Parkin represses 6-hydroxydopamine-induced apoptosis via stabilizing scaffold protein p62 in PC12 cells

Aim:

Parkin has been shown to exert protective effects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in different models of Parkinson disease. In the present study we investigated the molecular mechanisms underlying the neuroprotective action of parkin in vitro.

Methods:

HEK293, HeLa and PC12 cells were transfected with parkin, parkin mutants, p62 or si-p62. Protein expression and ubiquitination were assessed using immunoblot analysis. Immunoprecipitation assay was performed to identify the interaction between parkin and scaffold protein p62. PC12 and SH-SY5Y cells were treated with 6-OHDA (200 μmol/L), and cell apoptosis was detected using PI and Hoechst staining.

Results:

In HEK293 cells co-transfected with parkin and p62, parkin was co-immunoprecipitated with p62, and parkin overexpression increased p62 protein levels. In parkin-deficient HeLa cells, transfection with wild-type pakin, but not with ligase activity-deficient pakin mutants, significantly increased p62 levels, suggesting that parkin stabilized p62 through its E3 ligase activity. Transfection with parkin or p62 significantly repressed ERK1/2 phosphorylation in HeLa cells, but transfection with parkin did not repress ERK1/2 phosphorylation in p62-knockdown HeLa cells, suggesting that p62 was involved in parkin-induced inhibition on ERK1/2 phosphorylation. Overexpression of parkin or p62 significantly repressed 6-OHDA-induced ERK1/2 phosphorylation in PC12 cells, and parkin overexpression inhibited 6-OHDA-induced apoptosis in PC12 and SH-SY5Y cells.

Conclusion:

Parkin protects PC12 cells against 6-OHDA-induced apoptosis via ubiquitinating and stabilizing scaffold protein p62, and repressing ERK1/2 activation.     

The roles of cyclic AMP–ERK–Bad signaling pathways on 6-hydroxydopamine-induced cell survival and death in PC12 cells

The roles of cyclic AMP (cAMP)–ERK1/2–Bad signaling pathways in 6-hydroxydopamine (6-OHDA)-induced cell survival and death were investigated. In PC12 cells, 6-OHDA (10–100 μM) concentration-dependently increased the intracellular levels of cAMP mediated by the Ca²⁺-CaMKII-adenylyl cyclase system. 6-OHDA at the non-toxic level (10 μM) induced transient ERK1/2 phosphorylation and BadSer112 phosphorylation, which maintained cell survival. In contrast, the high levels of cAMP induced by toxic levels (50 and 100 μM) of 6-OHDA induced sustained ERK1/2 phosphorylaton and BadSer155 phosphorylation. The cells then moved to cell death process through Bcl2 phosphorylation and caspase-3 activation. BadSer155 phosphorylation by 6-OHDA was inhibited by PKA (H89) and MEK (U0126) inhibitors, indicating that it was mediated via the cAMP–PKA-sustained ERK1/2 system. In SK-N-BE(2)C cells, the non-toxic level of 6-OHDA also showed transient ERK1/2 phosphorylation and BadSer112 phosphorylation, and toxic levels of 6-OHDA exhibited sustained ERK1/2 phosphorylation and BadSer155 phosphorylation. These results suggest that ERK1/2 phosphorylation by 6-OHDA shows biphasic functions on cell survival and death in PC12 cells. It is, therefore, proposed that the cAMP–ERK1/2–Bad signaling pathways incurred by toxic levels of 6-OHDA play a role in dopamine neuron death of animal models of Parkinson’s disease.     

