摘要
第一部分促肾上腺皮质激素释放激素及其受体在慢性斑块型银屑病皮损中表达的研究
背景
银屑病是一种常见的由多基因遗传决定的、多环境因素刺激诱导的慢性炎症性皮肤病,以表皮角质形成细胞(keratinocyte,KC)过度增殖和异常分化为主要特征。在银屑病的发生发展过程中,剧烈的精神刺激或应激反应被证实是银屑病发生和加重的重要诱因。银屑病的许多临床表现,如皮损对称分布、应激触发或加重皮损都提示神经系统在银屑病发病过程中具有重要作用,但是这种临床现象的发生机制有待阐明。
促肾上腺皮质激素释放激素(corticotropin-releasing hormone,CRH)由41个氨基酸组成,是下丘脑-垂体-肾上腺轴(hypothalamic-pituitary-adrenal axis,HPA axis)的重要组成成份。机体在应激状态下由下丘脑产生并释放CRH,刺激垂体后叶特定CRH受体(CRH receptors,CRH-Rs)来调节HPA轴。活化的CRH-Rs通过一系列信号途径引起阿黑皮素(proopiomelanocortin,POMC)来源的神经肽的产生和释放,包括促肾上腺皮质激素(adrenocorticotropic hormone,ACTH)、α-黑素细胞刺激素(α-melallocyte stimulating hormone,α-MSH)和β-内啡肽(β-endorphin,β-END)等。近年来研究发现,人的皮肤中也存在并表达HPA轴的组成成份,包括CRH、CRH受体1(CRH receptor1,CRH-R1)、POMC及随后产生的ACTH、α-MSH、β-END和相关的受体,并且能够进行功能性及细胞特异性调节。研究发现,中枢应激反应和外周应激反应之间存在交互通话,在局部神经免疫性应激反应中,皮肤占据中心地位。因此,学者们提出了皮肤应激系统(skin stress response system,SSRS)、皮肤HPA轴的概念。
在皮肤应激反应时,CRH作为生长因子/多效细胞因子,调节多种细胞的增殖、分化、免疫反应。皮肤中CRH和CRH-R1可以调节皮肤的多种功能,特别是在局部内环境稳定中起到重要作用。这些研究提示:外周CRH和CRH-R1可能在皮肤疾病,特别是以KC异常增殖和分化为病理特征的银屑病中,具有重要作用。
目前,对于CRH和CRH-R1在银屑病皮损中的表达研究较少。本研究拟通过免疫组织化学和蛋白印迹方法观察慢性斑块型银屑病患者皮损、非皮损和正常对照皮肤组织中CRH和CRH-R1的表达情况,探讨皮肤中CRH和CRH-R1在银屑病发病机制中的作用。
目的
用免疫组织化学和蛋白印迹法检测慢性斑块型银屑病患者皮损、非皮损和正常对照皮肤组织中CRH和CRH-R1的表达情况,初步探讨皮肤中CRH和CRH-R1与银屑病的关系。
方法
1.标本来源
26例银屑病患者均来自山东大学齐鲁医院皮肤科门诊就诊或者住院病人,所有病例均经临床确诊为慢性斑块型银屑病。所有患者近4周内未外用糖皮质激素、未接受糖皮质激素及其它免疫抑制剂系统治疗。取材部位:躯干部或四肢部位局麻下切取1.0 cm×0.5 cm大小皮损组织块,距皮损大于5 cm的未受累的部位切取1.0 cm×0.5 cm大小非皮损组织块。26例正常对照皮肤来自本院门诊手术和美容整形手术病例。
2.免疫组织化学检测
取银屑病患者皮损、非皮损以及正常对照皮肤组织进行CRH和CRH-R1免疫组织化学检测。用Image-Pro Plus 6图像分析软件(Media Cybemetics,USA)分析图像。抗CRH抗体(1:500;Chemicon International,Inc.,CA,USA),抗CRH-R1抗体(1:40;R&D Systems,Inc.,MN,USA)。
3.蛋白印迹检测
分别取银屑病患者皮损、非皮损以及正常对照皮肤组织提取总蛋白,经过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、转膜、蛋白印迹、显影、凝胶成像系统成像、Quantity One成像分析软件进行半定量分析等步骤,检测CRH和CRH-R1的表达。抗CRH抗体(1:2000;Chemicon International,Inc.,CA,USA),抗CRH-R1抗体(1:300;R&D Systems,Inc.,MN,USA)。
结果
1.银屑病患者皮损、非皮损及正常对照皮肤CRH表达的检测
免疫组织化学染色分析:阳性表达为棕色颗粒。银屑病患者皮损、非皮损及正常对照皮肤标本均有CRH的阳性表达,主要集中在表皮层。Image-Pro Plus 6图像分析软件分析图像:CRH在银屑病皮损的表达明显低于非皮损和正常对照皮肤(P＜0.01),CRH在银屑病非皮损和正常对照皮肤中的表达差别无统计学意义(P＞0.05)。
蛋白印迹检测结果表明:银屑病患者皮损、非皮损及正常对照皮肤均有CRH蛋白的表达。Quantity One成像分析软件进行半定量分析:银屑病患者皮损CRH水平较非皮损和正常对照皮肤显著降低(P＜0.01),非皮损和正常对照皮肤中的表达差别无统计学意义(P＞0.05)。
2.银屑病患者皮损、非皮损及正常对照皮肤CRH-R1表达的检测结果
