Study On Calcitonin Gene-Related Peptide And Mitogen-Activated Protein Kinase Pathway In The Pathogenesis Of Psoriasis

PART ONECalcitonin gene-related peptide regulates the proliferation and expression of vascular endothelial growth factor in human HaCaT keratinocytes by activation of MAP kinasesBackgroundPsoriasis is a common, chronic, inflammatory disease. The cause and pathogenesis of psoriasis is still unknown. The disease is characterized by keratinocyte hyperproliferation, vascular hyperplasia and ectasia, and infiltration of T lymphocytes, neutrophils, and other types of leucocytes in affected skin. Several studies have demonstrated that VEGF expression is increased in lesional psoriatic skin, that the serum levels of circulating VEGF protein are significantly elevated in patients with severe disease activity. In addition, the gene for VEGF is located on chromosome 6p.21, close to PSORS1. A major role of VEGF in the pathogenesis of psoriasis was further corroborated by the phonotype of transgenic mice with epidermis-specific overexpression of VEGF. VEGF transgenic mice show epidermal hyperplasia and altered epidermal differentiation and inflammatory skin lesions that histologically closely resemble lesions of human psoriasis. Moreover, VEGF transgenic mice show the characteristic Koebner phenomenon. These findings indicate VEGF might play an important role in the pathogenesis of psoriasis.Several facts suggested that psychic stress is very much associated with the exacerbation and development of psoriasis. However, the mechanism of stress exacerbating psoriasis remains unclear. By expressing neuropeptides and neuropeptide receptors, nevers, resident skin cells and infiltrating migratory immunocytes share a powerful neuropeptide "language", which enable an interdependent communication between the central nervous system and the skin immune systems on multiple levels.Calcitonin gene-related peptide (CGRP) is one of the most abundant neuropeptides in human and rodent skin. It is widely expressed in both the central and peripheral nervous system. It has been demonstrated that the expression of CGRP is elevated in psoriatic lesions and CGRP-containing neuropeptide nerve fibers are denser in the psoriatic epidermis. These findings suggest that CGRP may play a significant role in the pathophysiologic process of psoriasis. CGRP has been previously influence proliferation of several cell types (e.g. endothelial cells, Schwann cell, tracheal epithelial cells). More recently, it was demonstrated on gingival fibroblasts that CGRP mediates its mitogenic effect via activation of the mitogen-activated protein kinases (MAPK) pathway. MAPK plays a role in signal transduction associated with cell proliferation, differentiation and the production of cytokines. There are three well-characterized MAPK subfamilies in mammalian cells: extracellular-signal-regulated protein kinase 1/2 (ERK1/2), the p38 mitogen-activated protein kinases (p38 MAPK) and the c-Jun N-terminal kinase (JNK).We therefore asked if CGRP is able to induce the proliferation and VEGF production in epidermal keratinocytes and investigated the role of MAPK signal pathway in the proliferation and VEGF production in epidermal keratinocytes by CGRP.ObjectiveThis study was designed to investigate the effect of CGRP on cell proliferation and VEGF production at the protein and mRNA levels in the human keratinocyte cell line HaCaT, and to identify intracellular transduction pathways involved in the effect of CGRP.Materials and methods1.Cell cultureHuman keratinocyte cell line HaCaT cells were cultured with DMEM supplemented with 10% fetal calf serum (FBS) at 37℃in a humidified incubator containing 5% CO₂.2. MTT assay for cell proliferationHaCaT cells were incubated with CGRP or with the antagonist of CGRP receptor (CGRP8-37) or PD98059, SB203580, or SP600125, selective inhibitors of MAP kinase kinase (MEK, which is upstream from ERK1/2), p38 and JNK, respectively, and then followed by MTT assay.3. Real-time RT-PCRHaCaT cells were incubated with CGRP or with CGRP 8-37, PD 98059, SB 203580, or SP 600125. The total RNA was extracted from HaCaT cells. The reverse transcription of RNA to cDNA was performed. cDNA were amplified with Real-time PCR.4. VEGF ELISAAfter stimulation, supernatants of cultured cells were collected, and VEGF concentration was calculated using human VEGF ELISA kit.5. Western blot analysisThe phosphorylation of ERK1/2, p38, and JNK were detected by Western blot analysis and CGRP8-37 and all kinds of inhibitors were used to block MAPK pathway. Densitometric analysis of the band intensity was carried out using Totallab Image Analysis Software.Results1. CGRP increased HaCaT proliferation CGRP (0.1, 1, and 10 nM) induced significant increase in MTT activity by the HaCaT cells. Serum contains a variety of mitogenic substances.2. Effect of CGRP8-37, PD98059, SB203580, or SP600125 on CGRP-induced HaCaT cells proliferationHaCaT cells were pretreated for one hour with CGRP8-37, PD98059, SB203580, or SP600125, followed by 24-hour CGRP treatment and MTT assay. The CGRP induced HaCaT cells activity was attenuated by the addition of CGRP 8-37, PD98059 or SB 203580, but not SP 600125.3. CGRP induced the mRNA expression and production of VEGF VEGF mRNA expression was significantly increased by CGRP (0.1, 1,and 10 nM). VEGF production was measured from supernatants of CGRP-treated cultured HaCaT cells collected 12 h after the exposure.4. Effect of CGRP8-37, PD98059, SB203580, or SP600125 on CGRP-induced the mRNA expression and production of VEGF in HaCaT cellsHaCaT cell were treatment with the antagonist of CGRP receptor (1 uM CGRP 8-37) or PD98059, SB203580 or SP600125 at a concentration of 10 uM for 1 h prior to the addition of 10 nM CGRP. Either CGRP 8-37 or PD98059 