LMO4 Is A Disease-provocative Transcription Coregulator Activated By IL-23 In Psoriatic Keratinocytes

Background and Objective Psoriasis is a chronic autoimmune disorder of the skin.The most common histologic features of psoriatic skin lesions are epidermic hyperplasia and parakeratosis.Pathogenesis of psoriasis are mainly associated with environmental and genetic factors,which results in immune system abnormal,eventually leads to hyperproliferation and abnormal differentiation of keratinocytes(keratinocytes,KC).To date,the study of psoriasis are more concerned about genetic factors and immune abnormalities.The population with the genetic background of psoriasis,is affected by environmental factors such as stimulation or pathogen components,which excites immune cells secrete inflammatory cytokines such as IL-23,IL-1 beta and IL-6.These cytokines induce T cells activation and expansion to secrete IL-17,IL-22 and TNF-a,which are finally act on KCs and affect the proliferation and differentiation of keratinocytes.Among these cytokines,IL-23 plays a more significant role.It has been proved that T cells are related to activated IL-23 receptor /Jak2/(Akt)/STAT3 signaling pathway in skin tissue.The structure of normal skin can be divided into basal layer,spinous layer,granular layer and cornified layer.KCs are located in the basal layer,which regenerate the epidermis through proliferation and differentiation and the cycle of renewal is about 30 days.However,in the psoriatic lesions,the cytokines that secreted by the abnormal immune system acts on the KCs and promotes the proliferation and differentiation of the cells,which causes the terminal differentiation incomplete,epidermis thickening and a large amount of scales.Therefore,inflammatory factors activate KC and affect their proliferation and differentiation,which is the final stage of the development of psoriasis.However,the signal molecules regulating theproliferation and differentiation of KCs,signal pathways and target genes,especially the activation of intranuclear transcription factor,are rarely been studied.LMO4,as an important transcription factor in the nucleus,belongs to the LMO protein family.It mainly interacts with other transcription factors to form a multiprotein complex to regulate gene transcription.LMO4 plays an important role in cell fate determination,cell growth,differentiation,tissue and organ development.Studies have confirmed that LMO4 plays an important role in regulating skin epidermal formation and epidermal cell migration in the embryonic development.LMO4 deficient mice died of neural tube insufficiency closure,epidermal differentiation dysregulation and dyspoiesis of skin protective barrier.In normal skin tissues,LMO4 is mainly expressed in KC of basal layer.Therefore,LMO4 may play an important role in the proliferation and differentiation of KC,but it is not clear whether LMO4 is involved in the development of psoriasis.Meanwhile,signaling pathways and molecular mechanisms involved in regulation of LMO4 expression in KC have not been reported.Previously studies confirmed the expression of IL-23 in psoriatic lesions was remarkable higher than normal tissue.In the process of psoriasis,activation of STAT3 regulates KCs acquired of the immunocyte-like characteristics and secretes cytokines or inflammatory factors.Therefore,this study will focus on the last stage of the development of psoriasis,using the hyperproliferation and abnormal differentiation of KCs as a breakthrough-point,to explore the possible role of transcription factor LMO4 in the pathogenesis of psoriasis and its molecular mechanism.Revealing the influence of IL-23/Jak2/(Akt)/STAT3 signaling pathway activation on the expression of LMO4 in psoriatic keratinocytes and its interference methods,to provide new ideas for alleviating the local symptom of psoriatic lesions.Methods1.Histological level studying To investigate the correlation between the LMO4 and psoriasis,H&E staining of sections from tissues were used to observe the histological structure.The expression of LMO4 m RNA was detected by RT-q PCR;immunohistochemistry was used to detect the correlation between the expression of LMO4,Involucrin,Keratin 14 and psoriasis,and the relationship with keratinocyte differentiation.The changes of IL-23,phospho-Akt and phospho-STAT3 were detected to observe the activation of IL-23/Jak/Akt/STAT3 signaling pathway in psoriatic lesions.2.Cellular level studying The primary KCs or immortalized human KC lines were studied:(1)overexpressing or interfering with LMO4,MTT,EDU and three-dimensional culture were used to observe the effect of LMO4 expression on the proliferation of KCs.