降血脂藥物statin誘導環氧化脢之作用機轉,細胞種類及動物種類上的差異及在臨床治療上的意義

行政院國家科學委員會專題研究計畫 成果報告

降血脂藥物 statin 誘導環氧化脢之作用機轉,細胞種類及動

物種類上的差異及在臨床治療上的意義

計畫類別： 個別型計畫

計畫編號： NSC93-2314-B-002-266-

執行期間： 93 年 08 月 01 日至 94 年 07 月 31 日

執行單位： 國立臺灣大學醫學院家庭醫學科

計畫主持人： 黃國晉

計畫參與人員： 林琬琬、陳瑞菁

報告類型： 精簡報告

處理方式： 本計畫可公開查詢

中 華 民 國 94 年 10 月 17 日

UNCORRECTED PROOF

1

2 HMG–CoA reductase inhibitors upregulate heme oxygenase-1 expression

3

in murine RAW264.7 macrophages via ERK, p38 MAPK and protein

4

kinase G pathways

5

Jui-Ching Chen

, Kuo-Chin Huang

, Wan-Wan Lin

*

7 b

Department of Family Medicine of National Taiwan University Hospital, Taipei, Taiwan 8 Received 7 January 2005; received in revised form 10 March 2005; accepted 15 March 2005 9

10 Abstract

11 Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in heme catabolism, which confers cytoprotection against oxidative injury and 12 provides a vital function in maintaining tissue homeostasis. HMG – CoA reductase inhibitors (statins) possess several anti-inflammatory 13 mechanisms and may be beneficial in the treatment of inflammatory diseases. Our previous study has shown that statins can inhibit iNOS 14 gene expression in murine RAW264.7 macrophages. In this study, we showed that lovastatin, fluvastatin, atorvastatin, simvastatin, 15 mevastatin and pravastatin are able to upregulate the mRNA expression of HO-1 gene. This effect of lovastatin was attenuated by farnesyl 16 pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), a protein kinase G (PKG) inhibitor (KT5823), a soluble guanylyl cyclase 17 inhibitor (ODQ), a p38 MAPK inhibitor (SB203580), and MEK inhibitors (U0126 and PD98059), but not by inhibitors of protein kinase C 18 (PKC), protein kinase A (PKA), c-jun N-terminal kinase (JNK) and Rho kinase. Consistent with this notion, our previous study has reported 19 the ability of statins to activate ERK and p38 MAPK in RAW264.7 macrophages. Here we further found the participation of cyclic guanosine 20 monophosphate (cGMP)/PKG pathway for ERK activation in cells stimulated with statin and the ability of statin to induce AP-1 activity, 21 which is an essential transcription factor in the regulation of HO-1 gene expression. In addition, a Ras inhibitor (manumycin A) treatment 22 also caused a marked induction of HO-1 mRNA followed by a corresponding increase in HO-1 protein; instead, inhibition of Rho activity by 23 toxin B only led to a transient and weak induction of HO-1. The involvement of signal pathways in manumycin A-induced HO-1 gene 24 expression was associated with p38 MAPK, JNK and ERK activation. Taken together, these results demonstrate for the first time that statins 25 might activate PKG to elicit activations of ERK and p38 MAPK pathways and finally induce HO-1 gene expression, which provides a novel 26 anti-inflammatory mechanism in the therapeutic validity.

27 D 2005 Published by Elsevier Inc.

28

29 Keywords: HMG – CoA reductase inhibitor; HO-1; p38 MAPK; ERK; cGMP; PKG; Isoprenoid; RAW264.7 macrophages

30

31 1. Introduction

32 Heme oxygenase (HO) is the rate-limiting enzyme in the

33 oxidative degradation of heme into bilirubin, iron, and

34 carbon monoxide (CO). While HO-2 and HO-3 are

35 constitutively expressed, 1 is the inducible form.

HO-36 1 is expressed with low level under basal conditions and can

37 be highly induced in response to various agents causing

38 oxidative stress including hyperthermia, UV irradiation[3],

39 hydrogen peroxide [3], heavy metals [3], inflammatory

40 cytokines[4], endotoxin[5], hypoxia[6], hyperoxia[7], and

41 nitric oxide (NO) [8,9]. HO-1 induction provides

cytopro-0898-6568/$ - see front matterD 2005 Published by Elsevier Inc. doi:10.1016/j.cellsig.2005.03.016

Abbreviations: AP-1, Activator protein 1; 8BrcGMP, 8-Bromo-cyclic guanosine monophosphate; dbcgmp, Dibutyryl 3V,5V-cyclic guanosine monophosphate; CO, Carbon monoxide; ERK, Extracellular signal-regulated kinase; FPP, Farnesyl pyrophosphate; GGPP, Geranylgeranyl pyrophosphate; HMG – CoA, 3-Hydroxy-3-methylglutaryl – coenzyme A; HO-1, Heme oxygenase-1; iNOS, Inducible nitric oxide synthase; JNK, c-jun N-terminal kinase; LPS, Lipopolysaccharide; MAPK, Mitogen-acti-vated protein kinase; MEK, Mitogen-actiMitogen-acti-vated protein/ERK kinase; NO, Nitric oxide; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PDE, Phosphodiesterase; PKA, Protein kinase A; PKC, Protein kinase C; PKG, Protein kinase G; RT-PCR, Reverse transcription-polymerase chain reaction; sGC, Soluble guanylyl cyclase; SNP, Sodium nitroprusside.

