Acta Physiologica Sinica, June 25, 2003, 55(3): 265－272
Received 2002-11-01 Accepted 2003-01-22
This work was supported by the National Natural Sciences Foundation of China (No.30070280).
Corresponding author. Tel: +86-24-23056238; Fax: +86-24-23922184; Email: hanyal@mail.sy.ln.cn
Research Paper
Protein kinase C
and protein tyrosine kinase mediate lipopolysaccharide- and cytokine-induced
nitric oxide formation in vascular smooth muscle cells of rats
HAN Ya-Ling1,*, KANG Jian1, LI Shao-Hua2
1Department of Cardiology, General Hospital of Shenyang, The Institute of Cardiovascular Research, PLA, 110016; 2Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, New Jersey 08854, USA
Abstract: Rat aorta media, adventitia and cultured vascular smooth muscle cells (VSMCs) were used in this study to identify the source of nitric oxide (NO) generation from various cell types of vascular tissues and to elucidate the mechanisms involved in NO formation. Treatment of vascular media and VSMCs with lipopolysaccharide (LPS) or cytokines [tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)] resulted in a dose-dependent increase of NO release. Inducible nitric oxide synthase (iNOS) in the stimulated VSMCs was significantly upregulated as shown by Western blot analysis. Protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) prevented LPS-, TNF-α- and IL-1β-induced NO production, whereas N-(2-guanidinoethyl)-5-isoquinoline-sulfonamide (HA1004), an H7 analogue with little activity towards PKC, had no inhibition effect. The role of PKC in LPS- and cytokine-induced changes on NO formation was confirmed by using another structurally distinct PKC inhibitor chelerythrine. Treatment of VSMCs with protein tyrosine kinase (PTK) inhibitor genistein or tyrphostin AG18 also reduced the NO production evoked by LPS, TNF-α or IL-1β, which was associated with inhibition of iNOS protein expression. In contrast, PKC inhibitor chelerythrine did not affect iNOS expression. These results suggest that PTK mediates LPS- and cytokine-induced NO formation by upregulation of iNOS expression. PKC may be involved in the post-translational modification of iNOS or the regulation of the availability of iNOS substrates and cofactors.
Key words: nitric oxide; protein kinase C; protein tyrosine kinase; smooth muscle
蛋白激酶C和蛋白酪氨酸激酶介导脂多糖及细胞因子诱导大鼠血管平滑肌细胞一氧化氮的生成
韩雅玲1,*, 康建1, 李少华2
1沈阳军区总医院全军心血管内外科研究所心内科, 沈阳 110016; 2美国Robert Wood Johnson医学院病理与医学实验科,新泽西州 08854, 美国
摘要: 采用Sprague-Dawley大鼠胸主动脉中膜、 外膜和培养的血管平滑肌细胞(VSMCs)作材料, 鉴定不同类型的血管组织经炎性介质刺激后其一氧化氮(NO)的产生来源, 阐明蛋白激酶C (PKC)和蛋白酪氨酸激酶(PTK)介导大鼠VSMCs生成NO的调控机制。大鼠VSMCs经脂多糖(LPS)和细胞因子(TNF-α, IL-1β)处理后, 以剂量依赖方式促进NO释放。采用Western Blot证实经刺激的VSMCs伴有iNOS表达上调。进一步实验表明PKC和PTK参与LPS和细胞因子诱导NO生成的胞内信号转导。用PKC抑制剂H7与VSMCs共培育, H7能明显减少LPS、 TNF-α和IL-1β诱导细胞NO的形成。白屈菜赤碱亦可抑制NO的生成, 但HA1004对VSMCs 的NO生成无抑制作用, 提示PKC参与NO的生成与调控。PTK抑制剂genistein和tyrphostin AG18均能抑制由LPS、 TNF-α和IL-1β引发VSMCs 释放NO, 同时伴iNOS蛋白表达下调, 而PKC抑制剂不能阻断iNOS的表达。上述观察结果提示, PKC介导LPS和细胞因子诱导细胞合成NO可能是通过iNOS翻译后加工; 而PTK则以上调iNOS表达而促增NO生成。
关键词: 一氧化氮; 蛋白激酶C; 蛋白酪氨酸激酶; 平滑肌
中图分类号: Q555.7;R34
Nitric oxide (NO) possesses important physiological functions including vasodilation and inhibition of platelet aggregation, and therefore prevents thrombosis and restenosis after angioplasty[1].