免疫组织化学染色分析:阳性表达为棕色颗粒。银屑病患者皮损、非皮损及正常对照皮肤标本均有CRH-R1的阳性表达,主要集中在表皮层。Image-Pro Plus6图像分析软件分析图像:CRH-R1在银屑病皮损的表达明显低于非皮损和正常对照皮肤(P＜0.01),CRH-R1在银屑病非皮损和正常对照皮肤中的表达差别无统计学意义(P＞0.05)。
蛋白印迹检测结果表明:银屑病患者皮损、非皮损及正常对照皮肤均有CRH-R1蛋白的表达。Quantity One成像分析软件进行半定量分析:银屑病患者皮损部位CRH-R1水平较非皮损部位和正常对照皮肤显著降低(P＜0.01),非皮损部位皮肤和正常对照皮肤CRH-R1表达差别无统计学意义(P＞0.05)。
结论
1.免疫组织化学和蛋白印迹检测发现银屑病患者皮损、非皮损及正常对照皮肤均有CRH和CRH-R1蛋白的表达;
2.银屑病患者皮损中存在CRH和CRH-R1的异常表达:CRH和CRH-R1在银屑病患者皮损中的表达明显低于非皮损及正常对照皮肤,CRH和CRH-R1的表达在非皮损和正常对照组织中表达差别无统计学意义。
第二部分促肾上腺皮质激素释放激素及其受体/MAPKs信号传导途径对角质形成细胞表达VEGF和IL-18的影响
背景
促肾上腺皮质激素释放激素(corticotropin-releasing hormone,CRH)由41个氨基酸组成,是下丘脑-垂体-肾上腺轴(hypothalamic-pituitary-adrenal axis,HPA axis)的重要组成成份。CRH通过其特定的CRH受体(CRH receptors,CRH-Rs)发挥作用。最近研究发现,CRH和CRH-Rs在皮肤中均有表达,在人的表皮和真皮中主要表达CRH受体1(CRH receptor1,CRH-R1)。
我们在第一部分的研究中发现,慢性斑块型银屑病患者皮损中CRH和CRH-R1的表达均明显低于银屑病非皮损和正常对照皮肤,提示CRH和CRH-R1在银屑病患者皮损中存在异常表达。因此,我们设想CRH和CRH-R1在银屑病发病,特别是皮损形成过程中可能发挥了一定的作用。鉴于银屑病病理的主要特征是角质形成细胞(keratinocyte,KC)过度增殖、炎症细胞浸润、真皮浅层新血管形成,我们选择了血管内皮生长因子(vascular endothelial growth factor,VEGF)和白细胞介素-18(interleukin-18,IL-18)作为研究对象,观察CRH对人角质形成细胞株HaCaT细胞表达VEGF和IL-18的影响。
VEGF是一组功能强大且能产生多种效应的细胞因子,在生理和病理状态下均具有刺激内皮细胞增殖,诱导新血管形成、增加血管通透性的作用。在正常皮肤中,VEGF主要由KC表达和分泌。研究发现,在慢性斑块型银屑病患者皮损和非皮损组织中KC过表达VEGF,而且转基因动物模型研究进一步表明VEGF可能在银屑病发病过程中起到重要作用。IL-18是白细胞介素-1(interleukin-1,IL-1)超家族的成员,在神经系统、内分泌系统和免疫系统间是一种重要的介质,是慢性炎症和自身免疫性疾病中自身免疫和获得性免疫的重要调节因子。近年来研究发现,KC来源的IL-18参与了银屑病皮损局部的T辅助细胞1(T Helper 1,Th1)免疫反应的发生,其活性受到皮肤炎症反应的调节,在炎症反应中具有重要作用。
目前,CRH对KC表达VEGF的影响尚未见报道。因此,我们选择HaCaT细胞作为体外研究对象,探讨CRH对其分泌VEGF和IL-18的影响,并且选择了普遍存在于真核细胞中的丝裂原活化蛋白激酶(mitogen-activated proteinkinases,MAPKs)信号途径来研究其的可能作用机制。MAPKs是一类高度保守的丝氨酸(Ser)/苏氨酸(Thr)蛋白激酶,能将多种细胞外刺激产生的信号通过级联反应从细胞膜传递到细胞核内,在细胞增殖、分化、凋亡、应激、炎症以及免疫反应等多种生理和病理过程发挥着极其重要的作用。MAPKs主要包括3个经典的亚家族途径,即细胞外调节激酶1/2(extracellular signal-regulated kinase1/2,ERK1/2),p38 MAPK(p38 mitogen-activated protein kinase,p38 MAPK)和c-Jun氨基末端激酶(c-Jun amino-terminal kinases,JNK)。
本研究拟在体外培养的HaCaT细胞中,以主要由KC细胞分泌的VEGF和IL-18为主要指标,观察CRH对KC表达VEGF和IL-18的影响及其可能的细胞内信号传导机制。
目的
1.观察CRH对HaCaT细胞表达VEGF和IL-18的影响;
2.探讨MAPKs信号传导途径在CRH对HaCaT细胞表达VEGF和IL-18调控中的作用。
方法
1.HaCaT细胞的培养
永生化的角质形成细胞株HaCaT细胞用含10%FBS的DMEM置于37℃、5%CO_2培养箱中进行培养。
2.实验设计
(1)根据参考文献选择100 nM CRH孵育HaCaT细胞,不同时间点收集细胞,测定VEGF和IL-18 mRNA及蛋白的表达;
(2)HaCaT细胞给予不同浓度CRH孵育,测定VEGF和IL-18 mRNA及蛋白的表达;