* Corresponding author. Tel.: +886 2 23123456x8315; fax: +886 2 23915297.

E-mail address: wwl@ha.mc.ntu.edu.tw (W.-W. Lin).

Cellular Signalling xx (2005) xxx – xxx

www.elsevier.com/locate/cellsig

UNCORRECTED PROOF

ARTICLE IN PRESS

42 tection against oxidative stress and apoptosis and preserves 43 cellular homeostasis [1,2]. This action is demonstrated not 44 only in cultured cell systems[10]but also in in vivo studies 45 [11,12]. Although the mediators and mechanisms by which 46 HO-1 provides protection are not clear and depend on cell 47 types and stimuli, accumulating lines of evidence point the 48 important role of CO[13,14]. A low concentration of CO 49 can exert protection through a soluble guanylyl cyclase 50 (sGC) and cyclic guanosine monophosphate (cGMP) path-51 way[15].

52 Statins are inhibitors of the 3-hydroxy-3-methyl-glu-53 taryl – coenzyme A (HMG – CoA) reductase and are widely 54 used as lipid-lowering agents [16]. Besides the therapeutic 55 use in hyperlipidemia, the anti-inflammatory and immuno-56 modulatory benefits of statins have been recently reported in 57 many aspects, although mechanisms are not yet completely 58 defined [17]. Most identified anti-inflammatory benefits of 59 statins rely on the reduction of cellular levels of mevalonate, 60 the direct product of HMG – CoA reductase, and mevalo-61 nate-derived isoprenoids, farnesyl pyrophosphate (FPP) and 62 geranylgeranyl pyrophosphate (GGPP), which are involved 63 in post-translational modification of several small G 64 proteins, such as Rho, Rac, Cdc42, and Ras[18,19]. 65 Since the understanding and evaluation of the pharma-66 cological effects of statins are increasing and accelerating 67 their clinical importance and validity, in this study we 68 intended to identify the action of statins on HO-1 gene 69 expression in murine RAW264.7 macrophages. Using this 70 cell type we previously have demonstrated the abilities of 71 statins to block inducible nitric oxide synthase (iNOS) 72 induction caused by lipopolysaccharide (LPS) and inter-73 feron-g [20]. Intriguingly in the present study we demon-74 strated that statins are capable of inducing HO-1 gene 75 transcription in murine RAW264.7 macrophages and 76 elucidated the mechanisms involved.

77 2. Materials and methods 78 2.1. Materials

79 Dulbecco’s modified Eagle’s medium (DMEM), fetal 80 bovine serum (FBS), penicillin, and streptomycin were 81 obtained from Gibco BRL (Grand Island, NY). Rabbit 82 polyclonal antibodies specific for HO-1, h-actin, ERK, JNK 83 and p38 mitogen activated protein kinase (MAPK) were 84 purchased from Santa Cruz Biotechnology (Santa Cruz, 85 CA). Antibodies specific to the phosphorylated ERK, JNK 86 and p38 MAPK were purchased from Cell Signaling 87 Technology (Beverly, MA). The ECL detection agents were 88 purchased from Amersham Biosciences (Piscataway, NJ). 89 Toxin B from Clostridium difficile was obtained from 90 Calbiochem (San Diego, CA). The Ras inhibitor manumy-91 cin A, lovastatin, phenol-extracted LPS (L8274) from E. 92 coli, farnesyl pyrophosphate (FPP), geranylgeranyl pyro-93 phosphate (GGPP), KT5720, KT5823, Y27632,

1H-94 [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ),

anisomy-95 cin, 8-bromo-cyclic guanosine monophosphate (8BrcGMP),

96 dibutyryl 3V,5V-cyclic guanosine monophosphate (dBcGMP)

97 and other chemicals were purchased from Sigma Aldrich

98 (St. Louis, MO). GF109203X, SB203580, Ro 31-8220,

99 U0126, PD98059 and mevastatin were purchased from

100 Calbiochem (San Diego, CA). SP600125 was obtained from

101 Tocris Cookson (Ellisville, MO). Atorvastatin, fluvastatin,

102 pravastatin and simvastatin were respectively provided by

103 Pflizer Inc. (NY), Novartis (Basel, Switzerland), Sankyo

104 Co., Ltd. (Tokyo, Japan) and Merck and Co., Inc. (NJ). The

105 AP-1 luciferase construct was provided by Dr. G. Hageman

106 (Flanders Interuniversity Institute for Biotechnology and

107 University of Gent, Gent, Belgium). All materials for

108 sodium dodecyl sulfate-polyacrylamide gel electrophoresis

109 (SDS-PAGE) were obtained from Bio-Rad (Hercules, CA).

