Fluvastatin Upregulates the α 1C Subunit of CaV1.2 Channel Expression in Vascular Smooth Muscle Cells via RhoA and ERK/p38 MAPK Pathways

Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis. And this process has been related to remodeling of L-type calcium channel (LTCC). We attempted to investigate whether fluvastatin has any effect on VSMC proliferation and LTCCα 1C subunit (LTCCα 1C) expression as well as the potential mechanisms involved. The VSMCs proliferation was assayed by osteopontin immunofluorescent staining and [3H]-thymidine incorporation. The cell cycle was detected by flow cytometric analysis. The activity of RhoA was determined with pull-down assay. MAPK activity and LTCCα 1C expression were assessed by western blotting. We demonstrated fluvastatin prevented the VSMCs dedifferentiating into a proliferative phenotype and induced cell cycle arrest in the G0/G1 phase in response to PDGF-BB stimulation. Fluvastatin dose-dependently reversed the downregulation of LTCCα 1C expression induced by PDGF-BB. Inhibition of ROCK, ERK, or p38 MAPK activation largely enhanced the upregulation effect of fluvastatin (P < 0.01). However, blockade of JNK pathway had no effect on LTCCα 1C expression. We concluded LTCCα 1C was a VSMC contractile phenotype marker gene. Fluvastatin upregulated LTCCα 1C expression, at least in part, by inhibiting ROCK, ERK1/2, and p38 MAPK activation. Fluvastatin may be a potential candidate for preventing or treating vascular diseases.


Introduction
Vascular smooth muscle cells (VSMCs) show the unique ability of undergoing a well-known phenotypic switch from differentiated, contractile cells to a proliferating phenotype, a process essential for restenosis, atherosclerosis, and hypertension. VSMCs normally exist in a quiescent, differentiated state in the blood vessel wall expressing a unique repertoire of contractile proteins, calcium ion channels, and signaling molecules that are necessary for their contractile properties [1]. Under the pathological conditions, VSMCs lose the contractile phenotype, which is associated with the silencing of contractile marker gene expression and the upregulation of genes that facilitates other cellular functions, such as proliferation and migration [2]. L-type calcium channel (LTCC) serves as a critical pathway gating the influx of Ca 2+ into the cytoplasm from the extracellular space. LTCC are heteroligomeric complexes consisting of 5 subunits ( 1 , 2 , , , and ). The LTCC 1C (LTCC 1C ) subunit functions as a voltage sensor, drug receptor, and Ca 2+ -selective pore. An increase in [Ca 2+ ] i initiates VSMCs contraction, which is a key process of excitationcontraction coupling [3]. Convincing data suggests that cell proliferation abolishes the expression of LTCC and reexpression of LTCC 1C parallels the reappearance of contractile phenotype marker [4][5][6]. Substantial evidence shows that platelet derived growth factor (PDGF) induces VSMCs proliferation through the mitogen-activated protein kinase (MAPK) and the Rho associated protein kinase (ROCK) 2 Disease Markers pathways [7]. However, there is a paucity of information available on the specific signal pathway involved in the PDGF-mediated regulation of LTCC 1C expression.
It is well known that statins, HMG-CoA (3-hydroxy-3-methyl-glutaryl coenzyme A) reductase inhibitors, exert pleiotropic properties. And they can inhibit VSMCs proliferation [8]. Wagner et al. reported lovastatin could induce VSMC differentiation and prevented the downregulation of contractile protein expression [9]. And our previous studies indicated fluvastatin regressed resistant vessel remodeling, ameliorated vasodilatation function in rats [10], and inhibited VSMCs migration [11]. However, the direct evidence regarding the effects of fluvastatin on the expression of LTCC 1C in VSMCs and the exact mechanism involved is still unavailable. Therefore, we investigate the effect of fluvastatin on LTCC 1C expression in response to PDGF and further explore the potential underlying mechanisms.

Cell Culture and Treatment.
VSMCs were isolated from the thoracic aortas of spontaneously hypertensive rats by the modified explant technique of Champbell in our laboratory as described previously [12][13][14]. And the experimental procedures were approved by the Animal Care and Use Committee of Fujian Medical University. Briefly, cells were grown in DMEM (Dulbecco's modified Eagle medium). VSMCs were identified by immunostaining procedures using anti-smooth muscle -actin antibody. And the third passage cells were used for experiments. When cells reached 80%-90% confluence, they were put to serum-free starvation for 24 h to synchronize the cell cycle. To study the time-response of Flu on proliferation and LTCC 1C expression, quiescent VSMCs were incubated with 10 g/L PDGF-BB, in the absence or in the presence of 10 −5 M fluvastatin. Then VSMCs were collected every 4 h till 24 h. To investigate the dose response of Flu on cell proliferation and LTCC 1C expression, VSMCs were preincubated with Flu (gift from Novartis Pharma AG, Switzerland) at the graded concentrations (10 −4 M∼10 −8 M) for 0.5 h before the addition of PDGF and incubated for 24 h. To determine the role of MAPK pathways in PDGF-mediated effects on LTCC 1C expression, confluent quiescent VSMCs were pretreated with the specific ERK1/2 inhibitor (PD98059, 20 mol/L), p38 MAPK inhibitor (SB203580, 10 mol/L), JNK inhibitor (SP600125, 50 mol/L), or ROCK-I/II inhibitor (Y27632, 10 mol/L) for 1 h, and/or Flu (10 −5 M) for 0.5 h before the addition of PDGF and incubated for 24 h. To test whether Flu affected the MAPK activity in response to PDGF, VSMCs were cultured with or without fluvastatin (10 −5 M) for 24 h and then treated with PDGF (10 g/L) for 5, 15, 30, and 60 min.