Emerging data indicate that the inflammatory response after mechanical arterial injury correlates with the severity of neointimal hyperplasia in animal models and postangioplasty restenosis in humans. Nonspecific systemic stimulation of the innate immune system concurrently with arterial vascular injury facilitates neointimal formation, and conditions associated with increased inflammation may increase restenosis[2]. It has also been demonstrated that iNOS is expressed after in vivo balloon injury of rat carotid arteries and atherosclerotic lesions. iNOS induction in VSMCs may play a role in local vascular injury associated with atherosclerosis by inhibiting smooth muscle cell proliferation as well as limiting thrombus formation by preventing platelet adhesion and aggregation[3]. The fact that VSMCs can be induced to express NO suggests that it may also play a role in inhibiting the neointimal hyperplasia. It is hypothesized that inflammation-evoked NO release may exert vascular protective effects. It is therefore important to understand the signaling pathway leading to NO formation.
Inflammatory cytokines, such as interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) released during vascular injury process have been shown to induce the expression of inducible nitric oxide synthase (iNOS) and NO formation[4,5]. However, the source of NO produced in vascular tissue after cytokine stimulation is still unclear. In the present study, we separated the vascular media from the adventitia in rat aorta by microdissection and demonstrate the vascular media as the main source of NO production upon lipopolyssacharide (LPS) and cytokine stimulation. Moreover, we found that protein kinase C and protein tyrosine kinase were mediators of LPS- and cytokine-induced NO formation in VSMCs with the latter involved in the induction of iNOS expression and the former in the posttranslational modification of iNOS.HAN Ya-Ling et al: PKC and PTK Mediated NO Formation in VSMCsActa Physiol. Sin., June 25, 2003, 55(3):265－272
1 MATERIALS AND METHODS
1.1
Tissue culture. The
experimental protocols were approved by the Animal Care and Use Committee of
the Naval Medical Research Institute (USA) and were conducted according to the
principles set forth in the “Guide for the Care and Use of Laboratory Animals”,
Institute of Laboratory Animals Resources, National Research Council,
Department of Health and Human Services, Publication No.86-123 (1985) (
1.2 Cell culture. VSMCs were isolated from enzymatically dissociated rat thoracic aorta described previously[6]. Briefly, the thoracic aortas were quickly cleaned and inverted with both ends tied off. The aortas were then incubated with Ca2+ - and Mg2+- free Hanks' solution (in mmol/L): NaCl 137, KCl 5.4, KH2PO4 0.44, Na2HPO4 0.4 , NaHCO3 4.17, HEPES 10, dextrose 5.55, glutamine 2, and 0.2% bovine serum albumin containing 2 mg/ml collagenase type Ⅲ (100 U/mg, Worthington), 0.14 mg/ml elastase type Ⅲ 81 U/mg, Sigma), 0.4 mg/ml trypsin inhibitor type Ⅰ-S (Sigma) and 25 μmol/L phentolamine. The aortas were incubated at 35℃ under 100% O2 while being agitated at 50 cycles/min for 60 min. After the first incubation, aortas were rinsed with Ca2+ - and Mg2+-free Hanks' solution to remove endothelial cells and reincubated in the above digestion solution for 90 min. The dissociated VSMCs were collected by centrifugation and cultured with DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified 5% CO2-95% air atmosphere.
1.3 Nitrite assay. Nitrites was determined as described[7]. Briefly, 200 μl samples were incubated with equal amount of freshly prepared 2,3-diaminonaphthalene (50 μg/ml) and 40 μl EDTA (10 μg/ml) for 10 min. The reaction was terminated with 1 mol/L NaOH. The fluorescent intensity of the product, 2,3-diamino-naphthotriazole, formed from 2,3-diaminonaphthalene and NO－2 was measured at wavelength (nm) 365 nm and 450 nm on a fluorescence spectrophotometer (LS-3B, Perkin-Elmer, CT). Protein content of the cells was determined by bicinchoninic acid assay (Pierce, IL). The nitrite production is expressed as nmol/mg protein or wet tissue.
1.4 Western blot analysis. Protein expression was assessed by Western blot analysis of near confluent cells, as described previously[8]. Protein was resolved by electrophoresis through 12% (for smooth muscle SM α-actin) or 7.5% (for iNOS) SDS-polyacrylamide gels and then transferred onto PVDF membranes in Tris-glycine transfer buffer in a trans-blot cell (Bio-Rad).The membranes were blocked overnight at 4℃ with 5% nonfat dry milk in Tris-buffered saline (TBS). Primary antibodies used were mouse monoclonal antibodies raised against SM α-actin (Sigma) and iNOS (Transduction Laboratories, Lexington, KY, USA) diluted 1∶1000 in 1% nonfat dry milk and applied for 2 h at room temperature. After washing three times in TBS containing 0.1% Tween 20, specific bindings were detected with HPR-conjugated secondary antibodies and positive immunoreactivity was visualized using enhanced chemiluminescence reagents (ECL, Amersham) by exposure to photographic film.