110 2.2. Cell culture

111 Murine RAW264.7 macrophages obtained from

Ameri-112 can Type Culture Collection (Manassas, VA, U.S.A.) were

113 grown at 37 -C in 5% CO2using DMEM containing 10%

114 FBS, 100 U/ml penicillin and 100 Ag/ml streptomycin.

115 2.3. Protein preparation and Western blotting

116 After stimulation, cells were rinsed twice with ice-cold

117 PBS and 100 Al of cell lysis buffer (20 mM Tris – HCl,

118 pH7.5, 125 mM NaCl, 1% Triton X-100, 1 mM MgCl2, 25

119 mM h-glycerophosphate, 50 mM NaF, 100 AM Na3VO4, 1

120 mM PMSF, 10 Ag/ml leupeptin, 10 Ag/ml aprotinin) was

121 then added to each plate. Protein was denatured in SDS,

122 electrophoresed on 10% SDS/polyacrylamide gel, and

123 transferred to nitrocellulose membrane. Nonspecific binding

124 was blocked with TBST (50 mM Tris – HCl, pH7.5, 150

125 mM NaCl, 0.1% Tween 20) containing 5% non-fat milk for

126 1 h at room temperature. After incubation with the

127 appropriate first antibodies, membranes were washed three

128 times with TBST. The secondary antibody was incubated for

129 1 h. Following 3 washes with TBST, the protein bands were

130 detected with the ECL reagent.

131 2.4. Reverse transcription-polymerase chain reaction

132 (RT-PCR)

133 To amplify HO-1 mRNA, the specific primers for

RT-134 PCR analysis were synthesized. Macrophages treated with

135 indicated agents were homogenized with 1 ml of RNAzol B

136 reagent (Gibco) and total RNA was extracted by acid

137 guanidinium thiocyanate-phenol-chloroform extraction. RT

138 was performed using StrataScript RT-PCR Kit and 10 Ag of

139 total RNA was reverse transcribed to cDNA following the

140 manufacturer’s recommended procedures. RT-generated

141 cDNA encoding HO-1 and h-actin genes were amplified

142 using PCR. The oligonucleotide primers used correspond to

143 the mouse HO-1 (5V-GAG AAT GCT GAG TTC ATG-3V

J.-C. Chen et al. / Cellular Signalling xx (2005) xxx – xxx 2

UNCORRECTED PROOF

h-145 actin (GAC TAC CTC ATG AAG ATC CT-3V and 5V-146 CCA CAT CTG CTG GAA GGT GG-3V). PCR was 147 performed in a final volume of 50 Al containing: Taq 148 DNA polymerase buffer, all four dNTPs, oligonucleotide 149 primers, Taq DNA polymerase, and RT products. After an 150 initial denaturation for 2 min at 94 -C, 35 cycles of 151 amplification (94-C for 45 s, 65 -C for 45 s, and 72 -C for 2 152 min) were performed followed by a 10-min extension at 72 153 -C. PCR products were analyzed on 2% agarose gel. The 154 mRNA of h-actin served as an internal control for sample 155 loading and mRNA integrity.

156 2.5. Transfection and AP-1-luciferase assays

157 Using electroporation (280 V, 1070 AF, 30 ms time 158 constant), cells (2 107

cells/cuvette) were cotransfected 159 with 1 Ag of AP-1 promoter construct and 1 Ag h-160 galactosidase expression vector (pCR3lacZ; Pharmacia, 161 Sweden). After electroporation, cells were cultured in 24-162 well plate at 2 106

cells/well. After 24-h incubation, cells 163 were incubated with the indicated concentrations of agents. 164 After another 24-h incubation, the media were removed and 165 the cells were washed once with cold PBS. To prepare 166 lysates, 100 Al of reporter lysis buffer (Promega) was added 167 to each well and cells were scraped from dishes. The 168 supernatant was collected after centrifugation at 13,000 rpm 169 for 30 s. Aliquots of cell lysates (5 Al) containing equal 170 amounts of protein (10 – 20 Ag) were placed into the wells of 171 an opaque, black 96-well microplate. An equal volume of 172 luciferase substrate (Promega) was added to all samples and 173 the luminescence was measured in a microplate luminom-174 eter (Packard, Meriden, CT). Luciferase activity values were 175 normalized to transfection efficiency monitored by h-176 galactosidase expression and was presented as the percent-177 age of luciferase activity in control group without statin 178 treatment.