(3)HaCaT细胞分别经CRH-R1拮抗剂antalarmin、ERK1/2阻断剂PD98059、p38 MAPK阻断剂SB203580、JNK1/2阻断剂SP6001259预处理1 h后,给予100 nM CRH孵育,测定HaCaT细胞VEGF和IL-18 mRNA及蛋白的表达;
(4)HaCaT细胞给予100 nM CRH孵育,检测ERK1/2、p38 MAPK和JNK1/2的磷酸化;
(5)HaCaT细胞经CRH-R1拮抗剂antalarmin、ERK1/2阻断剂PD98059、p38 MAPK阻断剂SB203580、JNK1/2阻断剂SP6001259预处理1 h后,给予CRH孵育,测定ERK1/2、p38 MAPK、JNK1/2磷酸化改变。
3.实验方法
3.1.实时荧光定量聚合酶链式反应(real-time RT-PCR)检测VEGF和IL-18 mRNA的表达
将所收集的细胞提取总RNA,经逆转录反应得到cDNA,以管家基因β-actin作为参照,通过real-time RT-PCR技术检测VEGF和IL-18 mRNA的表达。
3.2.双抗体夹心ELISA法检测VEGF和IL-18蛋白表达
将所收集的细胞培养上清进行孵育、酶反应、显色等步骤,酶联免疫检测仪450nm处测量各孔吸光值,根据标准曲线计算出相应浓度。
3.3.Western blot检测ERK1/2、p38 MAPK、JNK1/2蛋白表达
将所收集的细胞提取总蛋白,经过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、转膜、蛋白印迹、显色、凝胶成像系统成像、Quantity One成像分析软件进行半定量分析,检测ERK1/2、p38 MAPK、JNK1/2蛋白的表达。
结果
1.CRH对HaCaT细胞表达VEGF的影响
1.1.不同时间点100 nM CRH对HaCaT细胞VEGF mRNA表达的影响
100 nM CRH在一定范围内时间依赖性降低VEGF mRNA表达,与正常对照组比较差异有统计学意义(P＜0.05),其中100 nM CRH孵育4 h时VEGF mRNA表达量最低。
1.2.不同浓度的CRH对HaCaT细胞VEGF mRNA表达和VEGF蛋白分泌的影响
CRH浓度依赖性降低VEGF mRNA和蛋白表达,与正常对照组比较差异有统计学意义(P＜0.05),其中CRH浓度为100 nM时VEGF mRNA和蛋白表达量最低。
1.3.CRH-R1拮抗剂antalarmin、ERK1/2阻断剂PD98059、p38 MAPK阻断剂SB203580、JNK1/2阻断剂SP6001259对CRH刺激的HaCaT细胞VEGFmRNA表达和VEGF蛋白的分泌影响
CRH-R1拮抗剂antalarmin预处理后,CRH诱导的HaCaT细胞VEGF mRNA及蛋白表达均明显高于单纯CRH孵育组(P＜0.01)。p38 MAPK阻断剂SB203580预处理组或JNK1/2阻断剂SP600125预处理组CRH诱导的HaCaT细胞VEGFmRNA及蛋白的表达均明显高于单纯CRH孵育组(P＜0.01);ERK1/2阻断剂PD98059预处理组VEGF mRNA及蛋白表达与单纯CRH孵育组无明显差别(P＞0.05)。
1.4.MAPKs信号途径在CRH降低HaCaT细胞表达VEGF中的作用
1.4.1.CRH对HaCaT细胞p38 MAPK、JNK1/2磷酸化的影响
100 nM CRH能促进p38 MAPK和JNK1/2磷酸化。p-p38 MAPK和p-JNK1/2蛋白表达于5min达到高峰后逐渐减弱(P＜0.05);T-p38 MAPK、T-JNK1/2蛋白表达各组之间差异无统计学意义(P＞0.05)。
1.4.2.CRH-R1拮抗剂antalarmin、p38 MAPK阻断剂SB203580、JNK1/2阻断剂SP600125对CRH诱导HaCaT细胞p38 MAPK、JNK1/2磷酸化的影响
CRH-R1拮抗剂antalarmin预处理组p-p38 MAPK和p-JNK1/2表达均明显低于单纯CRH孵育组(P＜0.05);p38 MAPK阻断剂SB203580预处理组p-p38MAPK明显低于单纯CRH孵育组(P＜0.05);JNK1/2阻断剂SP600125预处理组p-JNK1/2表达也明显低于单纯CRH孵育组(P＜0.05)。
2.CRH对HaCaT细胞表达IL-18的影响
2.1.不同时间点100 nM CRH对HaCaT细胞IL-18 mRNA表达的影响
100 nM CRH在一定范围内呈时间依赖性降低IL-18 mRNA表达,与正常对照组比较差异有统计学意义(P＜0.05),其中100nM CRH孵育4h时IL-18 mRNA表达量最低。
2.2.不同浓度CRH对HaCaT细胞IL-18 mRNA表达和IL-18蛋白分泌的影响
CRH浓度依赖性降低IL-18 mRNA和蛋白表达,与正常对照组比较差异有统计学意义(P＜0.05),其中CRH浓度为100 nM时IL-18的表达最低。
2.3.CRH-R1拮抗剂antalarmin、ERK1/2阻断剂PD98059、p38 MAPK阻断剂SB203580、JNK1/2阻断剂SP600125对CRH刺激的HaCaT细胞IL-18mRNA表达和IL-18蛋白的分泌影响
