Effects Of Keratinocyte KRT5 On Melanocyte Function Through Notch Signaling Pathway

ObjectiveKRT5 gene is a pathogenic gene for Dowling-Degos disease(DDD),but the mechanism of KRT5 expressed only in keratinocytes leads to pigmentation abnormalities is unknown.In this study,HaCaT cells,MNT-1 melanoma cells,human primary melanocytes,and lesions of DDD patients were selected as research objects,and lentivirus shRNA and CRISPR/Cas9 technology were used to construct HaCaT cell models of KRT5 gene silencing and site-directed mutation.By co-culture with MNT-1 melanoma cells and primary melanocytes,RNA-seq were constructed by high-throughput sequencing,and differential proteins related to Notch signaling pathway and melanin metabolism were screened to explore the changes.Notch inhibitors and agonists were used to confirm the changes of important molecules related to melanin metabolism.The Notch signaling pathway and molecules of melanin metabolism were verified in the skin lesions of DDD patients with KRT5 gene mutation,to clarify mechanism of DDD pigment abnormality caused by KRT5 gene mutation.Methods1.HaCaT cells with altered expression of KRT5 were obtained by lentivirus shRNA and CRISPR/Cas9,and the expression levels of Notch ligand and Notch downstream were detected by qPCR and WB.2.HaCaT cells with altered expression of KRT5 were co-cultured with primary melanocytes or MNT-1 melanoma cells,respectively.RNA-seq were constructed by high-throughput sequencing,and changes of Notch signaling pathway and melanin-metabolisation-related molecules were detected by qPCR and WB.The melanin production was detected by NaOH lysis,transmission electron microscopy and immunofluorescence.3.Notch signaling pathway inhibitor DAPT or sJAG1 peptide and activator valproic acid(VPA),and lentivirus overexpression of N1ICD molecules were used to intervene Notch signaling pathway when primary melanocytes or MNT-1 melanoma cells were cultured or co-cultured with HaCaT cells with KRT5 gene modification.qPCR,WB and immunofluorescence were used to verify the impact on melanin metabolism of melanocytes.4.In lesions of DDD patients with KRT5 gene mutation,immunohistochemical methods were used to verify the changes of Notch signaling pathway and melanin production-related molecules.Results1.In HaCaT cell model of KRT5 knockdown by lentivirus shRNA,KRT5 mRNA in all interference groups was significantly decreased(P<0.05),the relative expression of KRT5 protein decreased in all interference groups,and Sh-KRT5-3 decreased most obviously.Jag1 and N1ICD expression were decreased in sh-KRT5-3 group.In HaCaT cell model with c.1delA mutation of KRT5 gene constructed by CRISPR/Cas9,Sanger sequencing showed that heterozygous mutation(c.1delA)occurred in initiation codon of first exon of KRT5 gene in experimental group,the expression of KRT5 protein in experimental group was decreased,and the expression of Jag1,N1ICD and Hes1 decreased.2.HaCaT cell model of KRT5 knockdown by lentivirus shRNA were co-cultured with MNT-1 melanoma cells for 72h,and MNT-1 cells were obtained by flow cytometry for transcriptomic sequencing.KEGG analysis showed a trend of difference between Notch signal and melanin metabolism.Hesl mRNA in MNT-1 cells in experimental group was lower than that in control group,and the difference was statistically significant(P<0.05).The expression of N1ICD protein and Hesl protein decreased in experimental group.Expression of Fascin1 mRNA in experimental group was significantly lower than that in control group,with statistical significance(P<0.01).The expression of MITF,TYR,TYRP1,TYRP2,MYO5A and Rab27A protein increased,and Fascin1 protein decreased in experimental group.Transmission electron microscopy showed stage 3 and 4 melanosomes were increased in MNT-1 cells co-cultured with sh-KRT5.Expression of N1ICD decreased and Pmel17 increased in HEMn cells co-cultured with KRT5 c.1delA HaCaT cells.3.In HEMn cells stimulated with 20μM sJAG1,the expression levels of N1ICD and Hesl decreased,TYR increased,and Fascin1 decreased.In HEMn cells treated with 10μM DAPT,the expression levels of N1ICD and Hesl decreased,TYR increased,and Fascin1 decreased.When 0.1mm VPA was added in co-culture system of HaCaT cells and MNT-1 cells in sh-KRT5 group,the expressions of MITF,TYR and Rab27A in the selected MNT-1 melanoma cells decreased,while the expression of Fascinl reversed to a similar level to that of sh-NC group.In HEMn cells co-cultured with KRT5 c.1delA HaCaT cells,fluorescence expression intensity of Pmel17 in 0.1 mM VPA group was close to control group.4.In lesions of the two DDD patients with KRT5 gene mutation,immunohistochemical showed the expression of Notch ligand Jagl decreased,N1ICD and Hesl decreased,Fascinl decreased,while Pmel17 increased.Conclusion1.The HaCaT cell models of KRT5 silenced by lentivirus and KRT5 haploid deficiency with c.1delA were successfully constructed.Keratinocyte KRT5 affected the expression of N1ICD and transcription factor Hesl by interfering the expression of Notch ligand Jag1.2.The alteration of KRT5 in keratinocytes led to the increase of melanin synthesis by inhibiting Notch signaling pathway and activating pigment production-related proteins TYR,TYRP1,TYRP2 and MITF,and affects melanin transport by affecting Fascin1.3.Both sJAG1 and DAPT promoted melanin production and inhibit melanin transport by inhibiting the Notch signaling pathway of melanocyte.Activation of Notch signaling by VPA reverses the effects of keratinocyte KRT5 alteration on melanin metabolism in melanocytes.Overexpression of N1ICD activated melanin synthesis and affect melanin transport.4.Keratinocyte KRT5 led to abnormal melanin metabolism by influencing Notch signaling pathway.