179 2.6. Statistical evaluation

180 Values were expressed as the meanT S.E.M. of at least 181 three experiments, which was performed in duplicate. 182 Analysis of variance (ANOVA) was used to assess the 183 statistical significance of the differences and a ‘‘p’’ value 184 less than 0.05 is considered statistically significant.

185 3. Results

186 3.1. Statins transcriptionally induce HO-1 gene expression 187 in murine RAW264.7 macrophages

188 Murine RAW264.7 macrophages were chosen to inves-189 tigate the signal pathways of statin in HO-1 expression, an 190 anti-inflammatory gene. Treatment with lovastatin, fluvas-191 tatin and simvastatin (each at 30 AM) induced HO-1

192 protein expression. At basal state, a weak

immunoreactiv-193 ity of HO-1 protein was detected. The stimulating action of

194 30 AM lovastatin and fluvastatin displayed the

time-195 dependency, occurring after 3 h exposure, peaking at 12

196 h and maintaining for up to 24 h (Fig. 1a). The HO-1

197 expression induced by simvastatin also occurred after 3 h

198 exposure but peaked at 6 h and then disappeared. Next

199 concentration-dependency of this action of six statins was

200 examined. Cells were incubated with indicated

concen-Fig. 1. Time- and dose-dependent effects of statins on the gene transcription of HO-1. (a, c) Cells were treated with statins at concentrations indicated for different periods. (b, d) Cells were treated with different concentrations of statins for 6 h (b) or 90 min (d). After stimulation, cell lysate or RNA was prepared respectively for determining HO-1 and h-actin proteins with immunoblotting (a, b) or for determining mRNA levels with RT-PCR (c, d). The h-actin level was considered as an internal control. Data on HO-1 protein and mRNA levels were measured by densitometry, normalized to the level of h-actin, and calculated as percentages of the maximal response of lovastatin (30 AM). Traces shown are representative of three separate experiments and the meanT S.E.M. was shown in parentheses.

UNCORRECTED PROOF

ARTICLE IN PRESS

201 trations of statins for 6 h and all of them were compared 202 and showed different potencies (Fig. 1b). Lovastatin, 203 fluvastatin and approximately atorvastatin induced com-204 parable extents of HO-1 protein induction within similar 205 concentration range of 1 – 30 AM. Pravastatin did not 206 stimulate HO-1 protein expression until 100 AM. Due to 207 moderate cell toxicity for simvastatin and mevastatin with 208 10 AM (data not shown), we used lower concentrations 209 (0.1 – 1 AM) of both agents. Results indicated that HO-1 210 induction by 0.3 AM simvastatin and mevastatin was 211 comparable to that of 1 AM lovastatin and maximal 212 response was almost achieved around 1 AM.

213 Since HO-1 is an inducible gene product, we determined 214 whether the effect of statin results from increased gene 215 transcription. Using RT-PCR analysis, we found that HO-1 216 mRNA was time- and concentration-dependently increased 217 by 30 AM of lovastatin (Fig. 1c,d). The incubation period as 218 short as 30 min was sufficient for lovastatin (30 AM) to 219 increase HO-1 mRNA level, which was further increased 220 and maintained for at least for 4 h.

221 3.2. HO-1 stimulation by statin is dependent on protein 222 prenylation

223 To further identify the product of HMG – CoA reductase 224 reaction necessary for the effect of statins, we incubated 225 cells with FPP and/or GGPP in the presence of lovastatin. 226 FPP and GGPP are involved in farnesylation and geranyl-227 geranylation of small G proteins, respectively. Fig. 2a 228 showed that FPP (30 AM) as well as GGPP (30 AM) 229 partially reversed the effect of lovastatin on HO-1 induction 230 and simultaneous presence of FPP and GGPP led to an 231 additive inhibition on HO-1 protein expression. These 232 observations suggest that the effect of statins on HO-1 233 expression is ascribed to the reduction of protein isopreny-234 lation, which negatively regulates HO-1 gene expression.

235 As mentioned previously, HO-1 was a cGMP-inducible

236 protein [8] and sodium nitroprusside (SNP) could induce

237 HO-1 protein expression in murine RAW264.7

macro-238 phages [9]. To examine whether protein prenylation is

239 involved in the sGC/cGMP-mediated HO-1 induction, we

240 pretreated murine RAW264.7 macrophages with FPP and/or

241 GGPP (each at 30 AM) for 30 min. We found that both

242 isoprenoids did not reverse SNP or dBcGMP-induced HO-1

243 protein levels (Fig. 2b,c).

