible for formation of new bone, as a target for LPA, with mitogenic effects observed well within the range of LPA concentrations reported to be present in serum (13). In addition, LPA appears to be a very potent mitogen for osteoblastic cells, because the 3.25-fold increase it induced in DNA synthesis is greater than that we observed in response to known osteoblast growth factors, such as insulin-like growth factor I and epidermal growth factor in the same assay (29), and is about 80% of that induced by 5% FCS. The current results taken together with recent evidence that sphingosine-1-phosphate, another phospholipid with growth factor-like actions (30), induces proliferation in osteoblastic cells (31) suggest that phospholipids may exert important effects in skeletal tissue. What role, if any, LPA plays in skeletal physiology or pathophysiology, however, remains to be determined. LPA is known to be released by activated platelets (13) and to exert proliferative effects on several cell types that are involved in tissue repair and wound healing (4, 5, 6, 27), suggesting that it is involved in the tissue response to injury. Whether LPA plays a role in repair and/or remodeling of skeletal tissue is as yet unknown.
In several cell types LPA activates p42/44 MAP kinases (1), and the mitogenic signal induced by LPA has been considered to be mediated by these ubiquitous serine/threonine kinases (1, 2). To our knowledge, however, direct examination of the role of p42/44 MAP kinases in LPA-induced mitogenesis, using specific inhibitors of the upstream kinase MEK, has not previously been undertaken. Our data demonstrate that, at least in osteoblastic cells, activation of p42/44 MAP kinases is not required for the proliferative effect of LPA. Thus, neither of the structurally dissimilar specific MEK inhibitors, PD-98059 and U-0126, abrogated LPA-induced DNA synthesis in osteoblastic cells despite each potently inhibiting LPA-induced p42/44 MAP kinase phosphorylation. The LPA mitogenic signal in osteoblastic cells is therefore independent of p42/44 MAP kinases. Whether this observation is specific to osteoblastic cells or is generally true of LPA mitogenic signaling remains to be determined. However, our data are consistent with the finding that LPA is capable of activating serum-response factor-induced c-fos transcription in a MEK-independent fashion (32). p42/44 MAP kinases are involved in diverse cellular processes, including proliferation, differentiation, and survival (33); further work is required to define the biological consequences of activation of these kinases by LPA in osteoblastic cells.
The proliferative action of LPA in osteoblasts does depend in part upon functional Gi proteins, as we observed that pertussis toxin substantially inhibited LPA-induced DNA synthesis, a finding consistent with published reports in other cell types (3, 34, 35, 36). However, we were unable to demonstrate complete suppression of LPA-induced mitogenesis by pertussis toxin, even at a concentration substantially higher than that which completely inhibits LPA- induced DNA synthesis in fibroblasts (3). This finding suggests that the LPA mitogenic signal in osteoblastic cells is in part transduced by a pertussis toxin-insensitive G protein. This suggestion is consistent with the findings of Rosskopf et al. (7), who reported only partial inhibition by pertussis toxin of LPA-induced DNA synthesis in B lymphoblasts. Thus, there appear to be cell type-specific differences in the degree of involvement of Gi proteins in LPA mitogenic signaling.
Although activation of PKC signaling by LPA has been observed in several cell types (4, 27, 37, 38), to our knowledge there has been only one previous study that investigated the role of PKC in LPA-induced mitogenesis. Inhibition of PKC induced partial suppression of LPA-induced proliferation in vascular smooth muscle cells (38). Our data support the idea that PKC activation is important for LPA-induced mitogenesis and suggest that Gi proteins and PKC are part of a common, rather than a parallel, mitogenic pathway activated by LPA in osteoblastic cells. Another potential intracellular transducer of the LPA mitogenic signal is the phospholipid kinase, PI-3-kinase, which has been implicated in the proliferative response to LPA in fibroblasts (28). However, we found no effect on LPA-induced DNA synthesis in osteoblastic cells of the specific PI-3-kinase inhibitor, LY294002. Thus, the identities of the signaling intermediates downstream of Gi proteins and PKC that transduce the mitogenic signal initiated by LPA in osteoblastic cells are unknown at present.
The signaling pathway(s) that links G protein-coupled receptors to p42/44 MAP kinases are both cell type and receptor specific (39). Agonists that signal through receptors that couple to Gi proteins commonly activate p42/44 MAP kinases in a pertussis toxin-sensitive fashion (40). Some G protein-coupled receptor agonists also activate p42/44 MAP kinases by pertussis toxin-insensitive pathways that involve PKC and/or intracellular calcium fluxes (24, 41). LPA receptors couple to pertussis toxin-sensitive Gi proteins and pertussis toxin-insensitive Gq and G12/13 proteins (1). Our data suggest that at least three proximal signaling molecules are involved in LPA-induced p42/44 MAP kinase activation in osteoblastic cells: Gi proteins, cytosolic calcium, and PKC. Our observation that pertussis toxin abrogates the ability of LPA to activate p42/44 MAP kinases in osteoblastic cells is consistent with findings in other cell types, which have frequently demonstrated a role for Gi proteins in the coupling of LPA to MAP kinases (7, 38, 41, 42, 43). The inhibition of LPA-induced p42/44 MAP kinase activation by pertussis toxin in osteoblastic cells appears to be complete, suggesting that in this cell type the effect is transduced exclusively through Gi proteins.
In summary, the findings of the current study identify the osteoblast as a target of the naturally occurring phospholipid LPA. LPA potently induces osteoblast proliferation at periphysiological concentrations in vitro and rapidly induces phosphorylation of p42/44 MAP kinases in osteoblastic cells. The activation of p42/44 MAP kinases by LPA is dependent on functional Gi proteins, a calcium signal, and PKC. However, although LPA-induced DNA synthesis is also dependent at least in part upon functional Gi proteins and activation of PKC, neither p42/44 MAP kinases nor PI-3-kinases are required for this effect. Thus, LPA induces proliferation in osteoblastic cells via a signaling pathway that is independent of p42/44 MAP kinases.
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Note Added in Revision |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in RevisionReferences |
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Acknowledgments |
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Footnotes |
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Received July 18, 2000.
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A. Grey, Q. Chen, K. Callon, X. Xu, I. R. Reid, and J. Cornish The Phospholipids Sphingosine-1-Phosphate and Lysophosphatidic Acid Prevent Apoptosis in Osteoblastic Cells via a Signaling Pathway Involving Gi Proteins and Phosphatidylinositol-3 Kinase Endocrinology, December 1, 2002; 143(12): 4755 - 4763. [Abstract] [Full Text] [PDF]
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A. Tokumura, E. Majima, Y. Kariya, K. Tominaga, K. Kogure, K. Yasuda, and K. Fukuzawa Identification of Human Plasma Lysophospholipase D, a Lysophosphatidic Acid-producing Enzyme, as Autotaxin, a Multifunctional Phosphodiesterase J. Biol. Chem., October 11, 2002; 277(42): 39436 - 39442. [Abstract] [Full Text] [PDF]
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) and absence (
) of
inhibitor. *, P < 0.01 vs. LPA
alone.