Statins Augment Vascular Endothelial Growth Factor Expression in Osteoblastic Cells via Inhibition of Protein Prenylation
Abstract0p, http://www.100md.com
Statins such as simvastatin are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that inhibit cholesterol synthesis. We presently investigated statin effects on vascular endothelial growth factor (VEGF) expression in osteoblastic cells. Hydrophobic statins including simvastatin, atorvastatin, and cerivastatin–but not a hydrophilic statin, pravastatin–markedly increased VEGF mRNA abundance in nontransformed osteoblastic cells (MC3T3-E1). Simvastatin (10-6 M) time-dependently augmented VEGF mRNA expression in MC3T3-E1 cells, mouse stromal cells (ST2), and rat osteosarcoma cells (UMR-106). According to heterogeneous nuclear RNA and Northern analyses, 10-6 M simvastatin stimulated gene expression for VEGF in MC3T3-E1 cells without altering mRNA stability. Transcriptional activation of a VEGF promoter-luciferase construct (-1128 to +827), significantly increased by simvastatin administration. As demonstrated by gel mobility shift assay, simvastatin markedly enhanced the binding of hypoxia-responsive element-protein complexes. These results indicate that the stimulation of the VEGF gene by simvastatin in MC3T3-E1 cells is transcriptional in nature. VEGF secretion into medium was increased in MC3T3-E1 by 10-6 M simvastatin. Pretreating MC3T3-E1 cells with mevalonate or geranylgeranyl pyrophosphate, a mevalonate metabolite, abolished simvastatin-induced VEGF mRNA expression; manumycin A, a protein prenylation inhibitor, mimicked statin effects on VEGF expression. The effect of simvastatin was blocked by pretreatment with wortmannin and LY294002, specific phosphatidylinositide-3 kinase inhibitors. Simvastatin enhanced mineralized nodule formation in culture, whereas coincubation with mevalonate, geranylgeranyl pyrophosphate, LY294002, or VEGF receptor 2 inhibitor (SU1498) abrogated statin-induced mineralization. Thus, statins stimulate VEGF expression in osteoblasts via reduced protein prenylation and the phosphatidylinositide-3 kinase pathway, promoting osteoblastic differentiation.
Introductionvaa, 百拇医药
COMPETITIVE INHIBITORS OF 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, also called statins, act at a rate-limiting step in cholesterol synthesis by blocking conversion of HMG-CoA to mevalonate (1). Statins, including simvastatin and atorvastatin, are highly effective cholesterol-lowering drugs that are widely considered to reduce morbidity from coronary artery disease (2, 3). Besides lowering serum lipids, statins can decrease platelet aggregation and thrombus deposition (4); promote angiogenesis (5); decrease ß-amyloid peptide, related to Alzheimer’s disease (6, 7); and suppress T lymphocyte activation (8). By modulating the initial part of the cholesterol synthesis pathway, statins decrease availability of several important intermediate compounds, including isoprenoid, that contain geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP). Isoprenoids are lipids attached by post-translational modification to some proteins such as the small G proteins, including Ras and Ras-like proteins (Rho, Rap, Rab, and Ral) (9).
Integrity of the skeleton requires a dynamic balance between bone formation and bone resorption, which are controlled by calcitropic hormones and cytokines. When bone resorption exceeds bone formation, diseases of bone metabolism such as postmenopausal osteoporosis can result (10). Communication between bone-forming osteoblasts and bone-resorbing osteoclasts is very close, and coupling of bone resorption to bone formation is necessary for maintenance of healthy bone. Osteoblasts arise from mesenchymal stem cell precursors and undergo differentiation in response to a number of factors including bone morphogenetic proteins (BMPs), TGF-ß, IGF-I, vascular endothelial growth factor (VEGF), and glucocorticoids (11, 12, 13, 14, 15, 16, 17, 18). Osteoblasts are located in proximity to endothelial cells. Vascular invasion is a prerequisite for endothelial bone formation and fracture healing. Gerber et al. (16) demonstrated in young mice that blocking the action of endogenous VEGF inhibited bone formation and resorption. Important interplay takes place between endothelial cells and osteoblasts, and the angiogenic factor VEGF appears to be involved in this intercellular communication (19, 20, 21).
VEGF is a glycoprotein that acts as both a promoter of permeability and an angiogenic factor (22, 23, 24). VEGF also has mitogenic actions restricted to endothelial cells and is chemotactic for endothelial cells (25). VEGF is secreted by many cell types, including osteoblasts and smooth muscle cells; its expression is regulated by a variety of growth factors and cytokines (17, 26, 27, 28). Several splice variants of VEGF, as well as their receptors, are expressed in osteoblasts (29) in which this cytokine has been shown to induce alkaline phosphatase activity and enhance responsiveness to PTH (15). We recently have shown that simvastatin promotes osteoblast differentiation and mineralization (30). BMPs such as BMP-2 have been shown to induce osteoblast differentiation and ectopic bone formation (31). Statins induce BMP-2 expression in osteoblasts and stimulate bone formation (30, 32, 33, 34). We have searched for differentiation mechanisms in addition to BMP-2 that are induced by statins in osteoblasts. We report here that statins, including simvastatin, induced VEGF expression via inhibition of protein prenylation in MC3T3-E1 osteoblastic cells.
Materials and Methods81, 百拇医药
Cell culture81, 百拇医药
MC3T3-E1 cells (a clonal preosteoblastic cell line derived from newborn mouse calvaria) were grown in -MEM (ICN Pharmaceuticals, Inc. Aurora, OH) supplemented with 10% fetal bovine serum (FBS) as previously described (26, 30) until 3 d after confluence. Then, the culture medium was replaced for 24 h with differentiation medium [{alpha} -MEM containing a 50 µg/ml concentration of the phosphate ester of ascorbic acid (Wako Pure Chemical Industries Ltd., Osaka, Japan) and 10 mM ß-glycerophosphate (Wako Pure Chemical Industries Ltd.)]. Cells were exposed to statins to be tested such as 1 nM to 100 µM simvastatin (Calbiochem, San Diego, CA), 10–100 µM atorvastatin (Yamanouchi Pharmaceutical, Tokyo, Japan), 0.1–1 µM cerivastatin (Takeda Pharmaceutical, Osaka, Japan), and 100 µM pravastatin (Sankyo Co., Ltd., Tokyo, Japan) for the indicated time periods. In studies of inhibitors, serum-deprived cells were treated with 1 mM mevalonate (Sigma, St. Louis, MO), 20 µM GGPP (Sigma), 2–20 µM LY294002 or 100 nM wortmannin [Sigma; both being phosphatidylinositide-3 kinase (PI3K) inhibitors], or 5 µM manumycin A (Sigma; a protein prenylation inhibitor) in the absence or presence of 1 µM simvastatin for the times indicated. In some experiments, cells were exposed to 75 µM 5,6-dichlorobenzimidazole riboside (DRB, Sigma; a RNA polymerase II inhibitor, dissolved in dimethylsulfoxide) 8 h after exposure to 1 µM simvastatin or vehicle. Mouse bone marrow-derived stromal ST2 cells were supplied by the RIKEN Cell Bank (Tsukuba, Japan) and were cultured in RPMI 1640 (ICN Pharmaceuticals, Inc.) that contained 10% FBS. After the cells reached confluence, they were cultured in {alpha} -MEM containing 10% FBS for 6 d. Then, cells were cultured in differentiation medium without FBS for 24 h. Cells were treated with 1 µM of test substances such as simvastatin for the period indicated. Rat osteoblast-like osteosarcoma UMR-106 cells (CRL1661, American Type Culture Collection, Manassas, VA) were grown by routine methods in monolayer culture in DMEM (ICN Pharmaceuticals, Inc.) containing 5% FBS. At confluence, the culture medium was replaced with serum-free DMEM for 24 h (35). Cells were exposed to 1 µM simvastatin for the indicated time period.
Determination of VEGF and BMP-2 mRNAsa$-^.[, http://www.100md.com
VEGF mRNA abundance was determined by Northern blot analysis. To prepare the probe for Northern analysis, we isolated total RNA from MC3T3-E1 cells. RT-PCRs were performed using a Gene Amp RNA PCR kit (PE Applied Biosystems, Foster City, CA). A 410-bp probe against mouse VEGF gene was generated by PCR from an oligonucleotide-primed single-stranded cDNA using the following PCR primer sequences: 5'-CGAGACCCTGGTGGACATCT-3' and 5'-CACCGCCTCGGCTTGTCAC-3'. Resulting bands were cloned into a pCR 2.1 vector (Invitrogen, San Diego, CA) and sequenced to confirm sequence identity (GenBank accession no. NM 009505; Ref. 36). Total RNA was extracted from cells using guanidine thiocyanate. Total RNA was fractionated on 1.2% agarose gels containing formaldehyde and transferred to a nylon membrane (Hybond-XL; Amersham Biosciences, Buckinghamshire, UK). The membranes were hybridized with mouse VEGF cDNA probe labeled with [{alpha} -32P]deoxy-CTP (specific activity, 110 TBq/mmol; ICN Pharmaceuticals, Inc.) using a Megaprime DNA labeling kit (Amersham). Hybridization was performed at 42 C for 48 h, followed by washing with 0.1–2x SSPE [150 mM NaCl, 10 mM sodium phosphate, and 1 mM EDTA (pH 7.4)] with 0.1% SDS at 65 C. For standardization, blots were rehybridized with a cyclophilin cDNA probe. Membrane signal intensity was quantified with a Molecular Imager FX (Bio-Rad Laboratories, Inc. Hercules, CA), and the resulting images were analyzed using Quantity One 4.1.1 (Bio-Rad Laboratories, Inc.) image analysis software. RT-PCR was performed to clarify BMP-2 mRNA expression. One microgram of total RNA was copied into cDNA, and the synthesized cDNA was amplified using a sense primer (5'-ATCAACTAGAAGCCGTGGAG-3') and an antisense primer (5'-CAATGGCCTTATCTGTGACC-3') by PCR (22 cycles). The relative amount of the BMP-2 transcript was corrected for the amount of cyclophilin mRNA present.
Quantification of VEGF heterogeneous nuclear RNA (hnRNA);4[w\, http://www.100md.com
VEGF hnRNA was determined by RT-PCR using specific primers designed to amplify hnRNA from an intron to an exon, as described previously (35). Briefly, the nucleotide sequences containing exon 3, the interposed intron, and exon 4 of murine VEGF gene were determined and submitted to a database (GenBank accession no. AB086118). A sense primer (5'-TAACGATGAAGCCCTGGAGT-3'), spanning nucleotides 100–119 of exon 3 of the murine VEGF gene, and an antisense primer (5'-AGCAGATGGTCAATTCGTGG-3'), spanning nucleotides 375–394 of the intron between exons 3 and 4, were synthesized. Total RNA was extracted from MC3T3-E1 cells and treated with ribonuclease-free deoxyribonuclease I (Boeringer, Indianapolis, IN) to remove potentially contaminating DNA. One microgram of RNA was copied into cDNA using reverse transcriptase and random hexanucleotide primers. The newly synthesized cDNA was amplified by PCR through 21 cycles. PCR products were loaded onto a 1.2% agarose gel and transferred to a nylon membrane. Southern blotting was performed using a murine VEGF genomic DNA (the region of hnRNA) probe radiolabeled with [{alpha} -32P]deoxy-CTP. Amplified RNA from murine VEGF gene was corrected for the level of cyclophilin hnRNA by RT-PCR using the same synthesized cDNA. PCR was performed for 18 cycles. Signal intensity was quantified with the Molecular Imager FX.