244 3.3. Ras and Rho inhibition are involved in the upregulation

245 of HO-1 expression

246 To further verify the involvement of reduced

isopreny-247 lation of signaling proteins in statin-induced HO-1

expres-248 sion, we studied the effects of manumycin A. Manumycin A

249 is a potent and selective inhibitor of farnesyltransferase

250 (IC50= 5 AM) compared to geranylgeranyltransferase

251 (IC50= 180 AM) and acts as an inhibitor of Ras function

252

[21,22]. Our results showed that manumycin A (10 or 30 253 AM) caused HO-1 protein induction in a time- and

254 concentration-dependent manner (Fig. 3a,b). Likewise direct

255 inhibition of Rho family proteins with toxin B (400 pM)

256 caused HO-1 protein expression, while this action was slight

257 and transient (Fig. 3a). Because of cytotoxicity occurrence

258 at 100 AM manumycin A, we did not further determine the

259 HO-1 response of manumycin A at higher concentrations.

260 3.4. Signaling pathways of statin- and manumycin

A-261 induced HO-1 expression

262 Numbers of pathways have been implicated in

trans-263 mitting the extracellular signals to the nuclei for HO-1

264 gene expression. To investigate the signal transduction

265 pathway(s) involved in regulating HO-1 expression in

266 response to statin and manumycin A, we examined the

Fig. 2. HO-1 induction by statins was reversed by GGPP and FPP. GGPP (30 AM) and/or FPP (30 AM) was pretreated for 30 min prior to the incubation of lovastatin (30 AM, a), SNP (300 AM, b), or dBcGMP (200 AM, c) for 6 h. The protein levels of HO-1 were measured in the cell lysates by Western blot and calculated as percentages of the control response of each stimulus. The results are representative of three separate experiments and the meanT S.E.M. was shown in parentheses.

Fig. 3. Manumycin A and toxin B mimic statins’ effect on HO-1 induction. (a) Murine RAW264.7 macrophages were treated with manumycin A (10 and 30 AM), toxin B (400 pM) or fluvastatin (30 AM) for the indicated time periods. (b) Murine RAW264.7 macrophages were treated with different concentrations of manumycin A for 6 h. Protein levels of HO-1 were measured in the cell lysates by Western blot and calculated as percentages of the response of fluvastatin (30 AM, a) or manumycin A (30 AM, b). The results are representative of three separate experiments and the meanT S.E.M. was shown in parentheses.

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UNCORRECTED PROOF

268 HO-1 protein levels. As shown in Fig. 4, we found that 269 treatment of cells with the PKG inhibitor (KT5823), the 270 sGC inhibitor (ODQ), two MEK inhibitors (PD98059 and 271 U0126), and the p38 MAPK inhibitor (SB203580) reduced 272 lovastatin-induced HO-1 induction. In contrast to the 273 inhibition on lovastatin response, KT5823 and ODQ failed 274 to change the response of manumycin A. Otherwise, 275 inhibitors of MAPKs, including U0126, PD98059, 276 SB203580 and the JNK inhibitor (SP600125), inhibited 277 manumycin A-induced HO-1 expression. KT5720 [a 278 protein kinase A (PKA) inhibitor], Y27632 (a Rho kinase 279 inhibitor), Ro 31-8220 and GF109203X [two protein 280 kinase C (PKC) inhibitors] did not have any effects. These 281 results suggest the participation of PKG, ERK and p38 282 MAPK, but not JNK, PKC, PKA, Rho kinase or PI3K, in 283 HO-1 expression in cells treated with statins, while the 284 action of manumycin A is dependent on ERK, JNK and 285 p38 MAPK. To further understand the involvement of 286 intracellular cGMP, we conducted experiments using 287 phosphodiesterase (PDE) inhibitor zaprinast. As shown in

288

Fig. 4b, zaprinast (100 AM) is able to induce HO-1 and 289 enhance the response of lovastatin.

