Gas6/Axl-PI3K/Akt pathway plays a central role in the effect of statins on inorganic phosphate-induced calcification of vascular smooth muscle cells

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Abstract

Apoptosis is essential for the initiation and progression of vascular calcification. Recently, we showed that 3-hydroxy-3-methylglutaryl (HMG) CoA reductase inhibitors (statins) have a protective effect against vascular smooth muscle cell calcification by inhibiting apoptosis, where growth arrest-specific gene 6 (Gas6) plays a pivotal role. In the present study, we clarified the downstream targets of Gas6-mediated survival signaling in inorganic phosphate (Pi)-induced apoptosis and examined the effect of statins. We found that fluvastatin and pravastatin significantly inhibited Pi-induced apoptosis and calcification in a concentration-dependent manner in human aortic smooth muscle cells (HASMC), as was found with atorvastatin previously. Gas6 and its receptor, Axl, expression were downregulated in the presence of Pi, and recombinant human Gas6 (rhGas6) significantly inhibited apoptosis and calcification in a concentration-dependent manner. During apoptosis, Pi suppressed Akt phosphorylation, which was reversed by rhGas6. Wortmannin, a specific phosphatidylinositol 3-OH kinase (PI3K) inhibitor, abolished the increase in Akt phosphorylation by rhGas6 and eliminated the inhibitory effect of rhGas6 on both Pi-induced apoptosis and calcification, suggesting that PI3K-Akt is a downstream signal of the Gas6-mediated survival pathway. Pi reduced phosphorylation of Bcl2 and Bad, and activated caspase 3, all of which were reversed by rhGas6. The inhibitory effect of statins on Pi-induced apoptosis was accompanied by restoration of the Gas6-mediated survival signal pathway: upregulation of Gas6 and Axl expression, increased phosphorylation of Akt and Bcl2, and inhibition of Bad and caspase 3 activation. These findings indicate that the Gas6-mediated survival pathway is the target of statins' effect to prevent vascular calcification.

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).

Section snippets

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.

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