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*
Department of Geriatric Medicine, Graduate School of Medicine, Faculty of
Medicine, Kyoto University, Kyoto, Japan
Division of Artificial Kidneys, Kyoto University, Kyoto, Japan
Discovery Research Laboratory, Shionogi and Co., Ltd., Japan
§
Department of Biology, Faculty of Science, Kyushu University, Kyushu,
Japan.
Correspondence to Dr. Kenji Ishii, Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyoku, Kyoto 606-8507, Japan. Phone: +81 75 751 3465; Fax: +81 75 751 3574; E-mail: kishii{at}kuhp.kyoto-u.ac.jp
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-carboxylation. In mesangial cells, Gas6 and its receptor Axl were
expressed. In addition, exogenous Gas6 phosphorylated Axl, activated
extracellular signal-regulated kinase, and stimulated
[3H]-thymidine incorporation in mouse mesangial cells. This study
also examined whether endogenous Gas6 stimulates mesangial proliferation.
Conditioned medium (CM) from serum-starved mesangial cells could stimulate
[3H]-thymidine incorporation and phosphorylate extracellular
signal-regulated kinase, whereas CM in the presence of warfarin could not.
Simultaneous administration of vitamin K could cancel the inhibitory effect of
warfarin. These results suggest that vitamin K-dependent growth factors in the
CM are critical for mesangial proliferation. Addition of the extracellular
domain of Axl to the CM inhibited its mitogenic effect on mesangial cells,
suggesting that this vitamin K-dependent growth factor is Gas6. It is
concluded that Gas6 is an endogenous mitogen in mesangial cells, and warfarin
inhibits mesangial proliferation possibly by inhibiting -carboxylation
of Gas6. This study sheds light on the regulation of mesangial proliferation
and may lead to a new therapeutic strategy for glomerular diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-carboxylase in the presence of
vitamin K
(10,13).
This modification is selectively inhibited by warfarin
(13). Recently, it was
reported that -carboxylation of Gas6 is essential for its
receptor-binding and growth-potentiating activities in VSMC
(14,15),
and this characteristic is unique among other known growth factors.
In the present study, we studied the effect of warfarin on mouse mesangial
cell proliferation by focusing on Gas6, and demonstrated that Gas6 is a new
autocrine growth factor of mesangial cells. We also showed the possibility
that warfarin inhibited mesangial cell proliferation by inhibiting
-carboxylation of Gas6.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preparation of Recombinant Gas6 and Extracellular Domain of Axl
(Axl-ECD)
Recombinant rat Gas6 was purified from the culture medium of Chinese
hamster ovary cells transfected with the Gas6 expression plasmid as described
elsewhere (18). The
extracellular domain of Axl fused with IgG Fc portion was prepared as
described previously (19).
Gas6 Expression in Mesangial Cells
Gas6 expression in mouse mesangial cells was assessed by reverse
transcription-PCR according to manufacturer's instructions (Takara Shuzo,
Kyoto, Japan). RNA was prepared from serum-starved mouse mesangial cells by
the GTC-phenol method (Trizol, Life Technologies, Gaithersburg, MD). To detect
mRNA of Gas6, PCR was conducted with a primer pair
5'-TGAGCTGCAGCTTCGGTACAA-3' and
5'-GGGTGCAGAAATCACCGATAC-3', which would result in a product of
835 nucleotides. Expression and secretion of Gas6 in rat mesangial cells were
assessed by Western blotting. Rat mesangial cells (106) were
serum-starved for 48 h, and the conditioned medium (2 ml) was concentrated to
20 µl by Centricon 10 (Amicon, Beverly, MA). The sample was analyzed by
immunoblotting with anti-rat Gas6 polyclonal antibody
(18).
Assessment of Mesangial Cell Proliferation
Incorporation of [3H]-thymidine into DNA was measured to
evaluate mesangial cell proliferation. Mesangial cells were plated at 1.5
x 104 cells/well in 24-well dishes in Dulbecco's modified
Eagle's medium containing 20% F12 and 20% fetal calf serum (growth medium)
(20). After 24 h, cells were
serum-starved in Dulbecco's modified Eagle's medium containing 0.5% bovine
serum albumin (starving medium) with or without warfarin for 48 h. Then the
medium was replaced with the fresh starving medium including various
concentrations of agonist, or left untreated. After 22 h, cells were labeled
with [3H]-thymidine (1 µCi/ml) for 2 h and washed with medium,
and the incorporation of [3H]-thymidine into acid-precipitable
materials was determined.
Axl Phosphorylation
Mouse mesangial cells (106) were grown to confluence with growth
medium in 10-cm dishes, and the medium was changed to the starving medium.