Effects of asarinin on dopamine biosynthesis and 6-hydroxydopamine-induced cytotoxicity in PC12 cells

This study investigated the effects of asarinin on dopamine biosynthesis and 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in rat adrenal pheochromocytoma (PC12) cells. Treatment with asarinin (25–50 μM) increased intracellular dopamine levels and enhanced L-DOPA-induced increases in dopamine levels. Asarinin (25 μM) induced cyclic AMP-dependent protein kinase A (PKA) signaling, leading to increased cyclic AMP-response element binding protein (CREB) and tyrosine hydroxylase (TH) phosphorylation, which in turn stimulated dopamine production. Asarinin (25 μM) also activated transient phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Bad phosphorylation at Ser 112, both of which have been shown to promote cell survival. In contrast, asarinin (25 μM) inhibited sustained ERK1/2, Bax, c-Jun N-terminal kinase (JNK1/2) and p38 mitogen-activated protein kinase (p38MAPK) phosphorylation and caspase-3 activity, which were induced by 6-OHDA (100 μM). These results suggest that asarinin induces dopamine biosynthesis via activation of the PKA-CREB-TH system and protects against 6-OHDA-induced cytotoxicity by inhibiting the sustained activation of the ERK-p38MAPK-JNK1/2-caspase-3 system in PC12 cells.     

Effect of taurine on advanced glycation end products-induced hypertrophy in renal tubular epithelial cells

Mounting evidence indicates that advanced glycation end products (AGE) play a major role in the development of diabetic nephropathy (DN). Taurine is a well documented antioxidant agent. To explore whether taurine was linked to altered AGE-mediated renal tubulointerstitial fibrosis in DN, we examined the molecular mechanisms of taurine responsible for inhibition of AGE-induced hypertrophy in renal tubular epithelial cells. We found that AGE (but not non-glycated BSA) caused inhibition of cellular mitogenesis rather than cell death by either necrosis or apoptosis. There were no changes in caspase 3 activity, bcl-2 protein expression, and mitochondrial cytochrome c release in BSA, AGE, or the antioxidant taurine treatments in these cells. AGE-induced the Raf-1/extracellular signal-regulated kinase (ERK) activation was markedly blocked by taurine. Furthermore, taurine, the Raf-1 kinase inhibitor GW5074, and the ERK kinase inhibitor PD98059 may have the ability to induce cellular proliferation and cell cycle progression from AGE-treated cells. The ability of taurine, GW5074, or PD98059 to inhibit AGE-induced hypertrophy was verified by the observation that it significantly decreased cell size, cellular hypertrophy index, and protein levels of RAGE, p27^Kip1, collagen IV, and fibronectin. The results obtained in this study suggest that taurine may serve as the potential anti-fibrotic activity in DN through mechanism dependent of its Raf-1/ERK inactivation in AGE-induced hypertrophy in renal tubular epithelial cells.     

Oridonin-induced A431 cell apoptosis partially through blockage of the Ras/Raf/ERK signal pathway

We have reported that oridonin, a diterpenoid isolated from the plant Rabdosia rubescens, had apoptosis-inducing activities in many cell lines (e.g., human melanoma A375-S2, human cervical cancer HeLa, human breast adenocarcinoma MCF-7, and murine fibrosarcoma L929). In this study, we further investigated signaling events involved in oridonin-induced apoptosis in human epidermoid carcinoma A431 cells. It was found that the total tyrosine kinase activity was inhibited and the protein expressions of epidermal growth factor receptor (EGFR) and phosphorylated EGFR were decreased in oridonin-induced A431 cell apoptosis. Expression of EGFR downstream effector proteins, Grb2, Ras, Raf-1, and extracellular signal-regulated kinase (ERK), was also downregulated by oridonin. Moreover, the oridonin-induced apoptosis was augmented by the Ras inhibitor manumycin A, Raf-1 inhibitor GW5074, or ERK inhibitor PD98059, suggesting that inactivation of Ras, Raf, or ERK participates in oridonin-induced apoptosis. Taken together, oridonin-induced apoptosis in A431 cells might be through blocking EGFR and its downstream Ras/Raf/ERK signal pathway.     