Transient expression assay and gel mobility shift assay\#fqqou, http://www.100md.com
Assays of transient expression and gel mobility shift were performed as described previously (37). Briefly, a fragment comprised of -1128 to +827 of a mouse VEGF gene promoter was cloned upstream of the luciferase reporter gene in the pGL3-basic reporter vector. A mutated construct of the mouse VEGF gene promoter without the hypoxia responsive element (HRE; -1128/-928 and -906/+827) has also been cloned. The nucleotide sequence of the clone was confirmed by comparison with a published sequence of mouse VEGF gene (38). Transient transfections were performed using MC3T3-E1 cells in six-well plates. Cells were cotransfected with 1.0 µg luciferase reporter plasmid and 1.0 µg ß-galactosidase expression vector (pEFBOS-LacZ) as an internal control, using Gene Porter transfection reagent (Gene Therapy Systems, San Diego, CA). Transfected cells were incubated with {alpha} -MEM containing 10% FBS for 18 h. The cells treated with a vehicle or with 1 µM simvastatin were cultured in {alpha} -MEM containing 1% BSA for 24 h, and were then harvested for the luciferase assay. Luciferase activity was corrected for transfection efficiency as indicated by the ß-galactosidase activity. For the gel mobility shift assay, the MC3T3-E1 cells were treated with a vehicle and with 10 µM simvastatin for 12 h. Nuclear extracts from the cells were prepared as described previously (37). Nuclear extracts and radiolabeled oligonucleotides of the HRE of the VEGF gene (-921 to -901; Ref. 39) and its mutant were incubated for 20 min at 25 C in a solution containing 10 mM HEPES, 50 mM KCl, 0.1 mM EDTA, 3 mM Mg2Cl2, 1 mM dithiothreitol, 6% glycerol, and 0.1 mg/ml polydeoxyinosinic deoxycytidylic acid at pH 7.9. DNA protein complexes were resolved on nondenaturing, nonreducing, 4.5% polyacrylamide gels and were visualized by autoradiography.
VEGF concentration in conditioned medium|2, http://www.100md.com
Serum-deprived MC3T3-E1 cells were treated with vehicle or test substances such as simvastatin and manumycin A for the period indicated. Conditioned media were collected after 12 h, and VEGF concentrations were measured by a mouse VEGF quantitative sandwich enzyme immunoassay (R&D Systems, Minneapolis, MN). This assay measures primarily the 165-amino acid isoform of VEGF, the main soluble isoform. Fifty microliters of conditioned medium were used for the assay.|2, http://www.100md.com
Assay of mineralized matrix formation|2, http://www.100md.com
MC3T3-E1 cells in 24-well plates were cultured in differentiation medium containing 10% FBS and test substance such as simvastatin and SU1498 (Calbiochem; VEGF receptor 2 kinase inhibitor) for 14 d after confluence. After the medium was removed, cells were washed twice with PBS. The extent of mineralized matrix in the plates was determined by Alizarin Red S (AR-S) staining (30, 40). Briefly, cells were fixed in 70% ethanol for 1 h at room temperature, washed with PBS, and stained with 40 mM AR-S (pH 4.2) for 10 min at room temperature. Next, cell preparations were washed five times with deionized water and incubated in PBS for 15 min to eliminate nonspecific staining. The stained matrix was photographed using a 35-mm camera. AR-S staining was released from the cell matrix by incubation in 10% (wt/vol) cetylpyridinium chloride for 15 min. The amount of dye released was quantified by spectrophotometry at 562 nm.
Statistical analysissj+, http://www.100md.com
All values are expressed as the mean ± SEM of three or four measurements. Statistical analyses of the time-course study and the luciferase assay were performed using unpaired Student’s t test. For all other experiments, statistical analyses were performed using Dunnet’s test. Data were analyzed using SAS software (SAS Institute Inc., Cary, NC).sj+, http://www.100md.com
Resultssj+, http://www.100md.com
Induction of VEGF mRNA by statins in osteoblastic cellssj+, http://www.100md.com
We first tested the hypothesis that statins can alter VEGF mRNA expression in osteoblastic cells, using MC3T3-E1 nontransformed cells. This cell line was derived from mouse calvaria and shows many osteoblastic characteristics, such as expression of type I collagen and alkaline phosphatase, and mineralized nodule formation. In these cells, marked enhancement of VEGF mRNA expression was observed with 12-h exposure to hydrophobic statins, including simvastatin, atorvastatin, and cerivastatin, but not with hydrophilic statins such as pravastatin (Fig. 1). To confirm statin-induced expression of VEGF mRNA in osteoblasts, we examined the effect of simvastatin on the mRNA abundance in three osteoblast-like cell lines, including MC3T3-E1 cells, mouse stromal ST2 cells, and rat osteosarcoma UMR-106 cells (Fig. 2). The time course of the effect of simvastatin was determined in MC3T3-E1 cells, in which a significant increase in VEGF mRNA could be detected as early as 6 h after initiation of treatment with 10-6 M simvastatin. Stimulation was maximal at 12 h, resulting in a 2-fold increase above control values; by 24 h, mRNA concentrations in treated cells had decreased nearly to control values (Fig. 2A). Simvastatin (10-6 M) treatment of ST2 cells for 3 h resulted in a 7-fold increase of VEGF mRNA; induction was significant between 3 and 6 h. This stimulation was greater in ST2 cells than in other cell lines (Fig. 2B). In UMR-106 cells, 10-6 M simvastatin significantly increased VEGF mRNA between 6 and 12 h after initiation of treatment (Fig. 2C). In dose-response experiments in MC3T3-E1 cells, a concentration of simvastatin as low as 10-7 M significantly enhanced abundance of VEGF mRNA. Maximal elevation was obtained at simvastatin concentrations of 10-6 and 10-5 M (Fig. 3). However, a high concentration (10-4 M) of the statin reduced VEGF mRNA expression in MC3T3-E1 cells. Thus, statins induced VEGF mRNA expression in osteoblastic cells in a time- and concentration-dependent manner.
fig.ommitteedhk*hm, 百拇医药
Figure 1. Effects of statins on VEGF mRNA abundance in MC3T3-E1 murine osteoblastic cells. The cells were cultured in the medium (-MEM:FBS/9:1) until the third day after confluence. Thereafter, the medium was changed into serum-free differentiation medium (-MEM containing 50 µg/ml of the phosphate ester of ascorbic acid and 10 mM ß-glycerophosphate). Cells were exposed to vehicle, simvastatin (10-6 and 10-5 M), atorvastatin (10-5 and 10-4 M), cerivastatin (10-7 and 10-6 M), or pravastatin (10-4 M) for 12 h. Cells were harvested, and total RNA was extracted. Total RNA (25 µg) was subjected to Northern blot analysis. The estimated sizes of VEGF and cyclophilin (Cyclo) were 3.8 and 0.9 kb, respectively.hk*hm, 百拇医药
fig.ommitteedhk*hm, 百拇医药
Figure 2. Stimulation of VEGF mRNA expression by simvastatin in different osteoblastic cells. Three osteoblastic cell lines such as MC3T3-E1 nontransformed murine osteoblasts (A), mouse bone-marrow derived stromal ST2 cells (B), and rat osteoblast-like osteosarcoma cells (C) were cultured under serum-free conditions for 24 h and thereafter treated with vehicle () or 10-6 M simvastatin () for the indicated period. Total RNA (25 µg) was extracted and subjected to Northern blot analysis. VEGF mRNA abundance was determined densitometrically and normalized to that of cyclophilin (Cyclo.) mRNA. The data are expressed as the mean ± SEM of four determinations. *, P < 0.01 (compared with vehicle-treated control at each point).
fig.ommitteedtf, 百拇医药
Figure 3. Effect of simvastatin on VEGF mRNA abundance in MC3T3-E1 cells. Serum-depleted cells were treated with the indicated concentrations of simvastatin for 12 h. Total RNA from cells was subjected to Northern blot analysis. The abundance was expressed as a percentage of the vehicle-treated control. Each bar represents the mean ± SEM of four measurements. *, P < 0.05; **, P < 0.01 (compared with the vehicle-treated control).tf, 百拇医药
Effects of cholesterol-synthesis metabolites on statin-induced VEGF mRNA expressiontf, 百拇医药
To determine the mechanism of VEGF mRNA induction by statins, MC3T3-E1 cells first were coincubated with simvastatin in the presence of mevalonate or GGPP (Fig. 4A). We tested the effect of mevalonate, the immediate product of the reaction catalyzed by HMG-CoA reductase, to determine whether the effect of simvastatin results from inhibition of this enzyme. We incubated cells with 10-6 M simvastatin together with 1 mM mevalonate. Induction of VEGF mRNA by simvastatin was completely abolished by mevalonate. Mevalonate alone did not modulate VEGF mRNA expression. Next, involvement of the isoprenoid intermediate, GGPP, on modulation of VEGF mRNA by simvastatin was examined. As shown in Fig. 4A, cotreatment of cells with 20 µM GGPP completely abolished the induction of VEGF mRNA synthesis. Because GGPP is used in post-translational modification of several cell proteins, we evaluated the effect of an inhibitor of protein prenylation, manumycin A. VEGF induction with manumycin A alone was remarkable at a low concentration (5 µM) after 12 h of treatment (Fig. 4A). FPP also inhibited the stimulation of VEGF mRNA synthesis induced by simvastatin treatment (data not shown). These results implicated reduced protein prenylation caused by the HMG-CoA reductase inhibitor, simvastatin, in the enhancement of VEGF mRNA expression in osteoblastic cells. Because statins activate the PI3K pathway in several cell types (5), we studied whether stimulation of VEGF mRNA synthesis by simvastatin was mediated via the PI3K pathway in MC3T3-E1 cells. Treatment of cells with two PI3K inhibitors, wortmannin and LY294002, completely blocked VEGF mRNA induction by simvastatin (Fig. 4B). These results strongly suggested that activation of the PI3K pathway by simvastatin was required for gene activation of VEGF.