After 24 h, cells were treated with 500 ng/ml recombinant Gas6 for various
periods of time at 37°C, and were lysed with 1 ml of the lysis buffer (20
mM Hepes, pH 7.4, 50 mM NaCl, 1% Triton X-100, 20 mM leupeptin, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 mM sodium
orthovanadate, 50 mM sodium fluoride) on ice
(18). Immunoprecipitation was
performed at 4°C for 2 h using 1 µg of 4G10 mouse anti-phosphotyrosine
antibody (Up-state Biotechnology, Lake Placid, NY). Immune complexes were
collected with 50 mg of protein A-Sepharose CL-4B. Proteins were immunoblotted
with goat anti-mouse Axl antibody (sc-1097; Santa Cruz Biotechnology, Santa
Cruz, CA).
Activation of Extracellular Signal-Regulated Kinase
Activation of extracellular signal-regulated kinase (ERK) was examined by
detecting phosphorylated ERK. Confluent mesangial cells were serum-starved in
6-well dishes for 24 h and stimulated with recombinant rat Gas6 (500 ng/ml)
for the indicated time. After being washed with ice-cold phosphate-buffered
saline containing 1 mM sodium orthovanadate, the cells were lysed with 100
µl of the ice-cold lysis buffer. The lysates (20 µg) were immunoblotted
with anti-phospho p44/p42 mitogen-activated protein (MAP) kinase polyclonal
antibody (9101S; New England Biolabs, Beverly, MA) or anti-ERK1 (sc-093; Santa
Cruz Biotechnology).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Next, we examined expression of Gas6 receptors in mouse mesangial cells. Gas6 can bind to receptor tyrosine kinases Axl, Rse (also known as Sky), and Mer with dissociation constants (Kd) of 0.4, 2.7, and 29 nM, respectively (18,19). Immunoblotting of mesangial cell lysates with anti-Axl antibody showed a 140-kD protein corresponding to the full-length Axl as well as a smaller immunoreactive protein of 120 kD (Figure 1B) as described previously in tumor cell lines (21). The expression level of Axl in mouse mesangial cells was comparable to that in mouse NIH3T3 cells. We also examined expression of another Gas6 receptor, Rse, which has a lower affinity for Gas6 than Axl and is expressed mainly in the nervous system (18). Expression of Rse was not detected in mesangial cells by immunoblotting with anti-Rse antibody, although it was detected in the mouse whole brain extract as expected (Figure 1B).
Gas6 Is a Mitogen for Mouse Mesangial Cells
To examine whether Gas6 by itself can act as a mitogen for mesangial cells,
we measured [3H]-thymidine incorporation in mouse mesangial cells
after incubation with various concentrations of Gas6. The recombinant Gas6
stimulated dose-dependent incorporation of [3H]-thymidine at
concentrations of 50 to 500 ng/ml (0.7 to 7 nM), with a fivefold increase at
maximum (500 ng/ml) (Figure
2A). Numbers of mesangial cells were also increased
dose-dependently by the administration of Gas6 (data not shown). These results
indicate that Gas6 by itself is a mitogen for mouse mesangial cells. However,
the mitogenic effect of Gas6 was relatively weak compared to that of
platelet-derived growth factor (PDGF) (2 nM)
(Figure 2A). To clarify that
the mitogenic effect of Gas6 is specific for Gas6-Axl interaction, we used the
recombinant extracellular domain of Axl (Axl-ECD) as an inhibitor of Gas6.
Axl-ECD was shown to capture recombinant Gas6 and inhibit its binding to
endogenous cell surface receptors
(18), resulting in inhibition
of receptor-dependent signal transduction. Gas6 (500 ng/ml), which was
preincubated with various concentrations of Axl-ECD in the starving medium for
1 h, was added to the serum-starved mesangial cells, and thymidine
incorporation was measured. The Axl-ECD dose-dependently inhibited thymidine
incorporation, suggesting that the mitogenic effect of Gas6 was specific for
Gas6-Axl interaction (Figure
2B). This inhibitory effect of Axl-ECD was not observed when mouse
mesangial cells were stimulated with PDGF (2 nM) (data not shown).
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Gas6 Phosphorylates Axl and Activates ERK
As reported previously, Gas6 stimulates the Axl tyrosine kinase
(22) to exert its mitogenic
effect in NIH3T3 cells
(11,12,22).