CRH-R1拮抗剂antalarmin预处理后,CRH诱导的HaCaT细胞IL-18 mRNA及蛋白表达均明显高于单纯CRH孵育组(P＜0.05)。ERK1/2阻断剂PD98059预处理、p38 MAPK阻断剂SB203580预处理、JNK1/2阻断剂SP600125预处理后CRH诱导的HaCaT细胞IL-18 mRNA及蛋白的表达均明显高于单纯CRH孵育组(P均＜0.05)。
2.4.MAPKs信号途径在CRH降低HaCaT细胞表达IL-18中的作用
2.4.1.CRH对HaCaT细胞ERK1/2、p38 MAPK、JNK1/2磷酸化的影响
100 nM CRH能够促进ERK1/2、p38 MAPK和JNK1/2磷酸化。p-ERK1/2蛋白5min开始逐渐增强,10min达高峰后逐渐减弱(P＜0.05);p-p38 MAPK和p-JNK1/2蛋白表达于5min达到高峰后逐渐减弱(P均＜0.05)。T-ERK1/2、T-p38MAPK、T-JNK1/2蛋白表达各组之间差异无统计学意义(P＞0.05)。
2.4.2.CRH-R1拮抗剂antalarmin、ERK1/2阻断剂PD98059、p38 MAPK阻断剂SB203580和JNK1/2阻断剂SP600125对CRH诱导HaCaT细胞ERK1/2、p38 MAPK、JNK1/2磷酸化的影响
CRH-R1拮抗剂antalarmin预处理组p-ERK1/2、p-p38 MAPK和p-JNK1/2表达均明显低于单纯CRH孵育组(P均＜0.05);ERK1/2阻断剂PD98059预处理组p-ERK1/2表达明显低于单纯CRH孵育组(P＜0.05);p38 MAPK阻断剂SB203580预处理组p-p38 MAPK明显低于单纯CRH孵育组(P＜0.05);JNK1/2阻断剂SP600125预处理组p-JNK1/2表达明显低于单纯CRH孵育组(P＜0.05)。
结论
1.CRH抑制HaCaT细胞VEGF和IL-18 mRNA和蛋白的表达;
2.CRH能够活化HaCaT细胞ERK1/2、p38 MAPK、JNK1/2信号途径,CRH-R1参与了CRH对ERK1/2、p38 MAPK、JNK1/2信号途径的活化;
3.CRH-R1和p38 MAPK、JNK1/2信号途径参与了CRH对HaCaT细胞VEGFmRNA和蛋白表达的抑制作用;
4.CRH-R1和ERK1/2、p38 MAPK、JNK1/2信号途径参与了CRH对HaCaT细胞IL-18 mRNA和蛋白表达的抑制作用。
PART ONE Expression of corticotropin-releasing hormone and receptor1 in chronic plaque psoriasis
Background
Psoriasis is a chronic inflammatory disease characterized by epidermal keratinocytic hyperproliferation and abnormal differentiation.It is one of the most illustrative examples of the close relation between exacerbation of disease and the psychopathologic burden of patients.Many clinical observations of plaque symmetry and stress-induced onset and/or exacerbation suggest an important role of the nervous system in the pathologic features of psoriasis.However,the mechanism remains poorly understood.
Corticotrophin-releasing hormone(CRH) is a central component of the hypothalamic-pituitary-adrenal(HPA) axis and is an important coordinator of the systemic stress response.Typically,in response to systemic stress,CRH released from the hypothalamus regulates the HPA axis by stimulating the pituitary CRH receptor (CRH-R).Activated CRH-R further stimulates signal transduction pathways that enhance the production and secretion of proopiomelanocortin(POMC)-derived peptides,including adrenocorticotropic hormone(ACTH),α-melanocyte stimulating hormone(α-MSH) andβ-endorphin(β-END).