fig.ommitteed.r5q9e(, http://www.100md.com
Figure 4. Regulation of VEGF mRNA expression by the cholesterol synthetic pathway in MC3T3-E1 cells. A, Serum-depleted cells were treated with 1 mM mevalonate (Mev) or 20 µM GGPP for 1 h before incubation for 12 h with vehicle or 10-6 M simvastatin (Sim). A protein prenylation inhibitor, manumycin A (MaA), at a concentration of 5 µM was added to the cells and incubated for 12 h. B, Serum-depleted cells were treated with vehicle (Veh), 100 nM wortmannin (Wor), or 20 µM LY294002 (LY) for 1 h before incubation for 12 h with vehicle or 10-6 M simvastatin (Sim). Total RNA from cells was subjected to Northern blot analysis. VEGF mRNA concentrations normalized relative to cyclophilin mRNA was expressed as a percentage of the vehicle control value. Each bar represents the mean ± SEM of four determinations. *, P < 0.01 (compared with vehicle control). #, P < 0.01 (compared with simvastatin-untreated vehicle control by Student’s t test)..r5q9e(, http://www.100md.com
Transcriptional regulation of the VEGF gene by simvastatin.r5q9e(, http://www.100md.com
To determine whether simvastatin altered VEGF mRNA stability, the rate of VEGF mRNA degradation was tested in vehicle- and simvastatin-treated MC3T3-E1 cells. Cells were exposed to vehicle or 10-6 M simvastatin for 6 h, after which 75 µM DRB, a RNA polymerase II inhibitor, was added to cultures. Decay of VEGF mRNA, as assessed by Northern analysis (Fig. 5), occurred at similar rates in both vehicle- and simvastatin-treated cells, indicating that simvastatin did not alter stability of this transcript. The VEGF gene contains eight exons. We determined the sequence of 830 bp including parts of exon 3 and 4 and the intron, to measure VEGF hnRNA concentration. RT-PCR products of the 285-bp spanning exon 3 and the intron were used to quantify VEGF hnRNA expression (Fig. 6A). Because transcriptional regulation frequently leads to changes in hnRNA synthesis, concentrations of hnRNA were assessed in the presence or absence of simvastatin. Concentrations of VEGF hnRNA were increased in MC3T3-E1 cells exposed to 10-6 M simvastatin from 4–12 h. The increase in hnRNA associated with simvastatin treatment already was maximal at 6 h, and despite some decrease after 8 h, significant elevations were maintained for up to 12 h (Fig. 6B). To further clarify statin-induced transcriptional activation of the VEGF gene, we constructed a reporter plasmid by combining the luciferase gene with 5'-flanking regions and exon 1 of the mouse VEGF gene (the sequence between -1128 and +827, including the HRE). MC3T3-E1 cells were transiently transfected with this VEGF promoter-luciferase construct and were subsequently treated with 10-6 M simvastatin for 24 h. Simvastatin treatment resulted in a significant increase in VEGF promoter activity. A HRE-deleted mutation of the mouse VEGF gene promoter abrogated the simvastatin-stimulated activity of luciferase (Fig. 7A). To determine whether or not simvastatin causes a change in protein binding to the HRE of the VEGF gene promoter, gel mobility shift analysis was performed using a radiolabeled 21-bp HRE oligonucleotide probe and nuclear extract from MC3T3-E1 cells treated or not treated with simvastatin (Fig. 7B). The HRE probe formed a complex with the nuclear extract factors, as demonstrated by the presence of specific bands. In the band indicated by a closed arrowhead in Fig. 7B, we observed a marked increase in the binding intensity of the HRE probe with the nuclear extract from simvastatin-treated MC3T3-E1 cells. Binding of the protein to HRE was competitively inhibited by a 100-fold excess of unlabelled HRE. In addition, we mutated 3 bp within the HRE oligonucleotide (Fig. 7B), and found that the mutated oligonucleotide could not form specific DNA-protein complexes and did not compete with the HRE-protein complex. The results of this gel mobility shift assay provide evidence that specific factor(s) recognize the HRE sequence.
fig.ommitteed^#4, 百拇医药
Figure 5. Effect of simvastatin on VEGF mRNA half-life in transcription-arrested MC3T3-E1 cells. Serum-depleted cells were exposed to 10-6 M simvastatin () or vehicle () for 8 h before the addition of 75 µM DRB, a transcriptional inhibitor. At the indicated time points after the addition of DRB, total RNA from cells was subjected to Northern blot analysis. VEGF mRNA normalized relative to cyclophilin mRNA was quantified by densitometry. Values are the mean ± SEM of four measurements.^#4, 百拇医药
fig.ommitteed^#4, 百拇医药
Figure 6. Time course of the stimulation of VEGF hnRNA concentration by simvastatin in MC3T3-E1 cells. Serum-depleted cells were treated with 10-6 M simvastatin () or vehicle () for indicated periods. Total RNA from vehicle- and simvastatin-treated cultures was reverse-transcribed and amplified by PCR. A, Schematic representation of the mouse VEGF gene. Mouse VEGF gene is comprised of eight exons and demonstrates the open reading frame (closed boxes) and the noncoding region (open boxes). The nucleotide sequence of the 830-bp region of exons 3 and 4 and the intron was determined and was submitted to nucleotide sequence databases (accession no. AB086118). The 285-bp region was amplified by PCR for determination of VEGF hnRNA. B, Total RNA (1 µg) was subjected to RT-PCR, and products were analyzed by Southern blots shown in upper panels. Quantitative determination of VEGF hnRNA in cells treated with vehicle () and 10-6 M simvastatin () is shown at the bottom. The results were normalized relative to cyclophilin expression and are expressed as the mean ± SEM of four determinations. *, P < 0.01 (compared with vehicle-treated control at each point).
fig.ommitteed(}iy]), 百拇医药
Figure 7. Effect of simvastatin on mouse VEGF gene activation in MC3T3-E1 cells. A, Luciferase activity of the VEGF gene promoter (-1128 to +827). The cells transfected with a wild-type (WT) or mutated (Mut) VEGF promoter-luciferase construct were treated with or without 10-6 M simvastatin for 24 h. A mutated construct (-1128 to -928 and -906 to +827) had deletion of the HRE. Luciferase activity is expressed in units relative to the pGL3-basic vector. The data from triplicate measurements are expressed as the mean ± SEM. P < 0.05 (comparison between two groups). B, Gel mobility shift assay of the HRE of the mouse VEGF gene (-921 to -901). The assay was performed using a wild-type (WT) or mutated (Mut) HRE sequence in nuclear extracts of MC3T3-E1 cells treated with or without 10-6 M simvastatin (Sim) for 12 h. Competition experiments were performed with excess unlabeled, wild-type, and mutated HRE. The nucleotide sequences are described in the bottom of panel B. The specific protein-DNA complex affected by simvastatin is indicated by the closed arrowhead.(}iy]), 百拇医药
Stimulation of VEGF protein secretion by simvastatin in MC3T3-E1 cells
We examined VEGF in medium from simvastatin-treated MC3T3-E1 cells (Fig. 8A). A significant elevation in VEGF secretion was observed at 24 h of treatment with 10-6 M simvastatin. We next tested the effects of mevalonate and various inhibitors on VEGF secretion added together with 10-6 M simvastatin or vehicle for 24 h (Fig. 8B). Coincubation with 1 mM mevalonate and simvastatin abrogated the statin-induced secretion of VEGF. Furthermore, PI3K inhibitors such as wortmannin and LY294002 also abolished the effect of simvastatin. An inhibitor of protein prenylation, manumycin A, mimicked the increased secretion by simvastatin in osteoblasts. These results indicated that simvastatin augmented VEGF protein secretion in osteoblastic cells, suggesting that this stimulation involves reduced protein prenylation and is mediated via the PI3K pathway.:?v, 百拇医药
fig.ommitteed:?v, 百拇医药
Figure 8. VEGF protein secretion in MC3T3-E1. Cells were cultured in -MEM medium containing 10% FBS until the third day after confluence. A, Time course of the stimulation of VEGF protein secretion by simvastatin in MC3T3-E1 cells. These cells were further incubated in serum-free differentiation medium for 24 h and treated with vehicle (open columns) or 10-6 M simvastatin (closed columns) for the indicated periods. The conditioned media from a 12-h period were collected, and VEGF concentrations were measured by a mouse VEGF assay kit. B, Effects of mevalonate, manumycin A, and PI3K inhibitors on VEGF protein secretion in MC3T3-E1 cells. The cells were treated with 10-6 M simvastatin (Sim) or 5 µM manumycin A (MaA) for 24 h, and compounds including mevalonate (Mev), wortmannin (Wor), and LY294002 (LY) were added 1 h before vehicle and simvastatin treatment. VEGF concentrations in the conditioned media of the last 12 h period of the culture were determined. The data are expressed as the mean ± SEM of three determinations. *, P < 0.01 (compared with vehicle-treated control). #, P < 0.01 (compared with simvastatin-untreated vehicle control by Student’s t test).
Effect of simvastatin on BMP-2 and VEGF gene expression}y, 百拇医药
Because statins induce BMP-2 expression (32, 33, 34) and BMPs, including BMP-2, stimulate VEGF gene expression and its production in osteoblasts (41, 42), we aimed to clarify whether or not simvastatin enhances the gene expression of VEGF via increased production of BMP-2 in MC3T3-E1 cells. Noggin, BMP antagonist, prevents BMPs from binding to their receptors. Treatment of the cells with noggin (1.0 µg/ml) and simvastatin (10-6 M) did not affect the statin-induced expression of VEGF mRNA (Fig. 9). Because the expression of BMP-2 mRNA in MC3T3-E1 cells was low and not detected by Northern blot analysis, we used the more sensitive method, RT-PCR analysis, for determining BMP-2 mRNA (Fig. 10). Simvastatin (10-6 M) significantly increased BMP-2 mRNA expression in MC3T3-E1 cells 24 and 36 h after treatment. The simvastatin-induced increase in BMP-2 mRNA expression by simvastatin was delayed in comparison to that of VEGF mRNA expression. These results indicate that simvastatin-enhanced gene expression of VEGF is independent of BMP-2 expression stimulated by statins.
fig.ommitteed/gow6m, 百拇医药
Figure 9. Effect of noggin, BMP inhibitor, on simvastatin-induced expression of VEGF mRNA by MC3T3-E1 cells. The cells were treated with a vehicle (Veh) or with 10-6 M simvastatin (Sim) in the absence or presence of noggin (1 µg/ml) for the indicated period. Total RNA (25 µg) was extracted and subjected to Northern blot analysis. VEGF mRNA abundance was measured densitometorically and normalized to that of cyclophilin mRNA. The data are expressed as the mean ± SEM of four measurements. *, P < 0.05 (compared with vehicle-treated control at each point). #, P < 0.05 (compared with noggin-treated control at each point). No significant difference was observed between the noggin-treated and untreated groups in the presence of simvastatin./gow6m, 百拇医药
fig.ommitteed/gow6m, 百拇医药
Figure 10. Time course of the effect of simvastatin on BMP-2 mRNA expression in MC3T3-E1 cells. Serum-starved cells were treated with 10-6 M simvastatin () or vehicle () for the indicated period. Total RNA was subjected to RT-PCR. The PCR products were measured by Southern blot analysis, and BMP-2 mRNA abundance was corrected for the expression of cyclophilin mRNA. The data are expressed as the mean ± SEM of four determinations. *, P < 0.05 (compared with vehicle-treated control at each point).
Effect of simvastatin on mineralized nodule formation in MC3T3-E1 cells+@6of{, 百拇医药
We finally tested the effect of simvastatin on osteoblastic differentiation as monitored by mineralization (Fig. 11). MC3T3-E1 cells were cultured in differentiation medium with 10% FBS for 14 d after confluence in the presence and absence of 10-6 M simvastatin. Figure 11A shows an increase in mineralization in response to simvastatin; mevalonate and GGPP blunted this increase. A PI3K inhibitor, LY294002, also blocked this effect of simvastatin. Furthermore, a VEGF receptor 2 (fetal liver kinase 1; Flk-1) kinase inhibitor, SU1498, blunted simvastatin-induced mineralization in MC3T3-E1 cells (Fig. 11B).+@6of{, 百拇医药
fig.ommitteed+@6of{, 百拇医药
Figure 11. Effect of simvastatin (Sim) on mineralization of extracellular matrix by MC3T3-E1 cells. The cells were cultured in differentiation medium containing 10% FBS in the presence or absence of 10-6 M simvastatin for 14 d after confluence. A, Effects of mevalonate (Mev), GGPP, and LY294002 (LY), a PI3K inhibitor. B, Effect of SU1498 (SU), a VEGF receptor 2 kinase inhibitor. Cells were cultured in the medium containing vehicle, 1 mM mevalonate, 20 µM GGPP, 2 µM LY294002, or 10 µM SU1498 with vehicle or 10-6 M simvastatin for 14 d. AR-S staining was performed for the demonstration of mineralized nodule formation at the end of culture. Mineralized nodules stained with AR-S were photographed (bottom panels), and then AR-S was eluted from the matrix using cetylpyridinium chloride. AR-S concentrations were measured by spectrophotometry at 562 nm (top panels). The data are expressed as the mean ± SEM of four determinations. *, P < 0.01 (compared with vehicle control).
