C/Ebpα Is Involved In The Lesion Formation Of Psoriasis Vulgaris By Regulating The Proliferation And Differentiation Of Keratinocyte

Psoriasis, which is associated with a genetic background and abnormal immune response, is one of the most common clinical erythematous desquamative dermatosis in China with an incidence of0.123%-0.470%. Because of easy recurrence and difficult to cure, psoriasis seriously influences patients health and quality of life. However, until now, the exact etiology and pathogenesis of psoriasis have not yet been fully elucidated, which becomes one of the most urgent problems to be solved in dermatology field.In recent years, many studies have been conducted on psoriasis in both China and abroad with some progresses, nevertheless, the cause and pathogenesis of the psoriasis have not been completely clarified so far. The fundamental pathophysiology of the psoriasis is supposed to be related to the genetic factors, immune factors, infection factors, endocrine factors, neuropsychiatric factors, habits of life, drugs and environmental factors. All of these above factors lead to the abnormality for the proliferation, differentiation and apoptosis of keratinocyte and the abnormal neogenesis of the vascular endothelial cell in the dermal papilla layer, which result in the clinically characteristic skin lesions of psoriasis such as red patches covered with silvery white scale trifles, the phenomenon of membrane, the phenomenon of candle dripping and the Auspitz sign.CCAAT/enhancer binding protein a (C/EBPa), an important member of the transcription factor C/EBP family, plays a vital role in the synthesis and metabolism of phospholipids and the development, proliferation, differentiation of variable cell types, for example fat cells, bone marrow cells, liver cells, keratinocyte and alveolar cells. C/EBPa is mainly expressed in the keratinocytes from the basal layer of the epidermis and the overexpression of C/EBPa may cause cell cycle arrest. The high expression of C/EBPa in the epidermis of mice and human indicates that C/EBPa plays a transcription factor role in the differentiation of stratified squamous epithelium cells. The overexpression of oncogene Ras leads to the decrease of C/EBPa in immortalized keratinocytes. Consequently, the inhibition effect of cell proliferation by C/EBPa is reduced. Thus it can be seen that C/EBPa plays an important role in regulating the proliferation and differentiation of epidermal keratinocytes in normal skin. How dose C/EBPa express in the skin lesions of psoriasis vulgaris with abnormal proliferation and differentiation of keratinocyte? Whether does C/EBPa participate in the course of pathological changes of psoriasis vulgaris or not? Up to now, there is no literature reported on those relevant issues in China and abroad.In order to investigate the role of C/EBPa in the pathogenesis of psoriasis, the mRNA and protein expressions of C/EBPa in the skin lesions of psoriasis were detected. To explore the clinical significance of abnormal C/EBPa expression, the relationship of the expression of C/EBPa between the psoriasis area and severity index (PASI), the proliferation and differentiation of keratinocytes were investigated. To explore the effect of C/EBPa gene on the proliferation of keratinocytes, the expression of C/EBPa gene in primary human keratinocyte was silenced by short interfering RNA (siRNA) for C/EBPa. We applied the key cytokines IL-22, which was secreted by Thl7cells and Th22cells in the pathogenesis of psoriasis, to stimulate the keratinocyte,and then detected the expression of C/EBPa and the phosphorylation levels of JNK, ERK and p38, the key signaling molecules in MAPK signaling pathways. Through the above investigations, we wanted to make clear that if the expression of C/EBPa could participate in the formation of the skin lesions in psoriasis vulgaris through MAPK signaling pathways. Part I. The mRNA and protein expressions of C/EBPa in the lesions of psoriasis with the clinical significanceObjective:To detect the mRNA and protein expressions of C/EBPa in the lesions of psoriasis and investigate their relationships with the abnormal proliferations of keratinocytes and the PASI.Methods:1. Specimens Collection:Between January2011and December2011,30cases of paraffin-embedded specimens of psoriasis vulgaris were collected as the experimental group in the Pathological Lab of the Department of Dermatology in Qilu Hospital of Shandong University. Meanwhile,30cases of health skin specimens matched for ages, gender and site of sampling with the experimental group were collected as the control group in the Department of Dermatology of our hospital.2. Immunohistochemical method to detect the expression of C/EBPa and Ki-67:The expressions of C/EBPa, Ki-67and CK10were detected by two-step immunohistochemical method in the control and experimental groups. Semi-quantitative scores were recorded according to the number of positive cells and the degree of dyeing. The difference of expression between two groups was analyzed by Chi-square test.3. The correlations between the expression of C/EBPa and the proliferation index (PI) of keratinocytes and the PASI:The PI of keratinocytes was calculated according to the positive expression of Ki-67and the associations between the expression of C/EBPa and PI and PASI were analyzed by Pearson correlation analysis. 