1.5 Immunofluorescence microscopy． For
immunofluorescence analysis, VSMCs were
grown on glass coverslips and were fixed with 4% paraformaldehyde and
permeabilized by immersing the coverslips in －20℃ methanol-acetone (1∶1) for 15 sec.
Nonspecific binding sites were blocked by incubation in PBS with 5% goat serum
for 30 min at room temperature. Cells were then incubated for 2 h at room
temperature with a mouse anti-SM α-actin monoclonal antibody (Sigma, type
1A4).After being rinsed with PBS, the cells were incubated with fluorescence
isothiocyanate-conjugated goat anti-mouse antibody (Jackson Immunoresearch
Laboratories, PA,
1.6 Statistics. All data are expressed as mean±SE; n represents the number of experiments. Statistical analyses were performed by Students't test for paired or unpaired observations when appropriate, and more than two treatments were compared by one-way ANOVA followed by Student-Neuman-Keuls method for multiple comparison. A value of P<0.05 was considered statistically significant.
2 RESULTS
2.1 Source of NO production by arterial tissues in response to LPS and cytokines
To address if LPS and cytokine IL-1β and TNF-α directly stimulated NO synthesis in arterial tissues and what cell types in the vessel wall were the source of NO production, intact rat aortic rings, the aortic media with the endothelium and the adventitia removed under dissection microscope, and the pure adventitia were incubated with 1 μg/ml LPS, 100 U/ml TNF-alpha, or 10 U/ml IL-1beta for 16 h and the culture supernatants were collected for NO measurement. Figure 1 shows that LPS, TNF-α and IL-1β all induced a significant increase in NO formation in intact aortic rings with more prominent induction by LPS treatment. In comparison with the adventitia, the vascular media that contain pure smooth muscle cells produced much more NO upon LPS and the cytokine stimulation. NO－2 concentration increased from 0.113±0.0243 to 3.240±0.252 nmol/mg protein. NO production was also increased from 0.113±0.0243 to 2.687±0.117 nmol/mg protein after TNF-α stimulation and from 0.113±0.0243 to 0.861±0.106 nmol/mg protein after IL-1β treatment, suggesting that the smooth muscle is the major source of NO formation. No significant difference in NO formation was observed between using the intact aortic rings and the aortic rings with endothelium removed (data not shown), indicating the vascular endothelium is not a major NO producer after LPS and cytokine treatment.
Fig.1.Vascular smooth muscle is the major NO producer upon LPS, TNF-α, and IL-1β stimulation. Arterial vascular tissues were exposed to LPS (1 μg/ml), TNF-α (100 U/ml) or IL-1β(10 U/ml). Nitrite accumulation was measured in culture medium at end of 16 h. Data are mean±SE of eight replicates per treatment. *P<0.05 when compared with unstimulated control.
2.2 Identification of smooth muscle cells by Western blotting and immunocytochemistry
In order to study LPS- and cytokine-induced NO formation and its signaling mechanisms, VSMCs from rat aorta were isolated and cultured. Under phase contrast microscope, cultured VSMCs revealed a typical spindle-shaped morphology as shown in Fig. 2A. Western blot analysis revealed the expression of smooth muscle α-actin which assembled into filaments as evidenced by immunofluorescence microscopy.
Fig.2.Culture and identification of smooth muscle cells from rat aorta. A: Phase contrast image showing confluent VSMCs. B: VSMC monolayer on coverslips was permeabilized, fixed and stained for F- actin with FITC-phalloidin. C: Western blot of SM α-actin. Cellular lysates were separated on a 12% SDS-PAGE, followed by immunoblot analysis with anti-SM α-actin antibody.
2.3 Effect of protein kinase inhibitors on NO generation by stimulated VSMCs
To further characterize LPS- and cytokine-induced NO release, cultured rat VSMCs were incubated with different concentrations of LPS, TNF-α, or IL-1β for 16 h. Both LPS and cytokine TNF-α and IL-1β elicited a dose-dependent NO formation which maximized upon 10 μg/ml LPS (1000 U/ml TNF-α or 100 U/ml IL-1β) stimulation (Fig.3).