In recent years,evidence has suggested that endocrine stress responses under control of the central nervous system also occur in peripheral tissue,outside of the classical HPA axis.CRH and CRH-R gene expression has been found in rodent and human skin.The existence of CRH produced peripherally and other HPA axis components such as POMC,ACTH,α-MSH,andβ-END at extracranial sites including skin suggests the existence of local HPA systems.Research has indicated that peripheral CRH is synthesized by cutaneous cells and immune cells in human skin.Moreover,CRH and POMC-derived peptides are produced locally in the skin and are regulated by infiltrating inflammatory cells,as well as by autocrine mechanisms.
In peripheral sites,cutaneous CRH is believed to regulate various functions of the skin that are important for local homeostasis.These findings have shed new light on the role of CRH/CRH-R in peripheral cutaneous diseases,especially psoriasis, which is characterized by impaired keratinocyte differentiation and proliferation.
Objective
In the present study,we investigated by immunohistochemistry and western blot the expression of CRH and CRH-R1 in the lesions of chronic plaque psoriasis,as well as in perilesional skin and normal control skin,to explore the role of cutaneous CRH/CRH-R1 in the pathogenic mechanism associated with psoriasis.
Materials and methods
1.Tissue samples
All patients(n=26;mean age=32±12 years) with dermatologist-confirmed diagnosis of chronic plaque psoriasis were recruited from outpatients and inpatients at Qilu Hospital.The mean course of psoriasis was 58±102 months.Age-matched healthy individuals(n=26,mean age=31±7 years) undergoing cosmetic surgery were used as controls.Subjects were excluded if they had chronic diseases.None of the patients was under any regular systemic therapy,including PUVA,cyclosporine, cyclophosphamide,methotrexate,or topical steroids for psoriasis,for at least 4 weeks before the study.Patients underwent clinical assessment,and skin biopsies were taken from the middle of plaques and perilesional skin at least 5 cm away from psoriatic lesions in the arm,tight,back or buttock.