Therefore, we examined whether Gas6/Axl signaling pathways function to
stimulate cell proliferation in mouse mesangial cells. Cells were stimulated
with Gas6 (500 ng/ml), and whole cell lysates were immunoprecipitated with
anti-phosphotyrosine antibody and immunoblotted with anti-Axl antibody. As
shown in Figure 3A, Gas6
induced tyrosine phosphorylation of Axl. Tyrosine phosphorylation occurred as
early as 5 min after addition of Gas6, and persisted for 60 min with a slight
decrease. The amount of Axl in each sample was almost the same by
immunoblotting with anti-Axl antibody (data not shown).
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We next studied activation of downstream signal transduction pathways by Gas6. Activation of MAP kinases, especially ERK, is predominantly observed after growth factor stimulation of receptor tyrosine kinases, and appears to be a common and central component among various signal transduction pathways (23). Therefore, we examined effects of Gas6 on activation of ERK in mouse mesangial cells. Immunoblotting of cell lysates with anti-phospho ERK antibody revealed that Gas6 induced phosphorylation of ERK (Figure 3B, top panel). Activation of ERK was observed 5 min after Gas6 treatment and returned to the basal level at 60 min. Next, we treated serum-starved mouse mesangial cells with 500 ng/ml (7 nM) Gas6 that had been preincubated with 100 nM Axl-ECD in 500 µl of the starving medium (Figure 3C). Activation of ERK by Gas6 was completely inhibited by preincubation with Axl-ECD. This result suggests that Gas6 exerts its mitogenic effect on mouse mesangial cells through the MAP kinase cascade by activating the Axl receptor tyrosine kinase.
Endogenous Gas6 Contributes to the Proliferation of Mouse Mesangial
Cells
Given that mouse mesangial cells secrete Gas6, which acts as a growth
factor on mesangial cells, we examined the proliferation of mouse mesangial
cells by endogenous Gas6. We evaluated the proliferation of mouse mesangial
cells with or without medium replacement
(Figure 4A).
[3H]-Thymidine incorporation was low when the medium was replaced
with fresh starving medium after 48-h serum starvation. However,
[3H]-thymidine incorporation without medium replacement was 9 times
higher than that with medium replacement. This gap led us to speculate on the
presence of autocrine growth factors from mouse mesangial cells during the
first 48 h of serum starvation.
|
To examine the extent to which endogenous Gas6 contributes to mesangial proliferation, we next added Axl-ECD into the medium during serum starvation (Figure 4B). If endogenous Gas6 was released and stimulated [3H]-thymidine incorporation, Axl-ECD should capture the released Gas6, thereby inhibiting [3H]-thymidine incorporation. The addition of Axl-ECD dose-dependently inhibited the [3H]-thymidine incorporation, indicating that released endogenous Gas6 and Axl function in an autocrine signaling pathway and contribute to the proliferation of mouse mesangial cells.
Warfarin Inhibits the Proliferation of Mouse Mesangial Cells
Warfarin is known to inhibit vitamin K-dependent -carboxylation of
Gas6 (14), which is essential
for its biologic activities
(14,15).
Therefore, it was anticipated that the synthesis of active Gas6 would be
inhibited in the presence of warfarin. We therefore examined whether mesangial
proliferation due to endogenous Gas6 is inhibited in the presence of warfarin.
First, serum-starved mouse mesangial cells were treated with various
concentrations of warfarin without medium replacement, and
[3H]-thymidine incorporation was measured
(Figure 5A). Warfarin
dose-dependently inhibited [3H]-thymidine incorporation at
concentrations of 0.01 to 1.0 µM. This inhibitory effect was not due to
warfarin's cytotoxicity, because lactate dehydrogenase in the conditioned
medium of warfarin-treated cells on day 4 was not increased compared with that
of nontreated cells (data not shown). We also examined whether warfarin
treatment can affect the expression of gas6 and Axl. The expression
levels of Axl protein and mRNA of gas6 were not affected by the
treatment with warfarin (data not shown). We next examined whether the
inhibitory effect of warfarin can be canceled by the simultaneous
administration of vitamin K. The effect of warfarin on
[3H]-thymidine incorporation was partially counteracted by 1 µM
vitamin K (Figure 5A),
suggesting that warfarin's effect is at least in part due to its specific
inhibitory effect on vitamin K-dependent -carboxylation. Finally, we
investigated the ability of conditioned medium of mesangial cells to stimulate
phosphorylation of ERK in the presence or absence of warfarin and vitamin K.
