Study On Corticotropin-Releasing Hormone And Receptor1/Mitogen-Activated Protein Kinases Pathway In Pathogenesis Of Psoriasis

PART ONE Expression of corticotropin-releasing hormone and receptor1 in chronic plaque psoriasisBackgroundPsoriasis 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.ObjectiveIn 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 methods1.Tissue samplesAll 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.ImmunohistochemistryFor 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 blotThe 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).Results1.Expression of CRH in psoriatic lesions,perilesional skin and normal control skinImmunostaining 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 skinImmunostaining 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.Conclusions1.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 pathwayBackgroundCorticotropin-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. Objective1.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 methods1.Cell cultureThe immortalized human HaCaT keratinocytes were maintained at 37℃and 5% carbon dioxide(CO₂) 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 pretreatmentHaCaT cells were seeded at density 4×10⁴ 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.Methods3.1.Real-time RT-PCRAfter 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.ELISAAfter 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 blotThe 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).Results1.CRH attenuated VEGF release in HaCaT cells via the MAPKs pathway1.1.Effect of 100 nM CRH on VEGF production in HaCaT cellsVEGF 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 cellsVEGF 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 productionCRH 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 cells1.4.1.CRH activated p38 MAPK and JNK1/2 phosphorylation in HaCaT cellsCRH 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 ceilsPretreating 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 pathway2.1.Effect of 100 nM CRH on IL-18 production in HaCaT cellsIL-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 cellsIL-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 productionCRH 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 cells2.4.1.CRH activated ERK1/2,p38 MAPK and JNK1/2 phosphorylation in HaCaT cellsCRH 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 cellsPretreating 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.Conclusions1.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.