2.Immunohistochemistry
For CRH and CRH-R1 immunostaining,sections were incubated overnight at 4℃in a wet box with the following primary antibodies diluted in PBS:rabbit polyclonal anti-corticotropin releasing hormone antibody(1:500;Chemicon International,Inc.,CA,USA) and mouse monoclonal anti-human CRH-R1 antibody (1:40;R&D Systems,Inc.,MN,USA).Negative control sections were treated identically,except the primary antibody was substituted with isotype-matched non-specific negative-control antibodies for rabbit IgG(Neomarkers,Inc.,Fremont, CA) and mouse IgG(Abcam,Cambridge,UK).The expression and localization of CRH and CRH-R1 in psoriatic lesions compared with those in perilesional skin and normal control skin.Image-Pro Plus 6 software(Media Cybernetics,USA) was used for image analysis.
3.Western blot
The expression of CRH and CRH-R1 were detected by western blot analysis in psoriatic lesions,perilesional skin and normal control skin.Equal protein loading was confirmed by assessing the protein level ofβ-actin.CRH(1:2000;Chemicon International,Inc.,CA,USA);CRH-R1(1:300;R&D Systems,Inc.,MN,USA); humanβ-actin(1:5000;Santa Cruz,CA).The images were recorded by use of FluorChem9900(Alpha Innotech,CA,USA) and analyzed with use of Quantity One software(Bio-Rad Laboratories,Hercules,CA).
Results
1.Expression of CRH in psoriatic lesions,perilesional skin and normal control skin
Immunostaining revealed CRH localized in psoriatic lesions,perilesional and normal control skin.CRH expression was significantly lower in psoriatic lesions than in perilesional and normal skin,with no difference in CRH expression between psoriatic perilesional skin and normal control skin.
Western blot showed all skin specimens were positive for CRH.There was a significant decrease in the expression of CRH in psoriatic lesions compared with psoriatic perilesional skin and normal control skin,no significant change in psoriatic perilesional skin and normal control skin.
2.Expression of CRH-R1 in psoriatic lesions,perilesional skin and normal control skin
Immunostaining found that CRH-R1 localized in psoriatic lesions,perilesional and normal control skin.CRH-R1 expression was lower in psoriatic lesions than in perilesional and normal control skin,with no difference in CRH-R1 expression between psoriatic perilesional skin and normal control skin.
Western blot showed that all skin specimens were positive for CRH-R1.There was a significant decrease in the expression of CRH-R1 in psoriatic lesions compared with psoriatic perilesional skin and normal control skin,no significant change in psoriatic perilesional skin and normal control skin.
Conclusions
1.Immunohistochemistry and western blot revealed that all skin specimens were positive for CRH and CRH-R1,including psoriatic lesions,perilesional skin and normal control skin.
2.An aberrant cutaneous CRH/CRH-R1 system may exist in chronic plaque psoriasis.There was a significant decrease in the expression of CRH and CRH-R1 in psoriatic lesions compared with psoriatic perilesional skin and normal control skin,no significant change in psoriatic perilesional skin and normal control skin.
PART TWO Corticotropin-releasing hormone attenuates vascular endothelial growth factor and interleukin-18 release from human HaCaT keratinocytes via the mitogen-activated protein kinases pathway
Background
Corticotropin-releasing hormone(CRH),a 41-amino acid neuropeptide,is produced mainly in the hypothalamus and regulates endocrine and behavioral responses to stress through the activation of the hypothalamic-pituitary-adrenal(HPA) axis.CRH exerts its actions via interaction with specific CRH receptors(CRH-Rs). Recent research has indicated that CRH and CRH-Rs are expressed and have functions in the skin,in human skin;CRH-R1 is the major receptor in epidermis and dermis.
In part one,our research found that lesions from patients with chronic plaque psoriasis showed significantly lower CRH and CRH-R1 expressions compared with psoriatic perilesional skin and normal control skin.The results suggested that an aberrant cutaneous CRH/CRH-R1 system may play a role in the pathogenesis of chronic plaque psoriasis,especially the formation of plaque.
Psoriasis is a chronic inflammatory disease characterized by erythematous plaques with silvery scales.Psoriatic lesions exhibit proliferation of epidermal keratinocytes,inflammatory cell infiltration,and increased angiogenesis of the superficial dermal vessels.So we choose vascular endothelial growth factor(VEGF) and interleukin-18(IL-18) as objects to study the effect of CRH and its receptor CRH-R1 on the expression of VEGF and IL-18 in a human keratinocyte cell line, HaCaT.