It was predicted that conditioned medium (CM) and vitamin K-treated
conditioned medium (KCM) contain active Gas6, while conditioned medium with 1
µM warfarin (warfarin-treated conditioned medium [WCM]) contains an
inactive form of Gas6. Ten milliliters of conditioned medium was prepared from
serum-starved mesangial cells in the presence or absence of warfarin and
vitamin K for 3 d. These conditioned media were applied to starved mesangial
cells, and activation of ERK was then examined. CM and KCM could stimulate the
phosphorylation of ERK and the stimulatory effect was higher in KCM. However,
WCM could not stimulate the phosphorylation, whereas warfarin- and vitamin
K-treated conditioned medium (WKCM) could stimulate the phosphorylation at a
level similar to that of KCM (Figure
5B). These results indicate that serum-starved mesangial cells
secrete some autocrine growth factor, which stimulates the phosphorylation of
ERK. The activity of the factor was inhibited in the presence of warfarin, and
this inhibitory effect of warfarin was canceled by the simultaneous
administration of vitamin K. These results excluded the possibility that
warfarin has a direct effect on ERK activation.
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|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-carboxylation of Gas6. Our study has shown that exogenous Gas6 phosphorylated Axl (Figure 3A), stimulated ERK (Figure 3B), and increased [3H]-thymidine incorporation in mouse mesangial cells (Figure 2). Previously, it was reported that Gas6 can stimulate [3H]-thymidine incorporation in NIH3T3 cells (11), and Gas6 can potentiate [3H]-thymidine incorporation in the presence of G protein-coupled receptor agonist in VSMC (10). Therefore, we suggest that the effects of Gas6 in mesangial cells are similar to that in NIH3T3, rather than in VSMC.
We also suggest that endogenous growth factors contribute to mesangial
proliferation, because medium replacement decreased [3H]-thymidine
incorporation in mesangial cells (Figure
4A). These results indicate that serum-starved mesangial cells
secrete some autocrine growth factors to the conditioned medium
(1). This increased
[3H]-thymidine incorporation without medium replacement was
inhibited by warfarin by 70% (Figure
5A). The similar effect of warfarin was observed for ERK
activation (Figure 5B). These
results indicate that the serum-starved mesangial cells secrete some growth
factor that is inactivated in the presence of warfarin, an inhibitor of
vitamin K-dependent -carboxylation. [3H]-Thymidine
incorporation stimulated by CM was also inhibited in the presence of Axl-ECD
by 35% (Figure 4B). Taken
together, these results indicate that Gas6, a vitamin K-dependent protein, is
one of the autocrine growth factors for mesangial cells.
The antiproliferative effect of warfarin seems to be stronger than that of Axl-ECD (Figures 4B and 5A). This difference might result from different mechanisms of effect of these two agents. Axl-ECD is a competitive inhibitor that prevents endogenous Gas6 from binding the cell surface receptor Axl, whereas warfarin inhibits the synthesis of active Gas6. Another possibility is that warfarin might prevent synthesis of other unknown growth factors. The last possibility is that Axl-ECD might be degraded during the 2-d incubation period. This might explain that inhibition of effect of exogenous Gas6 by Axl-ECD (Figure 2B) is more potent than inhibition of endogenous growth factors (Figure 4B).
The mitogenic effect of Gas6 was less potent than that of PDGF (Figure 2A). However, Gas6 might be clinically important because it is a unique vitamin K-dependent growth factor for mesangial cells. Therefore, it could be a target of warfarin therapy.
Our results address the question of the significance of Gas6/Axl signaling pathways to the proliferation of mesangial cells in vivo. In a preliminary study, we have found that serum concentrations of Gas6 were correlated well with the severity of mesangial proliferation in experimental glomerulonephritis (M. Yanagita, K. Ishii, H. Arai, H. Ozaki, T. Nakano, K. Ohashi, K. Mizuno, T. Kita, and T. Doi, manuscripts in preparation). Furthermore, we investigated the effect of warfarin on the clinical course of experimental glomerulonephritis (M. Yanagita et al., manuscripts in preparation). Considering that our in vitro data showed the antiproliferative effect of warfarin at low concentrations, we hypothesized that lower concentrations of warfarin might be enough for the treatment of glomerulonephritis than those clinically used.
In summary, Gas6/Axl signaling pathways are potential mediators of
mesangial proliferation, and warfarin prevents proliferation of mesangial
cells possibly by inhibiting -carboxylation of Gas6. Additional studies
are required to elucidate in vivo functions of the Gas6/Axl pathway
and its relation to mesangial proliferation.
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Acknowledgments |
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This study was supported by research grants (08407026, 09044293, 09281104, 09281103, 09877219, 10470216) from a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, and by Takeda Medical Research Foundation (1998, 1999).
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-carboxylation and a sex hormone-binding globulin-like domain in
receptor-binding and in biological activities of Gas6. FEBS
Lett 408: 306-310,1997[Medline]
-carboxyglutamic acid residues for the biological
activities of Gas6. Biochem J 323:387
-392, 1997
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