To explore the role of PKC in LPS- and cytokine-induced NO production in VSMCs, PKC inhibitors were added to VSMC culture 30 min before LPS (1 μg/ml) or cytokine (100 U/ml TNF-α or 10 U/ml IL-1β) stimulation. PKC inhibitor H7 at 10 μmol/L concentration completely prevented LPS-induced NO formation and significantly blocked TNF-α- and IL-1β-stimulated NO production. In contrast, HA1004, which has a similar inhibition constant (Ki) toward protein kinase A, protein kinase G and myosin light chain kinase but a much higher Ki toward PKC, had no inhibition effect (Fig.4). Furthermore, another structurally distinct PKC inhibitor chelerythrine (1 μmol/L), which acts on the catalytic domain of PKC, also inhibited LPS and cytokine induced NO production, suggesting that PKC mediates LPS and cytokines induced NO formation in VSMCs.
The role of protein tyrosine kinase in LPS- and cytokine-mediated NO synthesis was evaluated using protein tyrosine kinase inhibitors. Protein tyrosine kinase specific inhibitor genistein (10 μmol/L) completely inhibited NO synthesis stimulated with LPS (1 μg/ml) from 103.05±2.324 to 6.917±0.715 nmol/mg, TNF-α (100 U/ml) from 85.433±3.411 to 4.983 ±0.929 nmol/mg or IL-1β (10 U/ml) from 64.767±1.658 to 10.550±1.039 nmol/mg in VSMCs. Tyrphostin AG18 (100 μmol/L), a structurally different broad-spectrum protein tyrosine kinase inhibitor, showed similar inhibition effect (Fig.5), which suggests that protein tyrosine kinase is also involved in LPS- and cytokine-induced NO formation.
Fig.3.LPS, TNF-α and IL-1β induce a dose-dependent NO formation in VSMCs. VSMCs were incubated with either LPS, TNF-α, or IL-1β. Nitrite accumulation was measured in culture medium at end of 16 h. Data are mean±SE of seven replicates per treatment.
Fig.4.LPS, TNF-α, and IL-1β-stimulated NO formation is abrogated by protein kinase C inhibition. VSMCs were preincubated with H7 (10 μmol/L), HA (10 μmol/L) and chelerythrine (1 μmol/L) for 30 min respectively. This was followed by adding to the culture media either control solution (DMEM), IL-1β, TNF-α or LPS for 16 h and analyzing nitrite concentration in the culture media. Data are means±SE from seven independent experiments. *P<0.05, **P<0.01 when compared with control VSMCs.
2.4 Effect of protein kinase inhibitors on iNOS expression by stimulated VSMCs
Since both protein kinase C and protein tyrosine kinase inhibitors inhibited LPS and cytokine-induced NO synthesis, we tested the hypothesis that the inhibition
Fig.5.Protein tyrosine kinase inhibitor genistein and tyrphostin AG18 inhibit LPS- and cytokine-induced NO formation. VSMCs were treated with either control solution (DMEM), genistein (10 μmol/L) or tyrphostin AG18 (100 μmol/L) as described in the legend to Fig.4. Incubations were followed by analysis of nitrite concentration in the culture media. Data are mean±SE of six replicates per treatment.*P<0.05 when compared with control VSMCs.
Fig.6.Protein tyrosine kinase but not protein kinase C mediates LPS and cytokine-induced expression of inducible nitric oxide synthase (iNOS). VSMCs were pretreated with various kinase inhibitors for 30 min and then incubated with control solution, IL-1β, LPS and TNF-α for 16 h, followed by Western blot analysis using anti-iNOS antibody. iNOS protein ran as a 130 kD band. Top: VSMCs were treated with LPS, IL-1β and TNF-α in the absence or presence of chelerythrine (1 μmol/L). Bottom: VSMCs were incubated with LPS in the absence or presence of genistein (10 μmol/L), or tyrphostin AG18 (100 μmol/L).
may be due to the blockade of iNOS expression. VSMCs in monolayer culture were treated with LPS (1 μg/ml), TNF-α (100 U/ml) or IL-1β (10 U/ml) alone or combined with chelerythrine (1 μmol/L), genistein (10 μmol/L), or tyrphostin AG18 (100 μmol/L) for 16 h. The cells were washed twice with cold phosphate-buffered saline and harvested. Equal amount of whole cells lysate proteins were run on SDS-PAGE and analyzed for iNOS expression by immunoblotting. As shown in Fig.6, no iNOS protein was detected by Western analysis in control VSMCs. LPS, TNF-α and IL-1β treatment induced marked iNOS expression which was not affected by PKC inhibitor chelerythrine. In contrast, protein tyrosine kinase inhibitor genistein and tyrphostin AG18 prevented iNOS induction by LPS. A similar result was observed after IL-1β or TNF-α stimulation (data not shown).These results suggest that protein tyrosine kinase mediates LPS, IL-1β and TNF-α-stimulated NO formation by inducing iNOS expression, whereas PKC may act on post-translational modification of iNOS or the availability of substrates and cofactors involved in NO synthesis.