Involvement of Raf-1 in chronic delta-opioid receptor agonist-mediated adenylyl cyclase superactivation

Chronic delta-opioid receptor agonist treatment of Chinese hamster ovary (CHO) cells stably expressing the human delta-opioid receptor (hDOR/CHO) leads to increased cAMP formation after the removal of the agonist (adenylyl cyclase superactivation). We have previously found that at the same time, chronic delta-opioid receptor agonist treatment augments phosphorylation of the adenylyl cyclase VI isoenzyme. Since phosphorylation of adenylyl cyclase VI by Raf-1 protein kinase was recently shown, we tested the role of Raf-1 in adenylyl cyclase superactivation in hDOR/CHO cells. We found that pretreatment of the cells with the selective Raf-1 inhibitor GW5074 (3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one) (10 microM, 30 min) attenuates chronic deltorphin II-mediated increase in forskolin-stimulated cAMP formation by 40% (n = 6, P < 0.05). Better understanding of the molecular mechanism of adenylyl cyclase superactivation should aid in the development of analgesics that act longer and have fewer side effects.     

ERK1/2通路在4-AP诱导大鼠肺动脉收缩中的作用

目的探讨细胞外信号调节激酶-1/2(ERK1/2)通路在4-氨基吡啶(4-aminopyridione,4-AP)阻断正常大鼠肺动脉平滑肌细胞(PASMCs)膜上电压依赖性钾通道(KV)所引起的肺动脉收缩中的作用。方法取正常鼠肺动脉制作肺动脉环,分别加入4-AP(KV通道阻断剂),PD98059/U0126+4-AP,比较肺动脉收缩的变化。同时培养肺动脉平滑肌细胞进行Western blot分析4-AP对ERK1/2的影响。结果①在血管环试验中,4-AP引起的肺动脉收缩有浓度依赖性;加入20mmol.L-1PD98059或2μmol.L-1U0126可以抑制4-AP引起的肺动脉收缩。②4-AP可刺激PASMCs ERK1/2蛋白磷酸化;③U0126可抑制4-AP引起的ERK1/2蛋白磷酸化。结论ERK1/2通路参与4-AP阻断正常大鼠肺动脉平滑肌细胞膜上电压依赖性钾通道(KV)引起肺动脉收缩。     

芍药苷对6羟基多巴胺致PC12细胞损伤的保护作用

目的:探讨芍药苷对6羟基多巴胺(6-OHDA)致大鼠肾上腺嗜铬细胞瘤细胞(PC12)的细胞损伤保护作用及其可能机制.方法:体外培养PC12细胞,用6-OHDA建立细胞损伤模型.MTT和乳酸脱氢酶(LDH)活性测定存活率;RT-PCR检测Bcl-2、Bax的mRNA表达,Hochest33342/PI双染检测细胞凋亡率.结果:25、50、100 μmol·L-1芍药苷可显著减少6-OHDA诱导的PC12细胞凋亡,降低LDH漏出率,增加Bcl-2 mRNA和减少Bax mRNA表达.结论:芍药苷可显著减少6-OHDA诱导的PC12细胞凋亡,其作用机制可能与调节Bax、Bcl-2表达有关.     

Amelioration of central cardiovascular regulatory dysfunction by tropomyocin receptor kinase B in a mevinphos intoxication model of brain stem death

BACKGROUND AND PURPOSE

Little information exists on the mechanisms that precipitate brain stem death, the legal definition of death in many developed countries. We investigated the role of tropomyocin receptor kinase B (TrkB) and its downstream signalling pathways in the rostral ventrolateral medulla (RVLM) during experimental brain stem death.

EXPERIMENTAL APPROACH

An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos bilaterally into the RVLM of Sprague–Dawley rats was used, in conjunction with cardiovascular, pharmacological and biochemical evaluations.