Discussionz, 百拇医药
Mundy et al. (32) first reported that statins stimulated in vivo bone formation in rodents and increased new bone volume in cultures from mouse calvarium. The enhancing effect of statins on bone formation is associated with increased expression of BMP-2 via activation of the gene promoter (32, 33). Recently, we found that a relatively low dose of simvastatin (10-7 M) induced osteoblastic differentiation and markedlyincreased mineralization in MC3T3-E1 nontransformed osteoblasts (30). Furthermore, statins such as compactin and pitavastatin promoted differentiation of embryonic stem cells into osteoblasts (43) and up-regulated the gene expression for BMP-2 and osteocalcin by depletion of mevalonate and GGPP. Depletion, in turn, was followed by suppression of Rho-associated kinase activity (34). On the basis of these findings, BMP-2 clearly plays a role in the osteoblast maturation induced by statins. However, several factors influencing bone anabolism such as TGF-ß, fibroblast growth factor 2, IGF-I, and VEGF are involved in promoting differentiation of osteoblasts and stimulating bone formation. Thus, we investigated whether bone anabolic factors other than BMP-2 are induced by treatment of osteoblasts with statins. We found that statins such as simvastatin, atorvastatin, and cerivastatin markedly enhanced gene expression for VEGF in three osteoblastic cell lines.
Involvement of VEGF in endochondral bone formation has been demonstrated by VEGF inactivation, which restricts formation via inhibition of angiogenesis (16). Furthermore, in addition to vascular endothelial cells, osteoblasts produce VEGF whose expression is enhanced by factors including prostaglandin E1 (26, 44), IGF-I (17, 29, 45), TGF-ß (36, 46), fibroblast growth factor 2 (36), and 1,25-dihydroxyvitamin-D3 (47). Osteoblasts express VEGF receptors including VEGF receptor 2 (Flk-1), which binds VEGF-A, VEGF-C, and VEGF-D isoforms with high affinity (29). Exogenous or endogenous VEGF stimulates osteoblastic differentiation as monitored by new bone formation (29). Midy and Plouet (15) reported that, on a molar basis, VEGF was a much more potent inducer of osteoblastic differentiation than BMP-2. The present results indicate that hydrophobic statins such as simvastatin, atorvastatin, and cerivastatin clearly increase VEGF mRNA abundance in the MC3T3-E1 osteoblastic cell line and demonstrate that statin-induced mRNA expression also occurs in other osteoblast-like cells. ST2, a clone of marrow stromal cells, was isolated from the bone marrow of BC8 mice and shows characteristics of typical adipocytes (48). When ST2 cells were cultured in medium with ascorbic acid, they developed an osteoblastic phenotype (49). UMR-106 cells, which were derived from a rat osteosarcoma, show osteoblastic features such as PTH and 1,25-dihydroxyvitamin-D3 receptors (35, 50). The MC3T3-E1 cells were nontransformed preosteoblasts derived from mouse calvaria that differentiate exclusively into osteoblasts (51). Statins clearly augmented mRNA expression for VEGF by these various osteoblastic cell lines, indicating that statin-induced expression of VEGF is a characteristic of osteoblasts. However, high statin concentrations induce apoptosis of osteoblasts, as reported in studies of smooth muscle cells (52) and cultured cortical neurons (53). We confirmed this by demonstrating that 10-4 M simvastatin suppresses VEGF mRNA expression in MC3T3-E1 cells.
Data presented here demonstrate that a cellular signal evoked by statins is transmitted to the nucleus to stimulate VEGF gene expression. Simvastatin increased steady state levels of VEGF mRNA without modifying the half-life of this mRNA. Although hnRNA may reflect alterations in RNA processing, its abundance correlates well with transcription rates measured by nuclear runoff assays (54, 55). Abundance of hnRNA was significantly elevated as early as 4 h after treatment with simvastatin in MC3T3-E1 osteoblastic cells. Elevation of hnRNA preceded an increase in mRNA accumulation of the cells. In addition, we cloned the promoter region of the mouse VEGF gene and transiently transfected chimeric deletion constructs containing the 5'- flanking region (1128 bp) and the luciferase gene into MC3T3-E1 cells. Simvastatin markedly stimulated luciferase activity in transfected cells. The HRE of the human VEGF gene promoter (39) is present in the mouse gene, as demonstrated by sequence analyses performed by us and other researchers (38). Gel mobility analysis revealed that simvastatin clearly increased the binding of the HRE-protein complex in MC3T3-E1 cells. Our study of mutation also showed that HRE is crucial to simvastatin-stimulated promoter activity, determined by luciferase, and intact HRE is necessary to specific DNA-protein complex by gel mobility shift assay. These results indicate that mevalonate depletion resulting from addition of simvastatin, an HMG-CoA reductase inhibitor, up-regulates VEGF transcription in osteoblastic cells. Recent investigations (45) showed that hypoxia-inducible factor-2{alpha} mediated transcriptional activation of VEGF gene in response to hypoxia or IGF-I in human osteoblast-like cells. Statins may activate the VEGF gene through increased binding to specific elements of the VEGF promoter of activators for gene transcription such as hypoxia-inducible factor-2{alpha} . However, further studies are needed to elucidate the precise mechanisms underlying transcriptional activation of the VEGF gene by statins.
Many studies document that statins trigger various cellular events through their functions as HMG-CoA reductase inhibitors as well as other signaling pathways (1, 5, 6, 7, 8, 30, 32, 33, 34, 43, 52). By modulating the initial part of the cholesterol synthesis pathway, statins decrease concentrations of many important intermediate compounds, including isoprenoids that contain GGPP and FPP. Isoprenoids are lipids that are attached in post-translational modification to certain proteins including the c subunit of heterotrimeric G proteins, small G proteins (Ras), and Ras-like proteins such as Rho, Rap, Rab, or Ral (9). However, recent studies report that statins may have additional functions independent of inhibition of HMG-CoA reductase. For example, lovastatin binds to the I domain of {alpha} 1ß2 integrin to block interaction of this integrin with intercellular adhesion molecule-1; this blockade is not reversed by addition of mevalonate (56, 57). Accordingly, we confirmed involvement of the mevalonate pathway and isoprenoids in negative modulation of VEGF gene expression by demonstrating that the direct product of HMG-CoA reductase, mevalonate, as well as the isoprenoid, GGPP, reversed stimulation of VEGF gene expression in MC3T3-E1 osteoblastic cells. Furthermore, to assess whether protein prenylation may play a role in the induction of VEGF gene of osteoblasts, MC3T3-E1 cells were exposed to an inhibitor of protein prenyl transferase, manumycin A. This inhibitor, which selectively affects farnesyl transferase, a major prenyl transferase, clearly increased VEGF mRNA concentrations in osteoblastic cells. FPP also inhibited statin-induced expression of VEGF mRNA in these cells. Our results indicate that farnesylated and geranylgeranylated proteins are involved in the up-regulation of VEGF gene expression.
A newly developed statin, pitavastatin, has been shown to inhibit Rho-associated kinase activity by suppressing prenylation of small GTP-binding proteins in human osteoblasts. Increases then follow in gene expression for osteoblastic markers such as BMP-2 and osteocalcin (34). We therefore investigated mechanisms following statin-induced inhibition of protein prenylation that underlie up-regulation of VEGF production. A selective inhibitor of Rho kinase, Y-27632, did not mimic the effect of statin on VEGF mRNA expression in osteoblasts (our unpublished results), suggesting that inhibition of Rho kinase is not the major basis of this statin effect. According to Tokuda et al. (26), prostaglandin E1-induced VEGF synthesis in osteoblasts was mediated through activation of p38 MAPK. Because a specific inhibitor of p38 MAPK, SB203580 (58), could not abolish simvastatin-stimulated expression of VEGF mRNA (our unpublished results), p38 MAPK also is unlikely to be involved in this process. Statins such as simvastatin and atorvastatin have been shown to increase endothelial progenitor cell proliferation and survival in vitro (59, 60) and to promote angiogenesis in vivo via the PI3K/Akt pathway (5). Furthermore, IGF-I augmented the promoter activity of VEGF gene mediated by the PI3K/Akt pathway in osteoblastic cells (45). Involvement of PI3K as a mediator of statin action on VEGF gene expression was evaluated by using two specific inhibitors of the enzyme, LY294002 and wortmannin. Both inhibitors blunted the simvastatin action on VEGF mRNA accumulation in MC3T3-E1 osteoblastic cells. This finding strongly suggests that the stimulation of VEGF gene transcription induced by statins is mediated via the PI3K pathway.
Statins trigger different cellular events through their function as HMG-CoA reductase inhibitors, as well as other signaling pathways. The fact that manumycin A markedly stimulates VEGF mRNA expression at a low concentration (5 µM) suggests that inhibition of protein prenylation is of particular importance. Recent studies have demonstrated that the exogenous addition of VEGF into conditioned media of osteoblasts stimulates mineralized nodule formation in a dose-dependent manner (29, 61). We demonstrated that the inhibition of the VEGF signaling pathway by a VEGF receptor 2 (Flk-1) kinase inhibitor, SU1498, suppresses mineralization by simvastatin-treated MC3T3-E1 osteoblastic cells. Previous studies (29, 61), combined with our results, suggest that simvastatin promotes osteoblastic differentiation, at least in part, by stimulating VEGF expression. Interestingly, BMPs including BMP-2, elevated VEGF production by osteoblastic cells and increased angiogenesis in angiogenesis assays using bone explants (41). This process induced by BMPs in osteoblasts may be involved in activation of p70 S6 kinase (42). We demonstrated that simvastatin-induced expression of VEGF mRNA in osteoblastic cells is independent of BMP-2 expression. The inhibition of BMP-2 binding to specific receptors caused by noggin, BMP binding protein (62), had no effect on simvastatin-induced expression of VEGF mRNA in MC3T3-E1 cells. Furthermore, studies of the time course of the effects of simvastatin revealed that the simvastatin-induced increase in the expression of VEGF mRNA precedes the induction of BMP-2 mRNA expression in these cells. BMPs and VEGF interact to stimulate bone formation by osteoblasts. Thus, statins are likely to augment the expression of bone anabolic factors such as VEGF and BMP-2, subsequently accelerating bone formation. Accordingly, statins may be potentially useful drugs for the treatment of osteoporosis.
In summary, we demonstrated that hydrophobic statins such as simvastatin, atorvastatin, and cerivastatin augment expression of VEGF mRNA and protein in MC3T3-E1 murine osteoblastic cells. We also found that inhibition of protein prenylation elicited by these hydrophobic HMG-CoA reductase inhibitors increases transcription of the VEGF gene in osteoblastic cells. This is the first report showing that statins stimulate gene expression for a bone anabolic factor in osteoblasts other than BMP-2. Our results suggest that hydrophobic statins promote osteoblastic differentiation and mineralization at least in part by enhancing production of VEGF by osteoblasts. These findings raise a strong possibility that statins stimulate bone growth and repair in vivo by increasing angiogenesis via locally acting angiogenic factors such as VEGF.9d\}k, 百拇医药
Acknowledgments9d\}k, 百拇医药
We thank Dr. Hitoshi Saitoh at Fuji Gotemba Research Laboratory of Chugai Pharmaceutical Co. (Gotemba, Japan) for statistical analysis.