3 DISCUSSION
In the present study, we have shown that (1) the arterial media was the major source of NO in response to the LPS and cytokines stimulation although the vascular adventitia also contributed to NO production; (2) LPS and cytokines elicited a dose-dependent NO formation in VSMCs which was inhibited by structurally distinct PKC and PTK inhibitors, suggesting both types of protein kinases are involved in the stimulated NO release; and (3) LPS and cytokine IL-1β and TNF-α induced iNOS protein expression which was blocked by PTK inhibitor but not PKC inhibitor. This indicates that PTK mediates the stimulated NO release by induction of iNOS expression. Whereas PKC acts on the downstream of iNOS protein expression.
Increase of iNOS expression and NO release have been reported after vascular injury[4]. However, the source of NO has not been clearly demonstrated. Cultured vascular endothelial cells and fibroblasts express a small amount iNOS upon cytokine stimulation[9,10]. In the present study, we compared LPS- and cytokine-induced NO production in intact rat aortic rings, aortic rings without endothelium, aortic media with the adventitia and endothelium removed, and pure vascular adventitia. We found the vascular media that composes almost pure smooth muscle cells was the main NO producer. To a less degree the adventitia acted as a second NO source, probably contributed by fibroblasts and macrophages residing in the adventitia[11].
Inducible nitric oxide synthase expression and subsequent NO accumulation can be stimulated by LPS and cytokines in a variety of cell types especially macrophages[12]. This induced NO release has been proposed as a mechanism of hypotension and vascular hyporeactivity in endotoxic shock[13]. We found that LPS, IL-1β and TNF-α induced a dose-dependent increase in NO production in vascular smooth muscle cells. This dose-dependence and saturation at high concentrations suggest that it is mediated by specific receptors. Recently LPS was also shown to stimulate the production of cytokines and tissue factor by VSMCs[2,14]. Therefore, LPS may induce NO production both directly and indirectly via the synthesis of cytokines. LPS and cytokine stimulated NO synthesis is probably the result of iNOS induction since Western blot analysis showed induction and increased iNOS protein expression after LPS and cytokine treatment of VSMCs.
The intracellular signaling events involved in iNOS expression are not well understood, and the mechanisms involved in the control of NO synthesis in different cell types is a subject of current interest. We examined the role of PKC and PTK in the induction of iNOS expression and NO synthesis by using structurally distinct specific inhibitors. H7 has often been used in combination with protein kinase inhibitor HA1004 to assess the contribution of PKC to cellular processes, including the induction of gene expression. The use of H7 and HA1004 is based upon the fact that H7 is a much more potent PKC inhibitor compared to HA1004 in vitro assays. Thus, although both compounds are broad spectrum protein kinase inhibitors, inhibition by H7, but not by HA1004, has often been interpreted as evidence for the involvement of PKC in the cellular process under study. Our experiments showed that H7 but not HA1004 completely prevented LPS- and cytokine-induced NO formation, arguing PKC is a critical signal molecule involved in regulating NO synthesis. This hypothesis is supported by the inhibition of stimulated NO production by chelerythrine, another structurally distinct PKC inhibitor. However, chelerythrine did not block LPS- and cytokine-induced iNOS protein expression in VSMCs which suggests that it inhibits the NO formation by acting on either the post-translational modulation of iNOS or the availability of substrates and cofactors of iNOS[15]. Consistent with our result, PKC blockade with chelerythrine inhibited LPS-induced NO synthesis in rat cardiac myocytes but had no effect on the induction of iNOS protein expression[16].
PTK is an important regulator of iNOS activity in murine macrophages[17]. Our data also demonstrated that PTK played a critical role in LPS and cytokine stimulated NO synthesis. Both genistein and tyrphostin AG18, broad spectrum protein tyrosine kinase inhibitors[18], inhibited the LPS-induced NO production, probably by blocking iNOS gene expression. These findings are in agreement with observations in skin-derived dendritic cells showing that genistein suppresses the expression of iNOS activity induced by LPS[19]. In dendritic cells Jenus tyrosine kinase may mediate LPS-induced iNOS transcription.
Since NO relaxes vessels, suppresses VSMC proliferation and prevents neointimal formation, elucidation of the molecular mechanisms by which PKC and PTK mediate NO production in VSMCs is of major importance and might have implication for the design and execution of clinical strategies.
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