Flow Cytometry
Analysis of Cell Cycle. Cells density was adjusted to 1.0 × 10 6 cells/cm 3 and preincubated with fluvastatin (10 −5 M) for 0.5 h before the addition of PDGF-BB (10 g/L) and incubated for 24 h. Cells incubated with PDGF-BB alone served as control. Then the cells were harvested and fixed with 70% ethanol and incubated with RNase A (20 mg/L) and propidium iodide (50 mg/L) for 1 h in the dark. The stained cells were determined using a flow cytometer in combination with Flow Jo software.

RhoA
Pull-Down Assay. Active RhoA was isolated using the Rho activity binding domain of rhotekin as described by Ren and Schwartz [15]. Briefly, cells were preincubated with fluvastatin (10 −5 M) for 0.5 h before the addition of PDGF-BB (10 g/L) and treated for 24 h. Then those cells were lysed with buffers and incubated with 40 g of GST-RBD (Cytoskeleton, Acoma Street, Denver, USA) for 1 hour. After binding, the samples were washed with lysis buffer three times. Pulleddown proteins that are activated Rho (GTP-bound Rho) were fractionated on 12% SDS-PAGE and immunoblotted with polyclonal Ab against RhoA (Santa Cruz Biotechnology, CA, USA). The total cell lysates were also blotted with Ab for RhoA as a loading control. The level of activated RhoA was determined after normalization with the total RhoA present in the same cell lysates.
2.6. Statistical Analysis. All data was expressed as the mean ± SEM unless otherwise indicated. Group comparisons were performed with Student's -test (2-sample test) or one-way analysis of variance using SPSS 13.0. All reported probability values were 2-tailed, and a value of 0.05 was considered as statistical significance.

Fluvastatin Inhibited the Phenotype Switching and Proliferation of VSMC Elicited by PDGF.
VSMCs were adherently cultured using Petri dishes (Figure 1(a)). As depicted by immunofluorescent staining, -smooth muscle-actin was positive, suggesting those cells were smooth muscle cells ( Figure 1(b)). Alternatively, the fluorescence intensity of osteopontin (proliferate phenotype marker) was markedly enhanced in VSMCs treated with PDGF for 24 hs (Figure 1(c)), while it was attenuated by being cocultured with PDGF and 10 −5 M Flu (Figure 1(d)). Strikingly, VSMCs exposed to PDGF exhibited mostly flattened, fibroblastic appearance, nuclear division, and increased cell density.

Fluvastatin Induced Cell Cycle Arrest in VSMCs Stimulated by PDGF.
The proportion of VSMCs in the G0/G1 phase was decreased when incubated with 10 g/L PDGF for 24 h (33.8% ± 3.2% versus 66.7% ± 3.8% in vehicle VSMCs). Meanwhile, the percentage of cells in the S phase was increased in the PDGF incubation cells (49.2% ± 2.1% versus 29.6% ± 1.0% in vehicle VSMCs). Additionally, Flu significantly reversed the alterations of the VSMC cell cycle elicited by PDGF. The percentage of cells in G0/G1 increased to 53.8% ± 4.2%, while the cells in S phase decreased to 30.3% ± 2.8% when cotreated with 10 −5 M Flu and 10 g/L PDGF (Figure 2). This data indicated that Flu inhibited cell proliferation by inducing cell cycle arrest at the G0/G1 phase.

PDGF Time-Dependently Inhibited LTCC 1C Expression Which Could Be Prevented by Fluvastatin.
To determine whether the VSMC phenotype switching was associated with the alterations in LTCC 1C expression, the protein level of LTCC 1C was determined by western blot analysis. PDGF suppressed the LTCC 1C protein expression timedependently and the peak effect occurred after PDGF stimulation for 24 h (both < 0.01, Figure 3(a)). Exposure to PDGF for 24 h decreased LTCC 1C protein expression by 74.7%, as compared with vehicle cells. In addition, fluvastatin dose-dependently reversed the downregulation of LTCC 1C expression elicited by PDGF-BB. And the most efficient Flu concentration was at 10 −5 M. LTCC 1C protein expression was increased by 2.72-fold in VSMCs cotreated with PDGF and Flu, as compared to that in VSMCs incubated with PDGF ( Figure 3(b)).

