Gas6/Axl-PI3K/Akt pathway plays a central role in the effect of statins on inorganic phosphate-induced calcification of vascular smooth muscle cells
Introduction
Vascular calcification, such as coronary and aortic calcification, is clinically important in the development of cardiovascular disease (Eggen, 1968). Two distinct forms of vascular calcification are well recognized. One is medial calcification, which occurs between the cell layers of smooth muscle cells and is related to aging, diabetes and chronic renal failure (Neubauer, 1971, Goodman et al., 2000). The other is atherosclerotic calcification, which occurs in the intima during the development of atheromatous disease (Wexler et al., 1996). In diabetic patients, medial calcification has been shown to be a strong independent predictor of cardiovascular mortality (Everhart et al., 1988).
We recently demonstrated that atorvastatin prevented inorganic phosphate (Pi)-induced calcification by inhibiting apoptosis, one of the important processes regulating calcification. This was mediated by growth arrest-specific gene 6 (Gas6), a vitamin K-dependent protein (Son et al., 2006). Gas6 binds to Axl, the predominant receptor for Gas6, on the cell surface and transduces the signal by Axl autophosphorylation (Mark et al., 1996). Gas6-Axl interaction has been shown to be implicated in the regulation of multiple cellular functions (Yanagita et al., 2001, Goruppi et al., 1996, Nakano et al., 1997, Fridell et al., 1998). Especially, they are known to protect a range of cell types from apoptotic death (Goruppi et al., 1996, Goruppi et al., 1999, Healy et al., 2001). However, the downstream targets of Gas6-mediated signaling in Pi-induced apoptosis and the effect of statins on this pathway are poorly understood.
With respect to the targets of Gas6-Axl interaction, Lee et al. (2002) showed that activation of Akt is necessary for Gas6-dependent cell survival. Akt is an important mediator of metabolic and survival responses after growth factor stimulation. Akt is activated by phosphorylation, which is performed by phosphatidylinositol 3-OH kinase (PI3K), a kinase that is activated by Gas6-Axl interaction (Lee et al., 2002, Ming Cao et al., 2001). Activation of Akt leads to downstream signaling events including those associated with mitochondrial regulators of apoptosis such as Bcl2 and Bad.
In the present study, we examined the effect of statins using two different types: lipophilic fluvastatin and hydrophilic pravastatin. We investigated the effect of statins on Pi-induced apoptosis and calcification as well as on signaling components in this process. Consequently, we found that both statins restored the Gas6-mediated survival pathway, with upregulation of the expression of Gas6 and Axl, increased phosphorylation of Akt, Bcl2 and Bad; and finally inhibition of caspase 3 activation, resulting in the prevention of apoptosis and subsequent calcification in human aortic smooth muscle cells (HASMC).
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Materials
Pravastatin and fluvastatin were supplied by Sankyo Co. Ltd. and Tanabe Seiyaku Co., Ltd., respectively. Recombinant human Gas6 (rhGas6) was prepared as described previously (Ming et al., 2001). Wortmannin was purchased from Calbiochem. All other reagents were of analytical grade.
Cell culture
HASMC were obtained from Clonetics. They were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin at 37 °C in a
Statins inhibit Pi-induced apoptosis and calcification in HASMC
In HASMC, a high Pi level (≥ 2.6 mM), comparable to that of hyperphosphatemia in end-stage renal disease, significantly induced calcification. Fluvastatin showed an inhibitory effect on Pi-induced calcification at as high a concentration as 0.1 μM (26.1 ± 2.3% of control), while pravastatin showed the degree of effect at 50 μM (27.4 ± 3.1% of control) (Fig. 1A). An inhibitory effect on Ca deposition was also found by von Kossa's staining (Fig. 1B). Both statins prevented Pi-induced apoptosis at the
Discussion
In the present study, we found that both lipophilic fluvastatin and hydrophilic pravastatin protected against Pi-induced apoptosis and calcification in HASMC, as we found with atorvastatin previously. With regard to the different potency of statins, we found that the inhibitory effect of pravastatin was inferior to those of fluvastatin and atorvastatin, which exerted similar effects on calcification and apoptosis. This might relate to our previous finding that the inhibition of calcification by
Acknowledgements
This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (No. 15390239), Mitsui Sumitomo Insurance Welfare Foundation, Ono Medical Research Foundation, Kanzawa Medical Research Foundation, Novartis Foundation for Gerontological Research, and Takeda Research Foundation. We thank Yuki Ito for technical assistance.
References (28)
- et al.
Statins inhibit reoxygenation-induced cardiomyocyte apoptosis: role for glycogen synthase kinase 3β and transcription factor β-catenin
J. Mol. Cell. Cardiol.
(2004) - et al.
Gas6 induces Axl-mediated chemotaxis of vascular smooth muscle cells
J. Biol. Chem.
(1998) - et al.
Characterization of Gas6, a member of the superfamily of G domain-containing proteins, as a ligand for Rse and Axl
J. Biol. Chem.
(1996) - et al.
Identification of the product of growth arrest-specific gene 6 as a common ligand for Axl, Sky, and Mer receptor tyrosine kinases
J. Biol. Chem.
(1996) - et al.
Prevention of growth-arrest induced cell death of vascular smooth muscle cells by a product of growth arrest-specific gene, gas6
FEBS Lett.
(1996) - et al.
Cell adhesion to phosphatidylserine mediated by a product of growth arrest-specific gene 6
J. Biol. Chem.
(1997) - et al.
Gas6 regulates mesangial cell proliferation through Axl in experimental glomerulonephritis
Am. J. Pathol.
(2001) - et al.
Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L)
Cell
(1996) - et al.
Interleukin-3-induced phosphorylation of Bad through protein kinase Akt
Science
(1997) Relationship of calcified lesions to clinically significant atherosclerotic lesions
Ann. N. Y. Acad. Sci.
(1968)