KEY RESULTS

A significant increase in TrkB protein, phosphorylation of TrkB at Tyr⁵¹⁶ (pTrkB^Y516), Shc at Tyr³¹⁷ (pShc^Y317) or ERK at Thr²⁰²/Tyr²⁰⁴, or Ras activity in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Microinjection bilaterally into RVLM of a specific TrkB inhibitor, K252a, antagonized those increases. Pretreatment with anti-pShc^Y317 antiserum, Src homology 3 binding peptide (Grb2/SOS inhibitor), farnesylthioacetic acid (Ras inhibitor), manumycin A (Ras inhibitor) or GW5074 (Raf-1 inhibitor) blunted the preferential augmentation of Ras activity or ERK phosphorylation in RVLM and blocked the up-regulated NOS I/protein kinase G (PKG) signalling, the pro-life cascade that sustains central cardiovascular regulation during experimental brain stem death.

CONCLUSIONS AND IMPLICATIONS

Activation of TrkB, followed by recruitment of Shc/Grb2/SOS adaptor proteins, leading to activation of Ras/Raf-1/ERK signalling pathway plays a crucial role in ameliorating central cardiovascular regulatory dysfunction via up-regulation of NOS I/PKG signalling cascade in the RVLM in brain stem death. These findings provide novel information for developing therapeutic strategies against this fatal eventuality.     

6-羟基多巴胺诱导PC12细胞释放谷氨酸及死亡不受Ⅰ组亲代谢型谷氨酸受体配基的影响(英文)

目的:探讨Ⅰ组亲代谢型谷氨酸受体(mGluR)配基对6-羟基多巴胺(6-OHDA)诱导的PC12细胞死亡及谷氨酸(Glu)释放的影响。方法:培养PC12细胞,以100μmol/LⅠ组mGluR激动剂(RS)-3,5-dihydroxyphenylglycine(DHPG)和拮抗剂DL-2-amino-3-phosphonopropionic acid(DL-AP3)预先剌激细胞1h,再加入6-OHDA100μmol/L共孵育24h,显微镜下观察细胞形态变化,用TUNEL法检测凋亡细胞,用噻唑蓝(MTT)法检测细胞存活率,并用高效液相色谱检测Glu的释放量。结果:6-OHDA 降低PC12细胞存活率(P<0.01),其诱导的Glu释放呈浓度和时间依赖性。Ⅰ组mGluR配基不能减少6-OHDA引起的PC12细胞死亡,也不影响6OHDA引起的Glu释放量。结论:Ⅰ组mGluR配基对6-OHDA引起死亡的PC12细胞无保护作用。     

Regulatory B Subunits of Protein Phosphatase 2A Are Involved in Site-specific Regulation of Tau Protein Phosphorylation

Overexpression of amyloid precursor protein with the Swedish mutation causes abnormal hyperphosphorylation of the microtubule-associated protein tau. Hyperphosphorylated isoforms of tau are major components of neurofibrillary tangles, which are histopathological hallmarks of Alzheimer''s disease. Protein phosphatase 2A (PP2A), a major tau protein phosphatase, consists of a structural A subunit, catalytic C subunit, and a variety of regulatory B subunits. The B subunits have been reported to modulate function of the PP2A holoenzyme by regulating substrate binding, enzyme activity, and subcellular localization. In the current study, we characterized regulatory B subunit-specific regulation of tau protein phosphorylation. We showed that the PP2A B subunit PPP2R2A mediated dephosphorylation of tau protein at Ser-199, Ser-202/Thr-205, Thr-231, Ser-262, and Ser-422. Down-regulation of PPP2R5D expression decreased tau phosphorylation at Ser-202/Thr-205, Thr-231, and Ser-422, which indicates activation of the tau kinase glycogen synthase kinase 3 beta (GSK3β) by PP2A with PPP2R5D subunit. The level of activating phosphorylation of the GSK3β kinase Akt at Thr-308 and Ser-473 were both increased by PPP2R5D knockdown. We also characterized B subunit-specific phosphorylation sites in tau using mass spectrometric analysis. Liquid chromatography-mass spectrometry revealed that the phosphorylation status of the tau protein may be affected by PP2A, depending on the specific B subunits. These studies further our understanding of the function of various B subunits in mediating site-specific regulation of tau protein phosphorylation.     