Received July 5, 2002.+|-;z&:, http://www.100md.com
Accepted for publication November 4, 2002.+|-;z&:, http://www.100md.com
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Statins such as simvastatin are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that inhibit cholesterol synthesis. We presently investigated statin effects on vascular endothelial growth factor (VEGF) expression in osteoblastic cells. Hydrophobic statins including simvastatin, atorvastatin, and cerivastatin–but not a hydrophilic statin, pravastatin–markedly increased VEGF mRNA abundance in nontransformed osteoblastic cells (MC3T3-E1). Simvastatin (10-6 M) time-dependently augmented VEGF mRNA expression in MC3T3-E1 cells, mouse stromal cells (ST2), and rat osteosarcoma cells (UMR-106). According to heterogeneous nuclear RNA and Northern analyses, 10-6 M simvastatin stimulated gene expression for VEGF in MC3T3-E1 cells without altering mRNA stability. Transcriptional activation of a VEGF promoter-luciferase construct (-1128 to +827), significantly increased by simvastatin administration. As demonstrated by gel mobility shift assay, simvastatin markedly enhanced the binding of hypoxia-responsive element-protein complexes. These results indicate that the stimulation of the VEGF gene by simvastatin in MC3T3-E1 cells is transcriptional in nature. VEGF secretion into medium was increased in MC3T3-E1 by 10-6 M simvastatin. Pretreating MC3T3-E1 cells with mevalonate or geranylgeranyl pyrophosphate, a mevalonate metabolite, abolished simvastatin-induced VEGF mRNA expression; manumycin A, a protein prenylation inhibitor, mimicked statin effects on VEGF expression. The effect of simvastatin was blocked by pretreatment with wortmannin and LY294002, specific phosphatidylinositide-3 kinase inhibitors. Simvastatin enhanced mineralized nodule formation in culture, whereas coincubation with mevalonate, geranylgeranyl pyrophosphate, LY294002, or VEGF receptor 2 inhibitor (SU1498) abrogated statin-induced mineralization. Thus, statins stimulate VEGF expression in osteoblasts via reduced protein prenylation and the phosphatidylinositide-3 kinase pathway, promoting osteoblastic differentiation.
Introductionvaa, 百拇医药
COMPETITIVE INHIBITORS OF 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, also called statins, act at a rate-limiting step in cholesterol synthesis by blocking conversion of HMG-CoA to mevalonate (1). Statins, including simvastatin and atorvastatin, are highly effective cholesterol-lowering drugs that are widely considered to reduce morbidity from coronary artery disease (2, 3). Besides lowering serum lipids, statins can decrease platelet aggregation and thrombus deposition (4); promote angiogenesis (5); decrease ß-amyloid peptide, related to Alzheimer’s disease (6, 7); and suppress T lymphocyte activation (8). By modulating the initial part of the cholesterol synthesis pathway, statins decrease availability of several important intermediate compounds, including isoprenoid, that contain geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP). Isoprenoids are lipids attached by post-translational modification to some proteins such as the small G proteins, including Ras and Ras-like proteins (Rho, Rap, Rab, and Ral) (9).
Integrity of the skeleton requires a dynamic balance between bone formation and bone resorption, which are controlled by calcitropic hormones and cytokines. When bone resorption exceeds bone formation, diseases of bone metabolism such as postmenopausal osteoporosis can result (10). Communication between bone-forming osteoblasts and bone-resorbing osteoclasts is very close, and coupling of bone resorption to bone formation is necessary for maintenance of healthy bone. Osteoblasts arise from mesenchymal stem cell precursors and undergo differentiation in response to a number of factors including bone morphogenetic proteins (BMPs), TGF-ß, IGF-I, vascular endothelial growth factor (VEGF), and glucocorticoids (11, 12, 13, 14, 15, 16, 17, 18). Osteoblasts are located in proximity to endothelial cells. Vascular invasion is a prerequisite for endothelial bone formation and fracture healing. Gerber et al. (16) demonstrated in young mice that blocking the action of endogenous VEGF inhibited bone formation and resorption. Important interplay takes place between endothelial cells and osteoblasts, and the angiogenic factor VEGF appears to be involved in this intercellular communication (19, 20, 21).
VEGF is a glycoprotein that acts as both a promoter of permeability and an angiogenic factor (22, 23, 24). VEGF also has mitogenic actions restricted to endothelial cells and is chemotactic for endothelial cells (25). VEGF is secreted by many cell types, including osteoblasts and smooth muscle cells; its expression is regulated by a variety of growth factors and cytokines (17, 26, 27, 28). Several splice variants of VEGF, as well as their receptors, are expressed in osteoblasts (29) in which this cytokine has been shown to induce alkaline phosphatase activity and enhance responsiveness to PTH (15). We recently have shown that simvastatin promotes osteoblast differentiation and mineralization (30). BMPs such as BMP-2 have been shown to induce osteoblast differentiation and ectopic bone formation (31). Statins induce BMP-2 expression in osteoblasts and stimulate bone formation (30, 32, 33, 34). We have searched for differentiation mechanisms in addition to BMP-2 that are induced by statins in osteoblasts. We report here that statins, including simvastatin, induced VEGF expression via inhibition of protein prenylation in MC3T3-E1 osteoblastic cells.
Materials and Methods81, 百拇医药
Cell culture81, 百拇医药
MC3T3-E1 cells (a clonal preosteoblastic cell line derived from newborn mouse calvaria) were grown in -MEM (ICN Pharmaceuticals, Inc. Aurora, OH) supplemented with 10% fetal bovine serum (FBS) as previously described (26, 30) until 3 d after confluence. Then, the culture medium was replaced for 24 h with differentiation medium [{alpha} -MEM containing a 50 µg/ml concentration of the phosphate ester of ascorbic acid (Wako Pure Chemical Industries Ltd., Osaka, Japan) and 10 mM ß-glycerophosphate (Wako Pure Chemical Industries Ltd.)]. Cells were exposed to statins to be tested such as 1 nM to 100 µM simvastatin (Calbiochem, San Diego, CA), 10–100 µM atorvastatin (Yamanouchi Pharmaceutical, Tokyo, Japan), 0.1–1 µM cerivastatin (Takeda Pharmaceutical, Osaka, Japan), and 100 µM pravastatin (Sankyo Co., Ltd., Tokyo, Japan) for the indicated time periods. In studies of inhibitors, serum-deprived cells were treated with 1 mM mevalonate (Sigma, St. Louis, MO), 20 µM GGPP (Sigma), 2–20 µM LY294002 or 100 nM wortmannin [Sigma; both being phosphatidylinositide-3 kinase (PI3K) inhibitors], or 5 µM manumycin A (Sigma; a protein prenylation inhibitor) in the absence or presence of 1 µM simvastatin for the times indicated. In some experiments, cells were exposed to 75 µM 5,6-dichlorobenzimidazole riboside (DRB, Sigma; a RNA polymerase II inhibitor, dissolved in dimethylsulfoxide) 8 h after exposure to 1 µM simvastatin or vehicle. Mouse bone marrow-derived stromal ST2 cells were supplied by the RIKEN Cell Bank (Tsukuba, Japan) and were cultured in RPMI 1640 (ICN Pharmaceuticals, Inc.) that contained 10% FBS. After the cells reached confluence, they were cultured in {alpha} -MEM containing 10% FBS for 6 d. Then, cells were cultured in differentiation medium without FBS for 24 h. Cells were treated with 1 µM of test substances such as simvastatin for the period indicated. Rat osteoblast-like osteosarcoma UMR-106 cells (CRL1661, American Type Culture Collection, Manassas, VA) were grown by routine methods in monolayer culture in DMEM (ICN Pharmaceuticals, Inc.) containing 5% FBS. At confluence, the culture medium was replaced with serum-free DMEM for 24 h (35). Cells were exposed to 1 µM simvastatin for the indicated time period.
Determination of VEGF and BMP-2 mRNAsa$-^.[, http://www.100md.com
VEGF mRNA abundance was determined by Northern blot analysis. To prepare the probe for Northern analysis, we isolated total RNA from MC3T3-E1 cells. RT-PCRs were performed using a Gene Amp RNA PCR kit (PE Applied Biosystems, Foster City, CA). A 410-bp probe against mouse VEGF gene was generated by PCR from an oligonucleotide-primed single-stranded cDNA using the following PCR primer sequences: 5'-CGAGACCCTGGTGGACATCT-3' and 5'-CACCGCCTCGGCTTGTCAC-3'. Resulting bands were cloned into a pCR 2.1 vector (Invitrogen, San Diego, CA) and sequenced to confirm sequence identity (GenBank accession no. NM 009505; Ref. 36). Total RNA was extracted from cells using guanidine thiocyanate. Total RNA was fractionated on 1.2% agarose gels containing formaldehyde and transferred to a nylon membrane (Hybond-XL; Amersham Biosciences, Buckinghamshire, UK). The membranes were hybridized with mouse VEGF cDNA probe labeled with [{alpha} -32P]deoxy-CTP (specific activity, 110 TBq/mmol; ICN Pharmaceuticals, Inc.) using a Megaprime DNA labeling kit (Amersham). Hybridization was performed at 42 C for 48 h, followed by washing with 0.1–2x SSPE [150 mM NaCl, 10 mM sodium phosphate, and 1 mM EDTA (pH 7.4)] with 0.1% SDS at 65 C. For standardization, blots were rehybridized with a cyclophilin cDNA probe. Membrane signal intensity was quantified with a Molecular Imager FX (Bio-Rad Laboratories, Inc. Hercules, CA), and the resulting images were analyzed using Quantity One 4.1.1 (Bio-Rad Laboratories, Inc.) image analysis software. RT-PCR was performed to clarify BMP-2 mRNA expression. One microgram of total RNA was copied into cDNA, and the synthesized cDNA was amplified using a sense primer (5'-ATCAACTAGAAGCCGTGGAG-3') and an antisense primer (5'-CAATGGCCTTATCTGTGACC-3') by PCR (22 cycles). The relative amount of the BMP-2 transcript was corrected for the amount of cyclophilin mRNA present.
Quantification of VEGF heterogeneous nuclear RNA (hnRNA);4[w\, http://www.100md.com
VEGF hnRNA was determined by RT-PCR using specific primers designed to amplify hnRNA from an intron to an exon, as described previously (35). Briefly, the nucleotide sequences containing exon 3, the interposed intron, and exon 4 of murine VEGF gene were determined and submitted to a database (GenBank accession no. AB086118). A sense primer (5'-TAACGATGAAGCCCTGGAGT-3'), spanning nucleotides 100–119 of exon 3 of the murine VEGF gene, and an antisense primer (5'-AGCAGATGGTCAATTCGTGG-3'), spanning nucleotides 375–394 of the intron between exons 3 and 4, were synthesized. Total RNA was extracted from MC3T3-E1 cells and treated with ribonuclease-free deoxyribonuclease I (Boeringer, Indianapolis, IN) to remove potentially contaminating DNA. One microgram of RNA was copied into cDNA using reverse transcriptase and random hexanucleotide primers. The newly synthesized cDNA was amplified by PCR through 21 cycles. PCR products were loaded onto a 1.2% agarose gel and transferred to a nylon membrane. Southern blotting was performed using a murine VEGF genomic DNA (the region of hnRNA) probe radiolabeled with [{alpha} -32P]deoxy-CTP. Amplified RNA from murine VEGF gene was corrected for the level of cyclophilin hnRNA by RT-PCR using the same synthesized cDNA. PCR was performed for 18 cycles. Signal intensity was quantified with the Molecular Imager FX.