The prominence of dermal microvascular expansion in the psoriatic lesion demonstrates that psoriasis is an angiogenesis-dependent disease.VEGF is a crucial regulator of angiogenesis and vascular permeability in both physiological and pathological conditions such as tumor growth and chronic inflammation.VEGF is expressed and secreted by epidermal keratinocytes in normal human skin. Keratinocytes overexpress VEGF in clinically involved and uninvolved skin of patients with chronic plaque psoriasis.In transgenic mice with epidermis-specific overexpression of VEGF and enhanced skin vascularity and vascular permeability; chronic transgenic delivery of VEGF to the skin induced inflammation and all characteristics of psoriasis spontaneously,and the VEGF antagonist reversed the phenotype.These findings suggested a causative role of VEGF in the pathogenesis of psoriasis.IL-18,a member of the IL-1 cytokine superfamily which shares structural features with IL-1βand functions primarily as an IFN-γinducer and promoter of Th1 responses in T cells,is now recognized as an important regulator of innate and acquired immune responses which is expressed at sites of chronic inflammation and in autoimmune diseases.IL-18 might be an important mediator in the communication between the nervous,the endocrine and the immune systems.Recent works have demonstrated that human keratinocyte-derived IL-18 participates in the development of the Th1 response in psoriatic lesions,and its bioactivity appears to be tightly regulated in cutaneous inflammation.IL-18 secreted by keratinocytes might play an important role in cutaneous inflammatory response,especially in Th1-mediated inflammatory diseases such as psoriasis.
However,little is known about the exact role of CRH in skin.We hypothesized that CRH may modulate VEGF and IL-18 expression.We examined whether this effect functioned via the mitogen-activated protein kinases(MAPKs) signal transduction pathway,particularly p38 mitogen-activated protein kinase(p38 MAPK), extracellular signal-regulated protein kinase 1/2(ERK1/2) and c-Jun N-terminal kinase(JNK).
In light of this,we performed a study to investigate the effect of CRH on the expression of VEGF and IL-18 on HaCaT cells,and examine whether this effect functioned via MAPKs signal transduction pathway.
Objective
1.Investigate the effect of CRH on the expression of VEGF and IL-18 on HaCaT cells;
2.Examine whether the effect of CRH on the expression of VEGF and IL-18 functioned via MAPKs signal transduction pathway.
Materials and methods
1.Cell culture
The immortalized human HaCaT keratinocytes were maintained at 37℃and 5% carbon dioxide(CO_2) in Dulbecco's Modified Eagle's Medium(DMEM) supplemented with 10%heat-inactivated fetal bovine serum,100 U/ml penicillin and 100μg/ml streptomycin.
2.Cell pretreatment
HaCaT cells were seeded at density 4×10~4 cells/ml,grown for 48 h until 70% confluence.The cells were pretreated with 10μM antalarmin,10μM PD98059,10μM SB203580,or 10μM SP600125 respectively and incubated for 1 h before application of CRH.
3.Methods
3.1.Real-time RT-PCR
After the experimental treatment,the total RNA was extracted from HaCaT cells. The reverse transcription of RNA to cDNA was performed,cDNA were amplified with real-time RT-PCR.VEGF mRNA and IL-18 mRNA expression were normalized to the expressed housekeeping gene humanβ-actin.Samples were tested in triplicate and the average values were used for quantification.
3.2.ELISA
After stimulation for 24 h,culture supernatants of cells were collected, centrifuged(15 000 rpm,5 min) and stored at -80℃until analysis.The concentrations of VEGF and IL-18 in the culture supernatant were measured by commercially available enzyme linked immunosorbent assay(ELISA) kits according to manufacturer's instructions.Each supernatant was analyzed in triplicate.
3.3.Western blot
The phosphorylation of ERK1/2,p38 MAPK,and JNK1/2 were detected by western blot analysis,antalarmin and all kinds of inhibitors were used to block MAPKs pathway.Densitometric analysis of the band intensity was carried out using Quantity One software(Bio-Rad Laboratories,Hercules,CA).
Results
1.CRH attenuated VEGF release in HaCaT cells via the MAPKs pathway
1.1.Effect of 100 nM CRH on VEGF production in HaCaT cells
VEGF mRNA expression and its production were significantly downregulated by 100 nM CRH in a time-dependent manner.