290 3.5. Manumycin A mediates phosphorylation of p38 MAPK,

291 JNK and ERK

292 Since in previous study conducted in the same cell line

293 we have demonstrated that statin is able to cause ERK and

294 p38 MAPK activation[23], we attempted to further confirm

295 the crucial roles of three MAPKs in HO-1 expression by

296 manumycin A. As shown in Fig. 5, treatment of murine

297 RAW264.7 macrophages with 30 AM manumycin A

298 resulted in a time-dependent phosphorylation of p38

299 MAPK, JNK and ERK. Compared with the rapid onset of

300 anisomycin for these events seen at 5 min, manumycin

A-301 induced p38 MAPK phosphorylation occurred after 60 min

302 of incubation and then declined to basal level at 180 min

303 (Fig. 5a). Furthermore, immunoblotting to reflect JNK

304 activation indicated a delayed but significant response after

305 120 min of stimulation and the response continued until 180

306 min (Fig. 5b). In contrast to the delayed action on p38

307 MAPK and JNK, the manumycin A-induced increase in

308 ERK phosphorylation began at 5 min, peaked at 60 – 120

Fig. 4. Effects of protein kinase inhibitors on lovastatin- and manumycin A-induced HO-1 expression. Cells were pretreated with KT5720 (1 AM), KT5823 (3 AM), ODQ (1 AM), Y27632 (30 AM), GF109203X (3 AM), SB203580 (10 AM), SP600125 (10 AM), Ro 31-8220 (3 AM), U0126 (1 AM) or PD98059 (30 AM) for 30 min, then stimulated with lovastatin (30 AM) or manumycin A (30 AM) for 6 h. Protein levels of HO-1 were measured in the cell lysates by Western blot. Data on protein levels were measured by densitometry and calculated as percentages of the respective basal response of lovastatin or manumycin A (lane 2). Traces shown are representative of three separate experiments and the meanT S.E.M. was shown in a table. Asterisks are used to indicate the significance of these effects. In (b), zaprinast (100 AM) and lovastatin (30 AM) were treated as indicated for 6 h and HO-1 protein level was measured and quantified from three individual experiments.

Fig. 5. Manumycin A activates p38 MAPK, JNK and ERK. Cell lysates prepared from cells following manumycin A (30 AM) or anisomycin (1 AM) stimulation for different periods were immunoblotted with antibody specific for total or phosphorylated p38, JNK and ERK. Data on protein levels were measured by densitometry and calculated as percentages of the 30-min response of anisomycin. The results are representative of three separate experiments.

UNCORRECTED PROOF

ARTICLE IN PRESS

309 min and then declined at 240 min (Fig. 5c). The protein 310 levels of p38, JNK and ERK were not affected by 311 manumycin A treatment.

312 3.6. GC/cGMP pathway mediates statin-induced ERK 313 activation

314 Since p38 MAPK, ERK and PKG activation have been 315 implicated for lovastatin-induced HO-1 gene expression, we 316 used immunoblotting to verify the possible signaling 317 cascades underlying the action of statin. To test whether 318 cGMP pathway is upstream for p38 MAPK and ERK 319 activation, we treated murine RAW264.7 macrophages with 320 dBcGMP and 8BrcGMP, analogues of cGMP. Fig. 6a 321 showed the stimulating effects of dBcGMP (200 AM) on 322 ERK phosphorylation. dBcGMP treatment for a period as 323 short as 5 min was sufficient to activate ERK and this action 324 lasted at least for 4 h. In contrast, dBcGMP failed to alter the 325 phosphorylation of p38 MAPK and JNK (data not shown). 326 dBcGMP and 8BrcGMP, each at 200 AM, had the similar 327 efficacy to induce ERK activation at 30 min (Fig. 6b). Next 328 to further understanding the role of PKG, we examined the 329 effect of PKG inhibitor.Fig. 6b showed KT5823 incubation 330 indeed could block the ERK activation in response to 331 dBcGMP, 8BrcGMP and statin, but not to manumycin A. 332 These results suggest that cGMP/PKG-dependent signaling 333 pathway is involved in statin-induced ERK activation. 334 3.7. Statin induces AP-1 transactivation

335 Since AP-1 was shown as the major transcription factor 336 involved in HO-1 gene transcription [5,24 – 26], we

337 explored the effect of lovastain on the transactivity of

AP-338 1. As assessed by transfection with reporter gene driven by

339 AP-1 binding, we found lovastatin within 1 – 30 AM was

340 able to increase AP-1 activation in a

concentration-depend-341 ent manner (Fig. 7). Furthermore, the stimulating effect of