Transient expression assay and gel mobility shift assay\#fqqou, http://www.100md.com
Assays of transient expression and gel mobility shift were performed as described previously (37). Briefly, a fragment comprised of -1128 to +827 of a mouse VEGF gene promoter was cloned upstream of the luciferase reporter gene in the pGL3-basic reporter vector. A mutated construct of the mouse VEGF gene promoter without the hypoxia responsive element (HRE; -1128/-928 and -906/+827) has also been cloned. The nucleotide sequence of the clone was confirmed by comparison with a published sequence of mouse VEGF gene (38). Transient transfections were performed using MC3T3-E1 cells in six-well plates. Cells were cotransfected with 1.0 µg luciferase reporter plasmid and 1.0 µg ß-galactosidase expression vector (pEFBOS-LacZ) as an internal control, using Gene Porter transfection reagent (Gene Therapy Systems, San Diego, CA). Transfected cells were incubated with {alpha} -MEM containing 10% FBS for 18 h. The cells treated with a vehicle or with 1 µM simvastatin were cultured in {alpha} -MEM containing 1% BSA for 24 h, and were then harvested for the luciferase assay. Luciferase activity was corrected for transfection efficiency as indicated by the ß-galactosidase activity. For the gel mobility shift assay, the MC3T3-E1 cells were treated with a vehicle and with 10 µM simvastatin for 12 h. Nuclear extracts from the cells were prepared as described previously (37). Nuclear extracts and radiolabeled oligonucleotides of the HRE of the VEGF gene (-921 to -901; Ref. 39) and its mutant were incubated for 20 min at 25 C in a solution containing 10 mM HEPES, 50 mM KCl, 0.1 mM EDTA, 3 mM Mg2Cl2, 1 mM dithiothreitol, 6% glycerol, and 0.1 mg/ml polydeoxyinosinic deoxycytidylic acid at pH 7.9. DNA protein complexes were resolved on nondenaturing, nonreducing, 4.5% polyacrylamide gels and were visualized by autoradiography.
VEGF concentration in conditioned medium|2, http://www.100md.com
Serum-deprived MC3T3-E1 cells were treated with vehicle or test substances such as simvastatin and manumycin A for the period indicated. Conditioned media were collected after 12 h, and VEGF concentrations were measured by a mouse VEGF quantitative sandwich enzyme immunoassay (R&D Systems, Minneapolis, MN). This assay measures primarily the 165-amino acid isoform of VEGF, the main soluble isoform. Fifty microliters of conditioned medium were used for the assay.|2, http://www.100md.com
Assay of mineralized matrix formation|2, http://www.100md.com
MC3T3-E1 cells in 24-well plates were cultured in differentiation medium containing 10% FBS and test substance such as simvastatin and SU1498 (Calbiochem; VEGF receptor 2 kinase inhibitor) for 14 d after confluence. After the medium was removed, cells were washed twice with PBS. The extent of mineralized matrix in the plates was determined by Alizarin Red S (AR-S) staining (30, 40). Briefly, cells were fixed in 70% ethanol for 1 h at room temperature, washed with PBS, and stained with 40 mM AR-S (pH 4.2) for 10 min at room temperature. Next, cell preparations were washed five times with deionized water and incubated in PBS for 15 min to eliminate nonspecific staining. The stained matrix was photographed using a 35-mm camera. AR-S staining was released from the cell matrix by incubation in 10% (wt/vol) cetylpyridinium chloride for 15 min. The amount of dye released was quantified by spectrophotometry at 562 nm.
Statistical analysissj+, http://www.100md.com
All values are expressed as the mean ± SEM of three or four measurements. Statistical analyses of the time-course study and the luciferase assay were performed using unpaired Student’s t test. For all other experiments, statistical analyses were performed using Dunnet’s test. Data were analyzed using SAS software (SAS Institute Inc., Cary, NC).sj+, http://www.100md.com
Resultssj+, http://www.100md.com
Induction of VEGF mRNA by statins in osteoblastic cellssj+, http://www.100md.com
We first tested the hypothesis that statins can alter VEGF mRNA expression in osteoblastic cells, using MC3T3-E1 nontransformed cells. This cell line was derived from mouse calvaria and shows many osteoblastic characteristics, such as expression of type I collagen and alkaline phosphatase, and mineralized nodule formation. In these cells, marked enhancement of VEGF mRNA expression was observed with 12-h exposure to hydrophobic statins, including simvastatin, atorvastatin, and cerivastatin, but not with hydrophilic statins such as pravastatin (Fig. 1). To confirm statin-induced expression of VEGF mRNA in osteoblasts, we examined the effect of simvastatin on the mRNA abundance in three osteoblast-like cell lines, including MC3T3-E1 cells, mouse stromal ST2 cells, and rat osteosarcoma UMR-106 cells (Fig. 2). The time course of the effect of simvastatin was determined in MC3T3-E1 cells, in which a significant increase in VEGF mRNA could be detected as early as 6 h after initiation of treatment with 10-6 M simvastatin. Stimulation was maximal at 12 h, resulting in a 2-fold increase above control values; by 24 h, mRNA concentrations in treated cells had decreased nearly to control values (Fig. 2A). Simvastatin (10-6 M) treatment of ST2 cells for 3 h resulted in a 7-fold increase of VEGF mRNA; induction was significant between 3 and 6 h. This stimulation was greater in ST2 cells than in other cell lines (Fig. 2B). In UMR-106 cells, 10-6 M simvastatin significantly increased VEGF mRNA between 6 and 12 h after initiation of treatment (Fig. 2C). In dose-response experiments in MC3T3-E1 cells, a concentration of simvastatin as low as 10-7 M significantly enhanced abundance of VEGF mRNA. Maximal elevation was obtained at simvastatin concentrations of 10-6 and 10-5 M (Fig. 3). However, a high concentration (10-4 M) of the statin reduced VEGF mRNA expression in MC3T3-E1 cells. Thus, statins induced VEGF mRNA expression in osteoblastic cells in a time- and concentration-dependent manner.
fig.ommitteedhk*hm, 百拇医药
Figure 1. Effects of statins on VEGF mRNA abundance in MC3T3-E1 murine osteoblastic cells. The cells were cultured in the medium (-MEM:FBS/9:1) until the third day after confluence. Thereafter, the medium was changed into serum-free differentiation medium (-MEM containing 50 µg/ml of the phosphate ester of ascorbic acid and 10 mM ß-glycerophosphate). Cells were exposed to vehicle, simvastatin (10-6 and 10-5 M), atorvastatin (10-5 and 10-4 M), cerivastatin (10-7 and 10-6 M), or pravastatin (10-4 M) for 12 h. Cells were harvested, and total RNA was extracted. Total RNA (25 µg) was subjected to Northern blot analysis. The estimated sizes of VEGF and cyclophilin (Cyclo) were 3.8 and 0.9 kb, respectively.hk*hm, 百拇医药
fig.ommitteedhk*hm, 百拇医药
Figure 2. Stimulation of VEGF mRNA expression by simvastatin in different osteoblastic cells. Three osteoblastic cell lines such as MC3T3-E1 nontransformed murine osteoblasts (A), mouse bone-marrow derived stromal ST2 cells (B), and rat osteoblast-like osteosarcoma cells (C) were cultured under serum-free conditions for 24 h and thereafter treated with vehicle () or 10-6 M simvastatin () for the indicated period. Total RNA (25 µg) was extracted and subjected to Northern blot analysis. VEGF mRNA abundance was determined densitometrically and normalized to that of cyclophilin (Cyclo.) mRNA. The data are expressed as the mean ± SEM of four determinations. *, P < 0.01 (compared with vehicle-treated control at each point).
fig.ommitteedtf, 百拇医药
Figure 3. Effect of simvastatin on VEGF mRNA abundance in MC3T3-E1 cells. Serum-depleted cells were treated with the indicated concentrations of simvastatin for 12 h. Total RNA from cells was subjected to Northern blot analysis. The abundance was expressed as a percentage of the vehicle-treated control. Each bar represents the mean ± SEM of four measurements. *, P < 0.05; **, P < 0.01 (compared with the vehicle-treated control).tf, 百拇医药
Effects of cholesterol-synthesis metabolites on statin-induced VEGF mRNA expressiontf, 百拇医药
To determine the mechanism of VEGF mRNA induction by statins, MC3T3-E1 cells first were coincubated with simvastatin in the presence of mevalonate or GGPP (Fig. 4A). We tested the effect of mevalonate, the immediate product of the reaction catalyzed by HMG-CoA reductase, to determine whether the effect of simvastatin results from inhibition of this enzyme. We incubated cells with 10-6 M simvastatin together with 1 mM mevalonate. Induction of VEGF mRNA by simvastatin was completely abolished by mevalonate. Mevalonate alone did not modulate VEGF mRNA expression. Next, involvement of the isoprenoid intermediate, GGPP, on modulation of VEGF mRNA by simvastatin was examined. As shown in Fig. 4A, cotreatment of cells with 20 µM GGPP completely abolished the induction of VEGF mRNA synthesis. Because GGPP is used in post-translational modification of several cell proteins, we evaluated the effect of an inhibitor of protein prenylation, manumycin A. VEGF induction with manumycin A alone was remarkable at a low concentration (5 µM) after 12 h of treatment (Fig. 4A). FPP also inhibited the stimulation of VEGF mRNA synthesis induced by simvastatin treatment (data not shown). These results implicated reduced protein prenylation caused by the HMG-CoA reductase inhibitor, simvastatin, in the enhancement of VEGF mRNA expression in osteoblastic cells. Because statins activate the PI3K pathway in several cell types (5), we studied whether stimulation of VEGF mRNA synthesis by simvastatin was mediated via the PI3K pathway in MC3T3-E1 cells. Treatment of cells with two PI3K inhibitors, wortmannin and LY294002, completely blocked VEGF mRNA induction by simvastatin (Fig. 4B). These results strongly suggested that activation of the PI3K pathway by simvastatin was required for gene activation of VEGF.