IGF-1对6-OHDA诱导神经元氧化损伤的保护作用

目的 探讨胰岛素样生长因子-1（IGF-1）对6-羟基多巴胺（6-OHDA）诱导的PC12细胞氧化损伤的保护作用。方法 以25、50、100、150、200 μmol/L 的6-OHDA 处理PC12细胞,在12、24、48 h用MTT 法检测6-OHDA 对PC12细胞活性的影响,筛选最佳的实验浓度和观察时间。实验分为3组：对照组、6-OHDA组（150 μmol/L处理24 h）和IGF-1+6-OHDA组（IGF-1 100 nmol/L预处理2 h,后加入150 μmol/L 6-OHDA处理24 h）,MTT法检测各组细胞活性;免疫荧光染色法检测细胞活性氧（ROS）水平;Hoechst33342/PI双染法检测细胞凋亡。结果 随6-OHDA浓度的增加和作用时间的延长,PC12细胞的活性呈梯度降低,150 μmol/L 6-OHDA浓度和处理后24 h作为本研究的最佳的实验浓度和观察时间。与6-OHDA组比较,IGF-1+6-OHDA组PC12细胞活力增强、ROS水平下降、细胞凋亡减少。结论 IGF-1预处理能减少6-OHDA引起的PC12细胞氧化损伤及凋亡,增加细胞活性,为防治帕金森病提供了潜在的治疗策略。     

Activation of extracellular signal-regulated kinase during silibinin-protected, isoproterenol-induced apoptosis in rat cardiac myocytes is tyrosine kinase pathway-mediated and protein kinase C-dependent

Aim： To investigate the mechanism of silibinin-protected isoproterenol-induced apoptosis in rat cardiac myocytes. Methods： The viability of rat cardiac myocytes was measured by MTT method. The apoptotic ratio was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Protein kinase C （PKC） activity assay was carried out according to the instructions of the PepTag non-radioactive protein kinase C assay kit. Western blot analysis was used to evaluate the level of Ras, Raf-1 and mitogen-activated protein kinase （MAPK） expression. Results： The protective effects of silibinin were significantly sup- pressed by inhibitors, including genistein, manumycin A and GW5074 [inhibitors for protein tyrosine kinases （PTK）, Ras and Raf- 1, respectively]. The exposure of rat cardiac myocytes to isoproterenol alone caused decreased PKC activity, which was prevented by pretreatment with silibinin dose-dependently. Simultaneously, the increased expression of Ras and Raf- 1 activated by silibinin were blocked by the PKC inhibitor, stauroporine. In addition, the extracellularly responsive kinase （ERK） inhibitor, PD98059, suppressed silibinin-protected apoptosis, whereas the p38 MAPK inhibitor, SB203580, protected cardiac myocytes from isoproterenolinduced injury, and the c-Jun N-terminal kinase （JNK） inhibitor, SP600125 had no protective effects. Furthermore, Westem blot analysis showed that the expres- sion of phosphorylated ERK was increased by silibinin, the expression of phos- phorylated p38 MAPK was decreased and total ERK, p38, JNK and phosphorylated JNK MAPK did not change after treatment with both isoproterenol and silibinin. Furthermore, pretreatment of cardiac myocyte with PKC, Ras and Raf inhibitors significantly blocked ERK phosphorylation. Conclusion： Silibinin is suggested to protect isoproterenol-induced rat cardiac myocyte apoptosis by activating the tyrosine kinase pathway, PKC and MAPK pathways.     