342 lovastatin was inhibited by KT5823, ODQ, SB203580,

343 U0126 and PD98059, but not by SP600125.

344 4. Discussion

345 Accumulating evidence has indicated HO-1 functions as

346 a ‘‘therapeutic funnel’’. Induction of HO-1 is suggested to

347 have cytoprotective effect against oxidative injury and have

348 the potent anti-inflammatory properties. Modulation of gene

349 transcription is the principal mechanism by which HO-1 is

350 regulated. Based on these results, induction of HO-1 is a

351 therapeutic strategy for treating inflammatory diseases. In

352 this aspect, animal studies and cell cultures have implicated

353 the anti-inflammatory benefits of HO-1 expression in

354 atherosclerosis, now considered as a kind of chronic

355 inflammatory process [27 – 31]. In order to investigate new

356 strategies to modify the pathophysiology of atherosclerosis,

357 we have tested whether HMG – CoA reductase inhibitors

358 could regulate the expression of HO-1 and have explored

359 the signal pathway involved in this regulation in RAW264.7

360 macrophages. In this study we unexpectedly found the

361 ability of statins to induce HO-1 expression in macrophages

362 and this action is mediated by impeding prenylation of small

363 G proteins, for example Ras protein in particular.

Further-Fig. 6. cGMP/PKA pathway mediates ERK activation. Cells were stimulated with dBcGMP (200 AM) for different periods (a) or pretreated with KT5823 (3 AM) for 60 min, followed by the stimulation with dBcGMP (200 AM), 8BrcGMP (200 AM), lovastatin (30 AM) or manumycin A (30 AM) for 5, 30 or 60 min (b). Cell lysates were immunoblotted with antibody specific for total or phosphorylated ERK. Data on protein levels were measured by densitometry and calculated as percentages of the control response. Results are representative of three independent experiments and the meanT S.E.M. was shown in parentheses.

Fig. 7. AP-1 activation by lovastatin. Cells transfected with the AP-1 reporter gene and h-gal-lacZ plasmid were pretreated with different concentrations of lovastatin (left) or pretreated with each pharmacological inhibitor for 30 min (3 AM KT5328, 1 AM ODQ, 10 AM SB203580, 1 AM U0126, 30 AM PD98059, 10 AM SP600125) followed by lovastatin (30 AM) stimulation for 24 h. The luciferase activity derived from AP-1 activation was normalized to the transfection efficiency with h-gal-lacZ. The data represent the meanT S.E.M. from at least 3 independent experi-ments. *p < 0.05, indicating the significant activation by lovastain.

#_{p < 0.05, indicating the significant inhibition of lovastatin response.}

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UNCORRECTED PROOF

365 activation are required for statins-induced HO-1 regulation 366 in murine RAW264.7 macrophages. However, in our 367 preparation of this manuscript one recent study reported 368 similar HO-1 induction by simvastatin in human and rat 369 aortic smooth muscle cells[32].

370 Our observation that the inductive effect of statin was 371 overcome by FPP and GGPP demonstrated the specificity of 372 the HMG – CoA reductase inhibition. FPP and GGPP are 373 metabolites of HMG – CoA reductase and substrates for 374 protein isoprenylation, which takes place during C-terminus 375 processing of small G proteins and is essential for their 376 coupling with multiple effector systems to activate distinct 377 physiological responses. Thus, reversal effect of FPP and 378 GGPP on statin-elicited HO-1 induction suggests impeding 379 small G protein functions are involved. Alternatively, we 380 predict one or more small G proteins are exerting a negative 381 role in HO-1 gene expression in basal condition of macro-382 phages and this intracellular balanced environment is altered 383 by statins. To clarify this notion, we determined whether 384 inhibition of Ras isoprenylation by manumycin A, an 385 inhibitor of Ras farnesyl transferase [21,22], could mimic 386 HO-1 expression. As a result, manumycin A indeed induced 387 HO-1 protein expression in a time- and concentration-388 dependent manner. Like the necessity of isoprenylation of 389 Ras superfamily to achieve molecular function, Rho 390 proteins acting as molecular switches to control cellular 391 processes also require the attachment of geranylgeraniol, an 392 isoprenoid intermediate of the cholesterol biosynthesis 393 pathway. The fact that lovastatin blocks geranylgeraniol 394 synthesis also prompts us to propose the notion that Rho 395 proteins are possibly involve in the signaling regulation of 396 HO-1 expression. We examined clostridial toxin B, which is 397 a non-selective inhibitor of Rho proteins, Rho, Rac and 398 Cdc42 [33,34] and observed a weak, transient but signifi-399 cant induction of HO-1. These results together suggest that 400 interruption of the cellular activity of Ras in primary and 401 Rho proteins, to a lesser extent, is involved to initiate 402 stimulating signals for HO-1 protein expression in macro-403 phages. Except Ras and Rho family proteins, whether 404 additional G proteins are involved in the regulation of HO-1 405 gene expression needs future investigation.

406 In general, HO-1 gene expression is induced by stimuli 407 that activate MAPKs[26,35]. Three major subgroups of the 408 MAPK family identified to date include ERK, JNK and p38 409 MAPK. Depending on the stimuli specificity, contradictory 410 results on the regulatory role of different MAPK pathways 411 for HO-1 gene expression were observed. In this aspect 412 recent mechanistic studies on HO-1 induction have pointed 413 the critical intermediacy of the p38 MAPK cascade but not 414 ERK in the regulation of HO-1 expression by TGF-h [4], 415 hypoxia [6], cadmium[36], IL-10[37], and 15dPGJ2[38].