4. The mRNA and protein expressions of C/EBPa in lesions of psoriasis and health skin were detected by RT-PCR and western blot.Results:1. Clinical data of patients:The30cases of psoriasis vulgaris patients with a mean age of25.2years (range15to49years) included18males and12females. For the lesions among the30cases of psoriasis vulgaris,10cases were on the head and face,10cases on the trunk and10cases the on extremities.2. The expression of C/EBPa was localized dominantly in the cytoplasm of keratinocytes and the expression of C/EBPa was significantly lower in the lesions of psoriasis vulgaris than in the control group with a statistical difference (t=7.819, P<0.05).3. The expression of Ki-67was detected mainly in the nucleus of keratinocytes. The PI was significantly higher in the lesions of psoriasis vulgaris than in the control group with a statistical difference (t=4.535, P<0.05).4. According to the results of Pearson correlation analysis, the gray values of C/EBPa were positively correlated with the PI and PASI in the lesions of psoriasis vulgaris (r=0.654, P<0.05and r=0.693, P<0.05, respectively).5. According to the results of RT-PCR and western blot, the mRNA and protein expressions of C/EBPa were significantly lower in the lesions of psoriasis vulgaris than in control group with a statistical difference (P<0.05).Conclusions:The mRNA and protein expressions of C/EBPa were significantly reduced in the lesions of psoriasis vulgaris. The protein expression of C/EBPa was negatively correlated with the PI of keratinocytes and PASI of lesions of psoriasis vulgaris. It was speculated that C/EBPα, which could be used as an indicator reflecting the severity of psoriasis lesions, might play a role in the process of the abnormal proliferation of keratinocytes. Part Ⅱ. The gene expression of C/EBP α in keratinocytes and the effects on the proliferation and differentiation of keratinocytes by siRNA mediated C/EBP α gene silencingObjective:1. To detect the mRNA and protein expressions of C/EBPa in primary human keratinocytes.2. To investigate the effects of C/EBPα on the proliferation and differentiation of keratinocytes by siRNA mediated C/EBPα gene silencing.Methods:1. The culture of primary cells:The primary culture of human epidermal keratinocytes was used in the study. The skin specimens from the young children’s foreskin were obtained by the Department of Paediatric Urology of Shandong Province Hospital affiliated to Shandong University. The specimens were digested with0.25%trypsin at4℃for overnight to dissect epidermis from dermis. Then, the epidermis specimens were digested with0.25%trypsin-0.01%EDTA mixture to obtain single cell suspension, which was added into Epilife-HKGS culture solution. The adherent cells were gained by cultured in saturated humidity conditions with5%CO2at37℃and the medium was changed every other day. Following3-4stable passages, the cells were obtained for further study.2. The siRNA transfection for C/EBPα:The siRNA sequence for C/EBPaproduced by QIAGEN Company. The transfection efficiency, interference efficiency and transfection concentration for the siRNA had been verified in the pretest. The keratinocytes in the logarithmic phase were inoculated into6-well plates with2×105 cells/well. When the primary keratinocytes were fused to70%, siRNA was transfected into the cells by lipofectamine2000according to the manual strictly, as siRNA transfection group. Meanwhile, the empty vector transfection group and non-transfection group were also established.3. The detection of the interferential efficiency for C/EBPa gene:Total RNA was extracted after24h of transfection. The mRNA expression of C/EBP a was detected by RT-PCR. Total protein was extracted after72h of transfection. The protein expression of C/EBPa was detected by western blot.4. The effect of C/EBPa gene silencing on the proliferation of keratinocytes by CCK-8colorimetric analysis:KC-C and KC cells in the logarithmic phase were digested, counted and then inoculated into96-well plates with5×103cells/well. The proliferation of cells was detected at12h,24h,48h and72h by CCK-8colorimetric analysis.5. The expressions of the differentiation mark proteins, CK10and involucrin, in keratinocytes:Total protein was extracted after72h of transfection. The protein expressions of CK10and involucrin were detected by western blot.Results:1. The expression of C/EBPa was inhibited effectively by siRNA:The mRNA expression of C/EBPa was inhibited by47%compared with those in control group72h after siRNA transfection and the expressions of C/EBP a protein were also down-regulated significantly(P<0.05).2. CCK-8colorimetric analysis was used to detect the effect of C/EBPa gene silencing on the proliferation of keratinocytes. There was no significant difference in the proliferation of keratinocytes in the siRNA transfection group, empty vector transfection group and non-transfection group after12h inoculating. However, after inoculating for24h,48h and72h, the proliferation of keratinocytes in the siRNA transfection group was significantly decreased with a statistical difference compared to the empty vector transfection group and non-transfection group.