活性氧激活的ERK1/2通路在化学性缺氧损伤PC12细胞中的作用

目的探讨胞外信号调节激酶1/2(ERK1/2)在化学性缺氧损伤PC12细胞中的作用。方法应用化学性低氧模拟剂氯化钴(CoCl2)处理PC12细胞建立化学性缺氧损伤模型。应用细胞计数试剂盒-8(cell counting kit-8,CCK-8)比色法检测细胞存活率;罗丹明123(Rh123)染色荧光显微镜照像检测线粒体膜电位(MMP);流式细胞术检测凋亡细胞百分比;Western blot法测定caspase-3、ERK1/2和p38MAPK蛋白的表达水平。结果应用600μmol.L-1处理PC12细胞60 min可使磷酸化(p)ERK1/2的表达明显升高;在600μmol.L-1CoCl2处理PC12细胞前,应用500μmol.L-1N-乙酰半胱氨酸(NAC,为活性氧的清除剂)预处理1 h可抑制CoCl2对p-ERK1/2表达的上调作用;在600μmol.L-1CoCl2处理PC12细胞前,应用10μmol.L-1U0126(ERK1/2抑制剂)预处理2 h可保护PC12细胞对抗CoCl2引起的损伤,使细胞存活率升高,凋亡细胞数目和cleaved caspase-3表达及线粒体膜电位(MMP)丢失均减少;10μmol.L-1U0126预处理还能抑制CoCl2对p-p38MAPK表达的上调作用。此外,应用20μmol.L-1SB203580(p38MAPK抑制剂)预处理能抑制CoCl2对p-ERK1/2表达的上调作用。结论活性氧激活的ERK1/2通路介导CoCl2对PC12细胞的损伤作用,并与p38MAPK通路存在相互的的激活作用。     

Modulation of PC12 cell viability by forskolin-induced cyclic AMP levels through ERK and JNK pathways: an implication for L-DOPA-induced cytotoxicity in nigrostriatal dopamine neurons

The intracellular levels of cyclic AMP (cAMP) increase in response to cytotoxic concentrations of L-DOPA in PC12 cells, and forskolin that induces intracellular cAMP levels either protects PC12 cells from L-DOPA-induced cytotoxicity or enhances cytotoxicity in a concentration-dependent manner. This study investigated the effects of cAMP induced by forskolin on cell viability of PC12 cells, relevant to L-DOPA-induced cytotoxicity in Parkinson's disease therapy. The low levels of forskolin (0.01 and 0.1 μM)-induced cAMP increased dopamine biosynthesis and tyrosine hydroxylase (TH) phosphorylation, and induced transient phosphorylation of ERK1/2 within 1 h. However, at the high levels of forskolin (1.0 and 10 μM)-induced cAMP, dopamine biosynthesis and TH phosphorylation did not increase, but rapid differentiation in neurite-like formation was observed with a steady state. The high levels of forskolin-induced cAMP also induced sustained increase in ERK1/2 phosphorylation within 0.25-6 h and then led to apoptosis, which was apparently mediated by JNK1/2 and caspase-3 activation. Multiple treatment of PC12 cells with nontoxic L-DOPA (20 μM) for 4-6 days induced neurite-like formation and decreased intracellular dopamine levels by reducing TH phosphorylation. These results suggest that the low levels of forskolin-induced cAMP increased dopamine biosynthesis in cell survival via transient ERK1/2 phosphorylation. In contrast, the high levels of forskolin-induced cAMP induced differentiation via sustained ERK1/2 phosphorylation and then led to apoptosis. Taken together, the intracellular levels of cAMP play a dual role in cell survival and death through the ERK1/2 and JNK1/2 pathways in PC12 cells.     

Chronic morphine-mediated adenylyl cyclase superactivation is attenuated by the Raf-1 inhibitor, GW5074

The utility of morphine for the treatment of chronic pain is limited by the development of analgesic tolerance. Adenylyl cyclase (AC) superactivation, induced by chronic opioid agonist administration, is regarded as one of the molecular mechanisms leading to tolerance. In the present work, we tested the role of Raf-1 in morphine-mediated AC superactivation in CHO cells stably expressing the human μ-opioid receptor. We found that pretreatment of CHO cells stably expressing the human μ-opioid receptor with the selective Raf-1 inhibitor, 3-(3,5-dibromo-4-hydroxybenzylidene)-5-iodo-1,3-dihydroindol-2-one (GW5074, 10 μM, 60 min) completely abolished chronic morphine-mediated AC superactivation (P < 0.01). This finding indicates that Raf-1 may have a crucial role in compensatory feedback regulation of cellular cAMP levels by clinically important opioid analgesics.     