416 In contrast, both ERK and p38 MAPK pathways medicate 417 HO-1 gene transcription by sodium arsenite [39 – 41] and 418 NO[42]. Instead JNK mediates the induction of HO-1 gene 419 expression by the glutathione depletor phorone[43]. On the

420 other hand, HO-1 gene transcription after ischemia –

421 reperfusion involves ERK, JNK, and p38 MAPK pathways

422

[35]. In this study we found the effect of lovastatin on HO-1 423 induction is dependent on p38 MAPK and ERK, but not on

424 JNK. These observations are in line with our previous study

425 to show ERK and p38 MAPK activation by statin in

426 RAW264.7 macrophages [23]. Nevertheless, activation of

427 three MAPKs is involved in the action of manumycin A.

428 The discrepancy between both stimuli is possibly resulting

429 from the net outcome through the diverse effects of statins

430 on multiple isoprenylated proteins.

431 Confirming previous findings showing intracellular

sec-432 ond messenger cGMP as an intermediate to enhance HO-1

433 expression [8,9,42,44], the present investigation also

434 observed such phenomena in murine RAW264.7

macro-435 phages. We found dBcGMP, SNP (a direct sGC activator via

436 NO release) and zaprinast (a PDE inhibitor) could induce

437 HO-1 and ODQ (an inhibitor of sGC) could inhibit HO-1

438 response of lovastatin, suggesting the contribution of cGMP

439 signaling in this event. In addition, our data provide new

440 insight into the participation of PKG in this event, as in this

441 study we observed PKG inhibitor could attenuate HO-1

442 induction in response to lovastatin. However, in contrast to

443 lovastatin action, both sGC and PKG inhibitors did not

444 prevent the action of manumycin A, suggesting that

differ-445 ential signaling pathways are exerted by both HO-1 inducers.

446 Moreover, in this study we further provide new insight that

447 PKG-mediated ERK signaling pathway plays a crucial role

448 for HO-1 expression by statin. Even though PKG-dependent

449 ERK activation was reported to participate in various cell

450 functions [45,46], this signal cascade is for the first time

451 shown in the present study to regulate HO-1 induction. In

452 agreement with previous study detecting the ability of statins

453 to stimulate cGMP formation in PC12 cells [47], our data

454 with the use of pharmacological inhibitors point the essential

455 role of cGMP/PKG in statin’s action. To confirm this notion,

456 experiment in the measurement of intracellular cGMP level

457 was taken. Unfortunately we cannot detect significant cGMP

458 change in cells following lovastatin incubation for different

459 periods in macrophages (data not shown). Thus we speculate

460 that, despite no increase of intracellular cGMP, the basal

461 activity of PKG is sufficient to play a role in the modulation

462 of HO-1 induction. In addition, the differential dependency

463 of PKG pathway in statin- and manumycin A-mediated

HO-464 1 response again strengthens the distinct action mechanisms

465 underlying the HO-1 induction by these two inducers,

466 despite some mechanisms, for example ERK, that might be

467 in common.

468 A number of response elements in the mouse HO-1

469 promoter and 5V-flanking region have been identified. The

470 mouse HO-1 gene contains two inducible enhancers, E1 and

471 E2. E1 contains three stress response elements that

472 encompass the consensus motifs for AP-1 proteins. Deletion

473 and mutational analyses of regulatory element of HO-1 gene

474 indicate that AP-1 binding site plays an important role in

475 mediating HO-1 gene regulation and is a commonality in

UNCORRECTED PROOF

ARTICLE IN PRESS

476 response to multiple agents in the activation mechanism of 477 HO-1[5 – 7,24 – 26,48]. Thus far, even though the response 478 elements mediating cGMP-dependent transcriptional acti-479 vation of HO-1 are poorly defined, one previous study has 480 pointed the importance of AP-1 in cGMP-mediated HO-1 481 induction in rat hepatocytes[25]. Consistent with previous 482 studies showing the ability of statins for AP-1 activity 483 [49,50], our current data demonstrated that HMG – CoA 484 reductase inhibitors indeed could induce AP-1 activation in 485 macrophages, which, as previously indicated with strong 486 evidence, leads to the induction of HO-1 gene transcription. 487 Furthermore, pharmacological approaches coincidentally 488 indicated the upstream signaling pathways of cGMP/PKG, 489 ERK and p38 MAPK for AP-1 activation.

490 In conclusion, we are presenting novel data showing that 491 HMG – CoA reductase inhibitors and manumycin A are able 492 to induce HO-1 gene expression in murine RAW264.7 493 macrophages. This effect of statins is mediated through p38 494 MAPK, ERK, PKG pathways and involves AP-1 activation. 495 Since it is conceivable that an ideal inducer of HO-1 activity 496 being an appropriate therapeutic intervention, our data 497 strongly support the protective effects of statins in the 498 therapy of disorders associated with inflammation and 499 oxidative injuries.

500 Acknowledgement

501 This work was supported by research grants (NSC 93-502 2314-B-002-266 and NSC94-2314-B-002) from the 503 National Science Council, ROC.

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