fig.ommitteed.r5q9e(, http://www.100md.com
Figure 4. Regulation of VEGF mRNA expression by the cholesterol synthetic pathway in MC3T3-E1 cells. A, Serum-depleted cells were treated with 1 mM mevalonate (Mev) or 20 µM GGPP for 1 h before incubation for 12 h with vehicle or 10-6 M simvastatin (Sim). A protein prenylation inhibitor, manumycin A (MaA), at a concentration of 5 µM was added to the cells and incubated for 12 h. B, Serum-depleted cells were treated with vehicle (Veh), 100 nM wortmannin (Wor), or 20 µM LY294002 (LY) for 1 h before incubation for 12 h with vehicle or 10-6 M simvastatin (Sim). Total RNA from cells was subjected to Northern blot analysis. VEGF mRNA concentrations normalized relative to cyclophilin mRNA was expressed as a percentage of the vehicle control value. Each bar represents the mean ± SEM of four determinations. *, P < 0.01 (compared with vehicle control). #, P < 0.01 (compared with simvastatin-untreated vehicle control by Student’s t test)..r5q9e(, http://www.100md.com
Transcriptional regulation of the VEGF gene by simvastatin.r5q9e(, http://www.100md.com
To determine whether simvastatin altered VEGF mRNA stability, the rate of VEGF mRNA degradation was tested in vehicle- and simvastatin-treated MC3T3-E1 cells. Cells were exposed to vehicle or 10-6 M simvastatin for 6 h, after which 75 µM DRB, a RNA polymerase II inhibitor, was added to cultures. Decay of VEGF mRNA, as assessed by Northern analysis (Fig. 5), occurred at similar rates in both vehicle- and simvastatin-treated cells, indicating that simvastatin did not alter stability of this transcript. The VEGF gene contains eight exons. We determined the sequence of 830 bp including parts of exon 3 and 4 and the intron, to measure VEGF hnRNA concentration. RT-PCR products of the 285-bp spanning exon 3 and the intron were used to quantify VEGF hnRNA expression (Fig. 6A). Because transcriptional regulation frequently leads to changes in hnRNA synthesis, concentrations of hnRNA were assessed in the presence or absence of simvastatin. Concentrations of VEGF hnRNA were increased in MC3T3-E1 cells exposed to 10-6 M simvastatin from 4–12 h. The increase in hnRNA associated with simvastatin treatment already was maximal at 6 h, and despite some decrease after 8 h, significant elevations were maintained for up to 12 h (Fig. 6B). To further clarify statin-induced transcriptional activation of the VEGF gene, we constructed a reporter plasmid by combining the luciferase gene with 5'-flanking regions and exon 1 of the mouse VEGF gene (the sequence between -1128 and +827, including the HRE). MC3T3-E1 cells were transiently transfected with this VEGF promoter-luciferase construct and were subsequently treated with 10-6 M simvastatin for 24 h. Simvastatin treatment resulted in a significant increase in VEGF promoter activity. A HRE-deleted mutation of the mouse VEGF gene promoter abrogated the simvastatin-stimulated activity of luciferase (Fig. 7A). To determine whether or not simvastatin causes a change in protein binding to the HRE of the VEGF gene promoter, gel mobility shift analysis was performed using a radiolabeled 21-bp HRE oligonucleotide probe and nuclear extract from MC3T3-E1 cells treated or not treated with simvastatin (Fig. 7B). The HRE probe formed a complex with the nuclear extract factors, as demonstrated by the presence of specific bands. In the band indicated by a closed arrowhead in Fig. 7B, we observed a marked increase in the binding intensity of the HRE probe with the nuclear extract from simvastatin-treated MC3T3-E1 cells. Binding of the protein to HRE was competitively inhibited by a 100-fold excess of unlabelled HRE. In addition, we mutated 3 bp within the HRE oligonucleotide (Fig. 7B), and found that the mutated oligonucleotide could not form specific DNA-protein complexes and did not compete with the HRE-protein complex. The results of this gel mobility shift assay provide evidence that specific factor(s) recognize the HRE sequence.
fig.ommitteed^#4, 百拇医药
Figure 5. Effect of simvastatin on VEGF mRNA half-life in transcription-arrested MC3T3-E1 cells. Serum-depleted cells were exposed to 10-6 M simvastatin () or vehicle () for 8 h before the addition of 75 µM DRB, a transcriptional inhibitor. At the indicated time points after the addition of DRB, total RNA from cells was subjected to Northern blot analysis. VEGF mRNA normalized relative to cyclophilin mRNA was quantified by densitometry. Values are the mean ± SEM of four measurements.^#4, 百拇医药
fig.ommitteed^#4, 百拇医药
Figure 6. Time course of the stimulation of VEGF hnRNA concentration by simvastatin in MC3T3-E1 cells. Serum-depleted cells were treated with 10-6 M simvastatin () or vehicle () for indicated periods. Total RNA from vehicle- and simvastatin-treated cultures was reverse-transcribed and amplified by PCR. A, Schematic representation of the mouse VEGF gene. Mouse VEGF gene is comprised of eight exons and demonstrates the open reading frame (closed boxes) and the noncoding region (open boxes). The nucleotide sequence of the 830-bp region of exons 3 and 4 and the intron was determined and was submitted to nucleotide sequence databases (accession no. AB086118). The 285-bp region was amplified by PCR for determination of VEGF hnRNA. B, Total RNA (1 µg) was subjected to RT-PCR, and products were analyzed by Southern blots shown in upper panels. Quantitative determination of VEGF hnRNA in cells treated with vehicle () and 10-6 M simvastatin () is shown at the bottom. The results were normalized relative to cyclophilin expression and are expressed as the mean ± SEM of four determinations. *, P < 0.01 (compared with vehicle-treated control at each point).
fig.ommitteed(}iy]), 百拇医药
Figure 7. Effect of simvastatin on mouse VEGF gene activation in MC3T3-E1 cells. A, Luciferase activity of the VEGF gene promoter (-1128 to +827). The cells transfected with a wild-type (WT) or mutated (Mut) VEGF promoter-luciferase construct were treated with or without 10-6 M simvastatin for 24 h. A mutated construct (-1128 to -928 and -906 to +827) had deletion of the HRE. Luciferase activity is expressed in units relative to the pGL3-basic vector. The data from triplicate measurements are expressed as the mean ± SEM. P < 0.05 (comparison between two groups). B, Gel mobility shift assay of the HRE of the mouse VEGF gene (-921 to -901). The assay was performed using a wild-type (WT) or mutated (Mut) HRE sequence in nuclear extracts of MC3T3-E1 cells treated with or without 10-6 M simvastatin (Sim) for 12 h. Competition experiments were performed with excess unlabeled, wild-type, and mutated HRE. The nucleotide sequences are described in the bottom of panel B. The specific protein-DNA complex affected by simvastatin is indicated by the closed arrowhead.(}iy]), 百拇医药
Stimulation of VEGF protein secretion by simvastatin in MC3T3-E1 cells
We examined VEGF in medium from simvastatin-treated MC3T3-E1 cells (Fig. 8A). A significant elevation in VEGF secretion was observed at 24 h of treatment with 10-6 M simvastatin. We next tested the effects of mevalonate and various inhibitors on VEGF secretion added together with 10-6 M simvastatin or vehicle for 24 h (Fig. 8B). Coincubation with 1 mM mevalonate and simvastatin abrogated the statin-induced secretion of VEGF. Furthermore, PI3K inhibitors such as wortmannin and LY294002 also abolished the effect of simvastatin. An inhibitor of protein prenylation, manumycin A, mimicked the increased secretion by simvastatin in osteoblasts. These results indicated that simvastatin augmented VEGF protein secretion in osteoblastic cells, suggesting that this stimulation involves reduced protein prenylation and is mediated via the PI3K pathway.:?v, 百拇医药
fig.ommitteed:?v, 百拇医药
Figure 8. VEGF protein secretion in MC3T3-E1. Cells were cultured in -MEM medium containing 10% FBS until the third day after confluence. A, Time course of the stimulation of VEGF protein secretion by simvastatin in MC3T3-E1 cells. These cells were further incubated in serum-free differentiation medium for 24 h and treated with vehicle (open columns) or 10-6 M simvastatin (closed columns) for the indicated periods. The conditioned media from a 12-h period were collected, and VEGF concentrations were measured by a mouse VEGF assay kit. B, Effects of mevalonate, manumycin A, and PI3K inhibitors on VEGF protein secretion in MC3T3-E1 cells. The cells were treated with 10-6 M simvastatin (Sim) or 5 µM manumycin A (MaA) for 24 h, and compounds including mevalonate (Mev), wortmannin (Wor), and LY294002 (LY) were added 1 h before vehicle and simvastatin treatment. VEGF concentrations in the conditioned media of the last 12 h period of the culture were determined. The data are expressed as the mean ± SEM of three determinations. *, P < 0.01 (compared with vehicle-treated control). #, P < 0.01 (compared with simvastatin-untreated vehicle control by Student’s t test).
Effect of simvastatin on BMP-2 and VEGF gene expression}y, 百拇医药
Because statins induce BMP-2 expression (32, 33, 34) and BMPs, including BMP-2, stimulate VEGF gene expression and its production in osteoblasts (41, 42), we aimed to clarify whether or not simvastatin enhances the gene expression of VEGF via increased production of BMP-2 in MC3T3-E1 cells. Noggin, BMP antagonist, prevents BMPs from binding to their receptors. Treatment of the cells with noggin (1.0 µg/ml) and simvastatin (10-6 M) did not affect the statin-induced expression of VEGF mRNA (Fig. 9). Because the expression of BMP-2 mRNA in MC3T3-E1 cells was low and not detected by Northern blot analysis, we used the more sensitive method, RT-PCR analysis, for determining BMP-2 mRNA (Fig. 10). Simvastatin (10-6 M) significantly increased BMP-2 mRNA expression in MC3T3-E1 cells 24 and 36 h after treatment. The simvastatin-induced increase in BMP-2 mRNA expression by simvastatin was delayed in comparison to that of VEGF mRNA expression. These results indicate that simvastatin-enhanced gene expression of VEGF is independent of BMP-2 expression stimulated by statins.
fig.ommitteed/gow6m, 百拇医药
Figure 9. Effect of noggin, BMP inhibitor, on simvastatin-induced expression of VEGF mRNA by MC3T3-E1 cells. The cells were treated with a vehicle (Veh) or with 10-6 M simvastatin (Sim) in the absence or presence of noggin (1 µg/ml) for the indicated period. Total RNA (25 µg) was extracted and subjected to Northern blot analysis. VEGF mRNA abundance was measured densitometorically and normalized to that of cyclophilin mRNA. The data are expressed as the mean ± SEM of four measurements. *, P < 0.05 (compared with vehicle-treated control at each point). #, P < 0.05 (compared with noggin-treated control at each point). No significant difference was observed between the noggin-treated and untreated groups in the presence of simvastatin./gow6m, 百拇医药
fig.ommitteed/gow6m, 百拇医药
Figure 10. Time course of the effect of simvastatin on BMP-2 mRNA expression in MC3T3-E1 cells. Serum-starved cells were treated with 10-6 M simvastatin () or vehicle () for the indicated period. Total RNA was subjected to RT-PCR. The PCR products were measured by Southern blot analysis, and BMP-2 mRNA abundance was corrected for the expression of cyclophilin mRNA. The data are expressed as the mean ± SEM of four determinations. *, P < 0.05 (compared with vehicle-treated control at each point).
Effect of simvastatin on mineralized nodule formation in MC3T3-E1 cells+@6of{, 百拇医药
We finally tested the effect of simvastatin on osteoblastic differentiation as monitored by mineralization (Fig. 11). MC3T3-E1 cells were cultured in differentiation medium with 10% FBS for 14 d after confluence in the presence and absence of 10-6 M simvastatin. Figure 11A shows an increase in mineralization in response to simvastatin; mevalonate and GGPP blunted this increase. A PI3K inhibitor, LY294002, also blocked this effect of simvastatin. Furthermore, a VEGF receptor 2 (fetal liver kinase 1; Flk-1) kinase inhibitor, SU1498, blunted simvastatin-induced mineralization in MC3T3-E1 cells (Fig. 11B).+@6of{, 百拇医药
fig.ommitteed+@6of{, 百拇医药
Figure 11. Effect of simvastatin (Sim) on mineralization of extracellular matrix by MC3T3-E1 cells. The cells were cultured in differentiation medium containing 10% FBS in the presence or absence of 10-6 M simvastatin for 14 d after confluence. A, Effects of mevalonate (Mev), GGPP, and LY294002 (LY), a PI3K inhibitor. B, Effect of SU1498 (SU), a VEGF receptor 2 kinase inhibitor. Cells were cultured in the medium containing vehicle, 1 mM mevalonate, 20 µM GGPP, 2 µM LY294002, or 10 µM SU1498 with vehicle or 10-6 M simvastatin for 14 d. AR-S staining was performed for the demonstration of mineralized nodule formation at the end of culture. Mineralized nodules stained with AR-S were photographed (bottom panels), and then AR-S was eluted from the matrix using cetylpyridinium chloride. AR-S concentrations were measured by spectrophotometry at 562 nm (top panels). The data are expressed as the mean ± SEM of four determinations. *, P < 0.01 (compared with vehicle control).