硫化氢抑制葡萄糖调节蛋白78减轻6-OHDA诱导的PC12细胞损伤

目的：探讨硫化氢（H2S）是否通过改变葡萄糖调节蛋白78（GRP78）的表达参与其对抗6-羟基多巴胺（6-OHDA）诱导PC12细胞损伤的保护作用。方法应用具有神经毒性的6-OHDA损伤PC12细胞为帕金森病细胞模型,以硫氢化钠（NaHS）作为H2S的供体;应用CCK-8比色法检测细胞存活率;DFCH-DA染色检测细胞内活性氧（ROS）的水平;Rh123染色检测细胞线粒体膜电位（MMP）;Western blot检测GRP78的表达。结果200μmol/L的6-OHDA引起PC12细胞的存活率显著降低,ROS生成增加及MMP降低,且诱导了GRP78的高表达。应用25~400μmol/L的NaHS预处理30 min,呈浓度依赖性抑制6-OHDA引起的细胞存活率降低,其中400μmol/L的NaHS作用最明显,此浓度也可以显著减少6-OHDA引起的ROS增多,提高MMP,同时明显抑制6-OHDA诱导的GRP78高表达。结论 H2S具有抗6-OHDA氧化应激损伤的PC12细胞保护作用,抑制内质网应激分子GRP78的表达可能是其机制之一。     

Mice lacking the Raf-1 kinase inhibitor protein exhibit exaggerated hypoxia-induced pulmonary hypertension

BACKGROUND AND PURPOSE

Increased pulmonary vascular remodelling, pulmonary arterial pressure and pulmonary vascular resistance characterize the development of pulmonary arterial hypertension (PAH). Activation of the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)1/2 is thought to play an important role in PAH and Raf-1 kinase inhibitor protein (RKIP), negatively regulates this pathway. This study investigated whether genetic deletion of RKIP (and hence ERK1/2 up-regulation) resulted in a pulmonary hypertensive phenotype in mice and investigated a role for RKIP in mitogen-regulated proliferative responses in lung fibroblasts.

EXPERIMENTAL APPROACH

Pulmonary vascular haemodynamics and remodelling were assessed in mice genetically deficient in RKIP (RKIP−/−) after 2 weeks of either normoxia or hypoxia. Immunoblotting and immunohistochemistry were used to examine phosphorylation of Raf-1, RKIP and ERK1/2 in mouse pulmonary arteries. In vitro, RKIP inhibition of mitogen signalling was analysed in CCL39 hamster lung fibroblasts.

KEY RESULTS

RKIP−/− mice demonstrated elevated indices of PAH and ERK1/2 phosphorylation compared with wild-type (WT) mice. Hypoxic RKIP−/− mice exhibited exaggerated PAH indices. Hypoxia increased phosphorylation of Raf-1, RKIP and ERK1/2 in WT mouse pulmonary arteries and Raf-1 phosphorylation in RKIP−/− mouse pulmonary arteries. In CCL39 cells, inhibition of RKIP potentiated mitogen-induced proliferation and phosphorylation of RKIP, and Raf-1.

CONCLUSIONS AND IMPLICATIONS

The lack of RKIP protein resulted in a pulmonary hypertensive phenotype, exaggerated in hypoxia. Hypoxia induced phosphorylation of RKIP signalling elements in WT pulmonary arteries. RKIP inhibition potentiated mitogen-induced proliferation in lung fibroblasts. These results provide evidence for the involvement of RKIP in suppressing the development of hypoxia-induced PAH in mice.