(P<0.05at24h,48h and72h, respectively)3. According to the results of western blot, the protein expressions of CK10and involucrin were significantly lower in the siRNA transfection group than in the empty vector transfection group and non-transfection group with a statistical difference (P<0.05).Conelusions:1. The siRNA for C/EBPa could inhibit the expression of C/EBPa in keratinocytes. After C/EBPa gene silencing, the proliferation rate of keratinocytes was increased and the differentiation level of keratinocytes was decreased.2. The C/EBPa gene was a potential gene to cure the diseases withgabnormal proliferation and differentiation in keratinocytes. Part Ⅲ:The effect of IL-22stimulation on MAPK signaling pathway and the expression of C/EBPa in keratinocytesObjective:1. To investigate the effect of IL-22on the phosphorylation of key proteins in MAPK signaling pathway in keratinocytes.2. To investigate the effect of IL-22on the gene expression of C/EBPa in keratinocytes.3. To investigate the effect of IL-22on the proliferation and differentiation of keratinocytes and its correlation with the gene expression of C/EBPa.Methods:1. The culture of primary cells referred to Part II.2. The stimulation of IL-22on keratinocytes:The primary cultured cells of keratinocytes were used as the research subjects. When the primary keratinocytes were fused to60%, human IL-22fusion protein was added into the cells with the final concentration of30ng/mL,60ng/mL,90ng/mL, respectively. And then the cells with treatment were collected at different time points for subsequent experiments.3. The detection of the phosphorylation level of key protein in MAPK signaling pathway by western blot:Total protein was extracted after72h of transfection. The protein expressions of JNK, p-JNK, p38, p-p38, ERK and p-ERK were detected by western blot.4. The effect of IL-22on the proliferation of keratinocytes by CCK-8colorimetric analysis:The keratinocytes cells in the logarithmic phase were digested, counted and then inoculated into96-well plates with5×103cells/well. Then, human IL-22fusion protein was added into every well with the final concentration of30ng/mL,60ng/mL and90ng/mL, respectively. The proliferation of keratinocytes was detected at12h,24h,48h and72h by CCK-8colorimetric analysis.5. The effect of IL-22on the expressions of the differentiation mark proteins, CK10and involucrin, in keratinocytes:After the stimulation of IL-22for48h, total protein was extracted. The protein expressions of CK10and involucrin were detected by western blot.6. The detections of the mRNA and protein expressions of C/EBPa by RT-PCR and western blot:After the stimulation of IL-22for48h, total RNA and total protein were extracted, respectively. The mRNA and protein expressions of C/EBPa were detected by RT-PCR and western blot, respectively.Results:1. The phosphorylation levels of JNK, ERK and p38were significantly increased by the stimulation of IL-22:After the stimulation of IL-22for60minutes, p-JNK, p-ERK and p-p38were all significantly increased compared with the control group with the statistical difference (P<0.05).2. The proliferation of keratinocytes was significantly increased by IL-22:According to the results of CCK-8colorimetric analysis, the cell proliferation was significantly increased, with a significant time and concentration dependent manner, from12h after the cells were inoculated into the plate.3. The differentiation of keratinocytes was significantly inhibited by IL-22:According to the results of western blot, the protein expressions of CK10and involucrin in keratinocytes were significantly inhibited by IL-22with a significant concentration-dependent manner.4. The mRNA and protein expressions of C/EBPa were significantly inhibited by IL-22:According to the results of RT-PCR and western blot, the mRNA and protein expressions of C/EBPa in keratinocytes were significantly inhibited by IL-22with a significant concentration-dependent manner.Conelusions:1. IL-22can significantly regulate the proliferation and differentiation of keratinocytes.2. IL-22can significantly regulate the MAPK signaling pathway and the expression of C/EBPa in keratinocytes.3. IL-22may regulate the proliferation and differentiation of keratinocytes through MAPK signaling pathway and the expression of downstream molecular, C/EBPa.