(2)Western blot and cell immunofluorescence were used to detect the changes of differentiation markers K14,K5 and IVL,and analyzed the effect of LMO4 expression on differentiation in KCs.(3)To explore the effect of activated state of IL-23/Akt/STAT3 signaling pathway on the proliferation and differentiation of KCs,the KCs were treated with recombinant cytokines of IL-23,and Western blot detected phospho-Akt,phospho-STAT3,LMO4,and the differentiation markers of K14,K5,IVL.The proliferation of KCs was analyzed by MTT and EDU.3.Molecular level research To investigate the molecular mechanism of LMO4 expression which regulated by activated IL-23/Jak2/(Akt)/STAT3 signaling pathway,the wild-type LMO4 promoter was PCR amplified from genomic DNA from Ha Cat cells,and inserted into the p GL3-Basic vector.Mutated the potential p-STAT3 binding sites in promoter region through PCR.The promoter activity of LMO4 was analyzed after exposure to IL-23.4.Animal models(1)Establishment of poriasiform animal model.To confirm that the activated IL-23/Jak2/(Akt)/STAT3 signaling pathway can promote the development of psoriasis via regulating LMO4 expression.Intradermal injection of IL-23 in the skin of ears of BALB/c mice induces psoriasiform changes.Observing and recording the changes of skin lesions and scales;measuring the thickness of skin,analyzing H&E staining of tissue structure.To confirm the psoriasiorm animal model was successfully established.IHC was used to detect LMO4,and analyzed the activity of p-Akt and p-STAT3.(2)To study the effect of LMO4 interference on psoriasiform lesions in mice.Lentiviral particles containing sh RNA of LMO4 was smeared in the lesions of mice.Observing and recording the changes of skin lesions and scales;measuring the thickness of skin,analyzing H&E staining of tissue structure;IHC was used to detect the expression of LMO4;the expression of IVL and K14 was detected by RT-q PCR.To study the therapeutic effect of LMO4 interference in vivo in inhibiting epidermal thickening and reducing scales.Results1.Histological studies confirmed that LMO4 and IL-23 were highly expressed in psoriatic lesions,and the IL-23/Jak2/(Akt)/STAT3 signaling pathway was activated.(1)H&E staining confirmed that the structure of psoriatic lesions samples were consistent with histological features of psoriasis.(2)RT-q PCR assays showed that LMO4 was expressed at a high level in all psoriatic lesions,whereas LMO4 was less expressed in perilesional tissues and could not be detected in normal skin.(3)The results of IHC further confirmed that LMO4 and IL-23 were highly expressed in psoriatic lesions at protein level,meanwhile p-Akt and p-STAT3 increased significantly in psoriatic lesions.(1)The results showed that LMO4 was only expressed in KCs which located in the basal layer of normal skin tissues.The expression of LMO4 increased in perilesional tissues compared to normal skin.However,LMO4 significantly increased in psoriatic lesions for whole epidermis.LMO4-positive cells indicated that the mean reached up to 45.91%,however in normal skin tissues the mean was only 9.21%.(2)Basal layer marker K14 and stratum corneum marker IVL were overexpressed in both dermis and epidermis tissues of psoriatic lesions,consistent with LMO4 overexpression in psoriatic lesions.(3)The results showed that IL-23 was highly expressed in psoriatic lesions compared with normal epidermis and perilesional tissues,and Akt and STAT3 were activated in the psoriatic lesions.2.Cell level studies confirmed that the expression of LMO4 was regulated by the activated IL-23/Akt/STAT3 signaling pathway and was involved in regulating the proliferation and differentiation of KCs.(1)KCs were treated with calcium(2.0 m M)to induce differentiation in vitro.Observing the morphological changes of cells,the cells had formed tight junctions,became compacted,and showed cobblestone-like morphology.Immunoblotting analysis showed that the expression of K5 and IVL increased because of calcium induction,LMO4 expression increased gradually,consistent with the pattern of K5 and IVL expression.(2)Overexpression LMO4 in KCs and cells were cultured in KGM without differentiation inducers,results showed that LMO4 overexpression induce KCs self differentiation.WB and IF showed that the expression of K5,K10 and IVL increased significantly after overexpression of LMO4.The results of MTT and EDU showed that LMO4 overexpression promotes the cells growth.