Effect of Fluvastatin on PDGF-Induced Activation of MAPK.
VSMCs are known to acquire proliferative characteristics through the MAPK pathway, of which extracellular signal regulated kinase (ERK) 1/2, p38 MAP kinase, and c-Jun Nterminal kinase (JNK) are key components that play critical roles in the VSMCs proliferation and cell cycle progression.
For this purpose, we tested whether Flu affected the activity of ERK1/2, p38 MAPK, and JNK in response to PDGF. VSMCs were cultured with or without fluvastatin (10 −5 M) for 24 h and then treated with PDGF (10 g/L) for 5, 15, 30, and 60 min. Upon PDGF-BB stimulation for 5-60 min, ERK1/2, p38 MAPK, and JNK activation was dramatically increased in VSMCs, and cotreatment with PDGF and 10 −5 M Flu significantly inhibited ERK1/2 and p38 MAPK activation in a timedependent manner (Figures 5(a)-5(b)). However, activation of JNK was not affected by Flu in PDGF-stimulated VSMCs ( Figure 5(c)). Additionally, the total ERK1/2, p38 MAPK, and JNK levels were not altered by Flu. Collectively, these data indicated that Flu could suppress the phosphorylation of ERK1/2 and p38 MAPK elicited by PDGF.

Discussion
In the present study, we found that (1) PDGF stimulation inhibited LTCC 1C expression in VSMCs by activating RhoA, ERK1/2, and p38 MAPK pathways; (2) fluvastatin promoted a more differentiated VSMC phenotype concurrent with the upregulation of LTCC 1C expression via inactivating RhoA, ERK1/2, and p38 MAPK pathways; and (3) LTCC 1C was downregulated in the proliferating smooth muscle cells. It was a contractile phenotype marker. To the best of our knowledge, our investigation is the first to report the direct effect and the underlying mechanism of fluvastatin treatment on LTCC 1C expression in vitro. Our results indicated that PDGF suppressed LTCC 1C expression, which was similar to the reports that L-type Ca 2+ channel was lost when quiescent VSMCs underwent a phenotypic switch to the proliferating/synthetic state [16,17]. Alternatively, our results were partly substantiated by the investigations that fluvastatin treatment prevented left atrium LTCC 1C subunit downregulation in atrial tachycardia dogs [18]. Our present findings seemingly contradicted our previous investigations that, in hypertrophic pulmonary arteries induced by monocrotaline, atorvastatin downregulated LTCC 1C expression [19]. The possible explanation for this discrepancy might be the lacking of self-homeostatic regulation, neurohumoral regulation, and vascular tone in vitro. This argument was supported by those reports that the expression of the pore-forming 1C subunit of the CaV1.2 channel was elevated in arteries of hypertensive animals compared to age-matched normotensive animals [20,21] while, in proliferating VSMCs in vitro, the LTCC 1C expression was downregulated [22]. The mechanistic insights into the regulating the phenotypic switch of VSMC have been intensely studied. Our data reveals that fluvastatin attenuates PDGF-induced LTCC 1C expression, at least in part, by inhibiting ERK1/2 and p38 MAPK activation. PDGF is known to induce cell proliferation via a pathway involving PKC and small GTPases (Rho, Rac) [23]; ROCK acts as an upstream regulator leading to the activation of the MAPKs family, including p38 MAPK and ERK [24]. PDGF represses the characteristic VSMC gene expression by activating ERK1/2 and p38 MAPK pathways in cultured VSMCs [5,25]. HMG CoA catalyses the conversion of HMG CoA to mevalonate which can then be further metabolized to cholesterol. However, mevalonate is also the precursor of the isoprenoids, farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (ggPP) which plays a key role in the lipid modification of small G proteins, such as Ras and Rho. Depletion of isoprenoids by statins results in accumulation of nonfunctional Rho GTPases in the cytoplasm [26]. Statins therefore not only block cholesterol synthesis but also may inhibit the Ras and Rho signaling pathways. Considering that MAPK was the downstream molecules of isoprenoid 8 Disease Markers pathway, it is not surprising that fluvastatin can upregulate LTCC 1C expression via inhibiting MAPK signal pathway. However, it should be noted that the inactivation of Rho or MAPK could not fully abolish the PDGF-induced downregulation of LTCC 1C expression, suggesting that other signaling pathway(s) might be involved in the regulation of LTCC 1C by PDGF.
In conclusion, our novel findings indicated that fluvastatin prevented the PDGF-induced downregulation of LTCC 1C expression through the suppression of RhoA, ERK1/2, or p38 MAPK signaling. Our data is encouraged to unravel why calcium channel blockers are effective in uninjured arteries and at the early stages of disease but subsequently lose efficacy as the disease progresses and VSMCs become progressively more dedifferentiated. Furthermore, our findings indicate that statins induce a more differentiated VSMC phenotype paralleling the increased L-type calcium channels expression, which provide a rationale for the synergistic effects of statins and calcium channel blockers to lower blood pressure in hypertensive patients [27,28]. Considering that posttranslation modifications can alter the function of ion channel without requiring the changes in protein expression, patch-clamp experiments will be necessary to further verify the role of fluvastatin in calcium channel properties. Additionally, further studies are needed to clarify the involvement of Rac1 and Cdc42 in the upregulation effects of fluvastatin on LTCC 1C expression by PDGF.