Discussionz, 百拇医药
Mundy et al. (32) first reported that statins stimulated in vivo bone formation in rodents and increased new bone volume in cultures from mouse calvarium. The enhancing effect of statins on bone formation is associated with increased expression of BMP-2 via activation of the gene promoter (32, 33). Recently, we found that a relatively low dose of simvastatin (10-7 M) induced osteoblastic differentiation and markedlyincreased mineralization in MC3T3-E1 nontransformed osteoblasts (30). Furthermore, statins such as compactin and pitavastatin promoted differentiation of embryonic stem cells into osteoblasts (43) and up-regulated the gene expression for BMP-2 and osteocalcin by depletion of mevalonate and GGPP. Depletion, in turn, was followed by suppression of Rho-associated kinase activity (34). On the basis of these findings, BMP-2 clearly plays a role in the osteoblast maturation induced by statins. However, several factors influencing bone anabolism such as TGF-ß, fibroblast growth factor 2, IGF-I, and VEGF are involved in promoting differentiation of osteoblasts and stimulating bone formation. Thus, we investigated whether bone anabolic factors other than BMP-2 are induced by treatment of osteoblasts with statins. We found that statins such as simvastatin, atorvastatin, and cerivastatin markedly enhanced gene expression for VEGF in three osteoblastic cell lines.
Involvement of VEGF in endochondral bone formation has been demonstrated by VEGF inactivation, which restricts formation via inhibition of angiogenesis (16). Furthermore, in addition to vascular endothelial cells, osteoblasts produce VEGF whose expression is enhanced by factors including prostaglandin E1 (26, 44), IGF-I (17, 29, 45), TGF-ß (36, 46), fibroblast growth factor 2 (36), and 1,25-dihydroxyvitamin-D3 (47). Osteoblasts express VEGF receptors including VEGF receptor 2 (Flk-1), which binds VEGF-A, VEGF-C, and VEGF-D isoforms with high affinity (29). Exogenous or endogenous VEGF stimulates osteoblastic differentiation as monitored by new bone formation (29). Midy and Plouet (15) reported that, on a molar basis, VEGF was a much more potent inducer of osteoblastic differentiation than BMP-2. The present results indicate that hydrophobic statins such as simvastatin, atorvastatin, and cerivastatin clearly increase VEGF mRNA abundance in the MC3T3-E1 osteoblastic cell line and demonstrate that statin-induced mRNA expression also occurs in other osteoblast-like cells. ST2, a clone of marrow stromal cells, was isolated from the bone marrow of BC8 mice and shows characteristics of typical adipocytes (48). When ST2 cells were cultured in medium with ascorbic acid, they developed an osteoblastic phenotype (49). UMR-106 cells, which were derived from a rat osteosarcoma, show osteoblastic features such as PTH and 1,25-dihydroxyvitamin-D3 receptors (35, 50). The MC3T3-E1 cells were nontransformed preosteoblasts derived from mouse calvaria that differentiate exclusively into osteoblasts (51). Statins clearly augmented mRNA expression for VEGF by these various osteoblastic cell lines, indicating that statin-induced expression of VEGF is a characteristic of osteoblasts. However, high statin concentrations induce apoptosis of osteoblasts, as reported in studies of smooth muscle cells (52) and cultured cortical neurons (53). We confirmed this by demonstrating that 10-4 M simvastatin suppresses VEGF mRNA expression in MC3T3-E1 cells.
Data presented here demonstrate that a cellular signal evoked by statins is transmitted to the nucleus to stimulate VEGF gene expression. Simvastatin increased steady state levels of VEGF mRNA without modifying the half-life of this mRNA. Although hnRNA may reflect alterations in RNA processing, its abundance correlates well with transcription rates measured by nuclear runoff assays (54, 55). Abundance of hnRNA was significantly elevated as early as 4 h after treatment with simvastatin in MC3T3-E1 osteoblastic cells. Elevation of hnRNA preceded an increase in mRNA accumulation of the cells. In addition, we cloned the promoter region of the mouse VEGF gene and transiently transfected chimeric deletion constructs containing the 5'- flanking region (1128 bp) and the luciferase gene into MC3T3-E1 cells. Simvastatin markedly stimulated luciferase activity in transfected cells. The HRE of the human VEGF gene promoter (39) is present in the mouse gene, as demonstrated by sequence analyses performed by us and other researchers (38). Gel mobility analysis revealed that simvastatin clearly increased the binding of the HRE-protein complex in MC3T3-E1 cells. Our study of mutation also showed that HRE is crucial to simvastatin-stimulated promoter activity, determined by luciferase, and intact HRE is necessary to specific DNA-protein complex by gel mobility shift assay. These results indicate that mevalonate depletion resulting from addition of simvastatin, an HMG-CoA reductase inhibitor, up-regulates VEGF transcription in osteoblastic cells. Recent investigations (45) showed that hypoxia-inducible factor-2{alpha} mediated transcriptional activation of VEGF gene in response to hypoxia or IGF-I in human osteoblast-like cells. Statins may activate the VEGF gene through increased binding to specific elements of the VEGF promoter of activators for gene transcription such as hypoxia-inducible factor-2{alpha} . However, further studies are needed to elucidate the precise mechanisms underlying transcriptional activation of the VEGF gene by statins.
Many studies document that statins trigger various cellular events through their functions as HMG-CoA reductase inhibitors as well as other signaling pathways (1, 5, 6, 7, 8, 30, 32, 33, 34, 43, 52). By modulating the initial part of the cholesterol synthesis pathway, statins decrease concentrations of many important intermediate compounds, including isoprenoids that contain GGPP and FPP. Isoprenoids are lipids that are attached in post-translational modification to certain proteins including the c subunit of heterotrimeric G proteins, small G proteins (Ras), and Ras-like proteins such as Rho, Rap, Rab, or Ral (9). However, recent studies report that statins may have additional functions independent of inhibition of HMG-CoA reductase. For example, lovastatin binds to the I domain of {alpha} 1ß2 integrin to block interaction of this integrin with intercellular adhesion molecule-1; this blockade is not reversed by addition of mevalonate (56, 57). Accordingly, we confirmed involvement of the mevalonate pathway and isoprenoids in negative modulation of VEGF gene expression by demonstrating that the direct product of HMG-CoA reductase, mevalonate, as well as the isoprenoid, GGPP, reversed stimulation of VEGF gene expression in MC3T3-E1 osteoblastic cells. Furthermore, to assess whether protein prenylation may play a role in the induction of VEGF gene of osteoblasts, MC3T3-E1 cells were exposed to an inhibitor of protein prenyl transferase, manumycin A. This inhibitor, which selectively affects farnesyl transferase, a major prenyl transferase, clearly increased VEGF mRNA concentrations in osteoblastic cells. FPP also inhibited statin-induced expression of VEGF mRNA in these cells. Our results indicate that farnesylated and geranylgeranylated proteins are involved in the up-regulation of VEGF gene expression.
A newly developed statin, pitavastatin, has been shown to inhibit Rho-associated kinase activity by suppressing prenylation of small GTP-binding proteins in human osteoblasts. Increases then follow in gene expression for osteoblastic markers such as BMP-2 and osteocalcin (34). We therefore investigated mechanisms following statin-induced inhibition of protein prenylation that underlie up-regulation of VEGF production. A selective inhibitor of Rho kinase, Y-27632, did not mimic the effect of statin on VEGF mRNA expression in osteoblasts (our unpublished results), suggesting that inhibition of Rho kinase is not the major basis of this statin effect. According to Tokuda et al. (26), prostaglandin E1-induced VEGF synthesis in osteoblasts was mediated through activation of p38 MAPK. Because a specific inhibitor of p38 MAPK, SB203580 (58), could not abolish simvastatin-stimulated expression of VEGF mRNA (our unpublished results), p38 MAPK also is unlikely to be involved in this process. Statins such as simvastatin and atorvastatin have been shown to increase endothelial progenitor cell proliferation and survival in vitro (59, 60) and to promote angiogenesis in vivo via the PI3K/Akt pathway (5). Furthermore, IGF-I augmented the promoter activity of VEGF gene mediated by the PI3K/Akt pathway in osteoblastic cells (45). Involvement of PI3K as a mediator of statin action on VEGF gene expression was evaluated by using two specific inhibitors of the enzyme, LY294002 and wortmannin. Both inhibitors blunted the simvastatin action on VEGF mRNA accumulation in MC3T3-E1 osteoblastic cells. This finding strongly suggests that the stimulation of VEGF gene transcription induced by statins is mediated via the PI3K pathway.
Statins trigger different cellular events through their function as HMG-CoA reductase inhibitors, as well as other signaling pathways. The fact that manumycin A markedly stimulates VEGF mRNA expression at a low concentration (5 µM) suggests that inhibition of protein prenylation is of particular importance. Recent studies have demonstrated that the exogenous addition of VEGF into conditioned media of osteoblasts stimulates mineralized nodule formation in a dose-dependent manner (29, 61). We demonstrated that the inhibition of the VEGF signaling pathway by a VEGF receptor 2 (Flk-1) kinase inhibitor, SU1498, suppresses mineralization by simvastatin-treated MC3T3-E1 osteoblastic cells. Previous studies (29, 61), combined with our results, suggest that simvastatin promotes osteoblastic differentiation, at least in part, by stimulating VEGF expression. Interestingly, BMPs including BMP-2, elevated VEGF production by osteoblastic cells and increased angiogenesis in angiogenesis assays using bone explants (41). This process induced by BMPs in osteoblasts may be involved in activation of p70 S6 kinase (42). We demonstrated that simvastatin-induced expression of VEGF mRNA in osteoblastic cells is independent of BMP-2 expression. The inhibition of BMP-2 binding to specific receptors caused by noggin, BMP binding protein (62), had no effect on simvastatin-induced expression of VEGF mRNA in MC3T3-E1 cells. Furthermore, studies of the time course of the effects of simvastatin revealed that the simvastatin-induced increase in the expression of VEGF mRNA precedes the induction of BMP-2 mRNA expression in these cells. BMPs and VEGF interact to stimulate bone formation by osteoblasts. Thus, statins are likely to augment the expression of bone anabolic factors such as VEGF and BMP-2, subsequently accelerating bone formation. Accordingly, statins may be potentially useful drugs for the treatment of osteoporosis.
In summary, we demonstrated that hydrophobic statins such as simvastatin, atorvastatin, and cerivastatin augment expression of VEGF mRNA and protein in MC3T3-E1 murine osteoblastic cells. We also found that inhibition of protein prenylation elicited by these hydrophobic HMG-CoA reductase inhibitors increases transcription of the VEGF gene in osteoblastic cells. This is the first report showing that statins stimulate gene expression for a bone anabolic factor in osteoblasts other than BMP-2. Our results suggest that hydrophobic statins promote osteoblastic differentiation and mineralization at least in part by enhancing production of VEGF by osteoblasts. These findings raise a strong possibility that statins stimulate bone growth and repair in vivo by increasing angiogenesis via locally acting angiogenic factors such as VEGF.9d\}k, 百拇医药
Acknowledgments9d\}k, 百拇医药
We thank Dr. Hitoshi Saitoh at Fuji Gotemba Research Laboratory of Chugai Pharmaceutical Co. (Gotemba, Japan) for statistical analysis.
Received July 5, 2002.+|-;z&:, http://www.100md.com
Accepted for publication November 4, 2002.+|-;z&:, http://www.100md.com
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