significantly inhibited the CGRP-increased VEGF mRNA expression and its production.5. Roles of MAPK in CGRP induced proliferation and the expression of VEGFHaCaT cells were treated with 10 nM of CGRP for 5, 10, 30, 60 minutes, followed by extraction of the cellular protein. The expressions of total and phosphorylated ERK1/2, p38, and JNK were determined by Western analysis. The CGRP induced increases in phospho-ERK1/2, phospho-p38 and phospho-JNK after addition of CGRP. There was no change in the expression of total p38, ERK1/2, and JNK.Furthermore, total and phosphorylated p38, ERK1/2, and JNK were determined using the cellular protein of HaCaT cells treated with CGRP for 24 hours in the presence or absence of CGRP8-37, SB203580, PD98059, or SP600125. Western analysis revealed that CGRP induced phosphorylation of ERK1/2, p38, and JNK were inhibited by CGRP8-37.ConclusionsOur results demonstrate that CGRP increases HaCaT cells proliferation. In addition, CGRP receptor and the activation of ERK1/2 and p38 play an important role in mediating the CGRP induced proliferation by HaCaT keratinocytes. CGRP induces the expression of an angiogenic factor, VEGF, which is involved in the pathogenesis of psoriasis, through CGRP receptor and the MAPK pathway. These findings suggest that CGRP which is elevated expressed in epidermis of psoriasis may involve in the pathogenesis of psoriasis by enhancing keratinocyte proliferation and upregulating the expression of VEGF.PART TWOExpression and localization of the activatedmitogen-activated protein kinase in lesional psoriatic skinBackgroundPsoriasis is a common, chronic, inflammatory skin disease. The cause and pathogenesis of psoriasis is still unknown. The disease is characterized by abnormal proliferation and differentiation of keratinocytes, infiltration of T lymphocytes, and vascular hyperplasia and ectasia. Abnormalities in several signaling pathways and in the expression or activation of different transcription factors in psoriatic keratinocytes have been hypothesized to play a role in the pathophysiology of psoriasis.The mitogen-activated protein kinase (MAPK) pathways are the best characterized of intracellular protein kinase cascades. They play well-known roles in cell proliferation, differentiation, gene expression, and inflammation. Alterations in gene expression and enzyme activity, induced by extracellular stimuli/stress, are mediated through the interplay of multiple signaling pathways. Among these are the highly conserved mitogen-activated protein kinase (MAPK) pathways, which are the central mediators that propagate signals from the membrane to the nucleus. Three relatively well-characterized MAPK signaling pathways are the extracellular-signal-regulated protein kinase (ERKs), the p38 mitogen-activated protein kinases (p38 MAPKs) and the c-Jun N-terminal kinase (JNKs). On activation by phosphorylation of both threonine and tyrosine residues, these kinases phosphorylate intracellular enzymes and transcription factors.ObjectiveIn the present study, we investigate MAPK expression and activation in lesional psoriatic skin, compared with nonlesional psoriatic skin and normal control skin.Materials and methods1. Tissue samplesSkin biopsies were obtained from involved and uninvolved skin of 12 typical psoriasis vulgaris patients, and 10 anatomic-matched healthy individuals undergoing reconstructive surgery in this study. All patients were off systemic immunosuppressive drugs such as corticosteroids or cyclosporine for at least a month and off topical corticosteroids for more than 1 week before their skin biopsy. The uninvolved skin was taken at lease 2 cm from the involved skin.2. Western blot analysisThe phosphorylation of ERK1/2, p38, JNK and the total ERK1/2, p38, JNK were detected by Western blot analysis in lesional psoriatic skin, non-lesional psoriatic skin and normal skin. Equal protein loading was confirmed by assessing the protein level of P-actin. Densitometric analysis of the band intensity was carried out using Totallab Image Analysis Software.3. ImmunohistochemistryThe expression and localization of activated ERK1/2, p38, and JNK in lesional psoriatic skin compared with non-lesional psoriatic skin and normal skin.Results1. Western blot analysis showed a significant increase in the levels of p-ERK1/2 and p-p38 in psoriatic lesions compared with nonlesional psoriatic skin and control skin, although no significant change in the total ERK1/2 and p38. In contrast, immunoblot analysis demonstrated there were no significant differences in both p-JNK and total JNK in psoriatic lesions compared with nonlesional psoriatic skin and control skin.2. The results of immunohistochemical analyses showed that p-ERK1/2 and p-p38 exhibited a clear nuclear localization throughout the entire epidermic part of lesional psoriatic skin. Furthermore, p-ERK1/2 and p-p38 were found to be strongly expressed in fibroblasts and vessel endothelial cells of dermic part. p-ERK1/2 expression in nuclear of keratinocytes was found in a total of 11/12(92%) psoriatic lesions, and p-p38 was found in 10/12 (83%). In contrast, p-JNK was detectable mainly in the cytoplasm of keratinocytes in these sections and there was no found p-JNK stainning in fibroblasts and vessel endothelial cells.In uninvolved skin of psoriatic patients and normal control skin, p-ERK1/2, p-p38, and p-JNK mainly exhibited a weaker, diffuse cytoplasmic staining through most of the epidermic layers.ConclusionsIn conclusion, the abnormally activated of the ERK1/2 and p38 MAPK pathway in lesional psoriatic skin may therefore be involved in epidermal hyperproliferation and inflammation those characterize the disease. Future studies will be necessary to test whether this pathway is a useful target for new antipsoriatic treatments.