(3)KCs were treated with IL-23(10 ng/ml)or calcium(2.0 m M)respectively to detect the expression changes of LMO4 and IL-23/Jak2/(Akt)/STAT3 signaling pathway.The results showed that calcium only increased the expression of LMO4.Immunoblotting showed that Akt and STAT3 were activated in KCs exposed to IL-23,coincident with upregulated LMO4 expression.KCs were respectively treated with the inhibitor of Jak2,Akt and STAT3 together with IL-23,resulting in a reduction of LMO4 expression,and alterations of p-Akt and p-STAT3 were not observed.(4)KCs were treated with IL-23(10 ng/ml),immunoblotting showed that IL-23 increased expression of IVL and K5,similar to the effects of calcium.The expression of LMO4 gradually increased after exposure to IL-23.The results of MTT and EDU showed that IL-23 promoted the proliferation of KCs.(5)Ha Cat cells were transduced with lentiviral particles containing sh RNA of LMO4 or sh RNA of a scrambled sequence of identical base composition to generate KC/sh RNALMO4 or KC/sh RNA-scramble cells.KCs were cultured in DMEM with calcium or IL-23.KC/sh RNA-LMO4 cells did not differentiate.Compared to KC/sh RNA-Scramble,the expression of IVL gradually decreased in DMEM with calcium of KC/sh RNA-LMO4,alterations of K14(K5)was not observed.KCs proliferation in the presence of IL-23 or calcium was significantly inhibited when LMO4 was silenced.KC/sh RNA-LMO4 cells could not form multicellular structures in three-dimensional organotypic culture systems.3.Molecular level research confirmed that IL-23/Jak2/(Akt)/STAT3 signaling pathway regulates the expression of LMO4 through p-STAT3 binding to LMO4 promoter.Bioinformatics analysis revealed that there were three potential p-STAT3 binding sites in the promoter region of the upstream of the LMO4 gene.The luciferase reporter gene recombinant vector was constructed by cloning wild type LMO4 promoter.After IL-23 treatment,the activity of luciferase was increased with the prolongation of the treatment time.After mutation of p-STAT3 binding site,the activity of LMO4 promoter did not change expose to IL-23.4.The study of psoriasiform mice model confirmed that the activation of IL-23/Jak2/(Akt)/STAT3 signaling pathway in the lesions consistent with LMO4 overexpression,and LMO4 interference can effectively reduce the thickness of lesions and inhibit cell differentiation.(1)IL-23 intradermal injection in the skin of ears of BALB/c mice to induce psoriasiform changes.Dermathemia and scales were observed in the site of injection.H&E staining of sections from ears of mice repeatedly injected with IL-23 revealed aceratosis,acanthosis,and dermal inflammatory infiltrates,same with human psoriatic lesions.Compared with the PBS-injected controls,ear thickness increased by more than 80 ?m after injecting IL-23.(2)Staining for p-Akt and p-STAT3 revealed very high counts of immunopositive cells.Immunohistochemistry showed that a higher numbers of LMO4-positive cells in hyperplastic epidermis,particularly in the nuclei of KCs from spinous layers to cornified layers.(3)Lentiviral particles containing sh RNA of LMO4 was smeared in the lesions of mice.Reduction of scales was observed.The increase in thickness of IL-23-injected ears was significantly inhibited by LMO4 sh RNA.H&E staining showed that the epidermis thickness decreased substantially after exposure to Lmo4 sh RNA compared with scrambled sh RNA.LMO4 staining of sections from IL-23-injected ears treated with Lmo4 sh RNA was significantly lower than that in ears treated with scrambled sh RNA.The expression of IVL in lesions that treated with LMO4 sh RNA was significantly lower than that in ears treated with scrambled sh RNA,and was consistent with the decrease of LMO4 expression.Conclusion1.The transcriptional factor LMO4 is highly expressed in the psoriatic lesions,regulates the proliferation and differentiation of KCs,which is associated with the increase of the thickness and the occurrence of scales.2.The activated IL-23/Jak2/(Akt)/STAT3 signaling pathway regulates the expression of LMO4 in KCs.The main molecular mechanism is the specific binding of LMO4 gene promoter with activated p-STAT3,which promotes the expression of LMO4.3.RNA interference technique can effectively inhibit the expression of LMO4,thus inhibit the proliferation and differentiation of KCs,and provide a new therapeutic targets for the treatment of psoriasis.