1.2.Effect of CRH on VEGF production in HaCaT cells
VEGF mRNA expression and its production were significantly downregulated by CRH in a dose-dependent manner.
1.3.CRH-R1 and MAPKs signaling pathway were involved in VEGF production
CRH downregulated VEGF mRNA and protein expression in HaCaT cells by CRH-R1 through p38 MAPK and JNK1/2 pathways,except ERK1/2.
1.4.The role of MAPKs pathway in the effect of CRH in HaCaT cells
1.4.1.CRH activated p38 MAPK and JNK1/2 phosphorylation in HaCaT cells
CRH induced a rapid phosphorylation of p38 MAPK and JNK1/2,with a peak at 5 min,and CRH-R1 was involved in CRH-induced phosphorylation of p38 MAPK and JNK1/2.
1.4.2.The effect of CRH-R1 antagonist antalarmin,p38 MAPK inhibitor SB203580,JNK1/2 inhibitor SP600125 on phosphorylation of p38 MAPK and JNK1/2 induced by CRH in HaCaT ceils
Pretreating HaCaT cells with the p38 MAPK inhibitor SB203580,the JNK1/2 inhibitor SP600125 or the CRH-R1 antagonist antalarmin significantly inhibited the CRH-induced phosphorylation of p38 MAPK and JNK1/2.These data indicated that CRH activated p38 MAPK and JNK1/2 phosphorylation in HaCaT cells,and CRH-R1 was involved in the CRH-induced phosphorylation of p38 MAPK and JNK1/2.
2.CRH attenuated IL-18 release in HaCaT cells via the MAPKs pathway
2.1.Effect of 100 nM CRH on IL-18 production in HaCaT cells
IL-18 mRNA expression and its production were significantly downregulated by 100 nM CRH in a time-dependent manner.
2.2.Effect of CRH on IL-18 production in HaCaT cells
IL-18 mRNA expression and its production were significantly downregulated by CRH in a dose-dependent manner.
2.3.CRH-R1 and MAPKs signaling pathway were involved in IL-18 production
CRH downregulated IL-18 expression in HaCaT cells by CRH-R1 through ERK1/2,p38 MAPK and JNK1/2 pathways.
2.4.The role of MAPKs pathway in the effect of CRH in HaCaT cells
2.4.1.CRH activated ERK1/2,p38 MAPK and JNK1/2 phosphorylation in HaCaT cells
CRH induced a rapid phosphorylation of ERK1/2 with a peak at 10 min;CRH induced a rapid phosphorylation of p38 MAPK and JNK1/2,with a peak at 5 min,and CRH-R1 was involved in CRH-induced phosphorylation of ERK1/2,p38 MAPK and JNK1/2.
2.4.2.The effect of CRH-R1 antagonist antalarmin,ERK1/2 inhibitor PD98059,p38 MAPK inhibitor SB203580,JNK1/2 inhibitor SP600125 on phosphorylation of p38 MAPK and JNK1/2 induced by CRH in HaCaT cells
Pretreating HaCaT cells with the CRH-R1 antagonist antalarmin,the ERK1/2 inhibitor PD98059,the p38 MAPK inhibitor SB203580,the JNK1/2 inhibitor SP600125 significantly inhibited the CRH-induced phosphorylation of ERK1/2,p38 MAPK and JNK1/2.These data indicated that CRH activated ERK1/2,p38 MAPK and JNK1/2 phosphorylation in HaCaT cells,and CRH-R1 was involved in the CRH-induced phosphorylation of ERK1/2,p38 MAPK and JNK1/2.
Conclusions
1.VEGF and IL-18 mRNA expression and protein production were significantly downregulated by CRH.
2.CRH activated ERK1/2,p38 MAPK and JNK1/2 phosphorylation in HaCaT cells,and CRH-R1 was involved in CRH-induced phosphorylation of ERK1/2,p38 MAPK and JNK1/2.
3.CRH downregulated VEGF expression in HaCaT cells by CRH-R1 through p38 MAPK and JNK1/2 pathways.
4.CRH downregulated IL-18 expression in HaCaT cells by CRH-R1 through ERK1/2,p38 MAPK,JNK1/2 pathways.
引文
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[7] Pisarchik A, Slominski A. Molecular and functional characterization of novel CRFR1 isoforms from the skin. Eur J Biochem 2004;271:2821-30.
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