Effects of Pemphigus Vulgaris Serum on the Expression of ATP2C1 and PKP3 in HaCaT Cells

Qiao-Lin Pan, Zhi-Min Xie, Xiang-Nong Dai, Yi Zhang, Xu-Cheng Shen, Qing-Qing Li,Xing-Dong Ye,?

1Department of Dermatology, Institute of Dermatology, Guangzhou Medical University, Guangzhou, Guangdong 510095, China;2Department of Dermatology, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510799,China;3Department of Dermatology, Guangzhou Institute of Dermatology, Guangzhou, Guangdong 510095, China.

Abstract Objective:To investigate the effects of serum frompatients with pemphigus vulgaris (PV) on the transcription and protein protein expression level of calcium-transporting ATPase type 2C member 1(ATP2C1)and plakophilin 3(PKP3)in HaCaT cells.Methods: The HaCaT cells were divided into four groups: PV sera group, anti-Dsg3 monoclonal antibody group(AK23, positive control group), normal healthy serum group, and blank cell group.The groups were treated with corresponding different conditions for 24 hours.Quantitative polymerase chain reaction and Western blot were used to detect mRNA and protein levels of ATP2C1 and PKP3.Results:Compared with the blank group,the mRNA level of the ATP2C1 and PKP3 genes in PV sera group was significantly increased by 384% and 404%, respectively (both P <0.001).The treatment of PV sera and anti-Dsg3 antibody increased PKP3 protein expression(P = 0.03 and P = 0.004)but decreased protein expression of ATP2C1 in HaCaT cells (both P <0.001).Conclusions: Our study indicates that serum from patients with PV promotes both ATP2C1 and PKP3 transcription in HaCaT cells, implying that the two genes may be involved in the pathological process of PV.

Keywords: pemphigus vulgaris, pathogenesis, ATP2C1, PKP3, desmoglein

Introduction

Pemphigus vulgaris (PV) is a chronic autoimmune blistering skin disease related to the development of serum autoantibodies (AuAbs) to desmogleins (Dsg) of keratinocytes,including both Dsg1 and Dsg3.1–3In recent years,proteomics techniques have identified multiple targets of pemphigus autoimmunity.The multiple-hit theory of the pathogenesis of pemphigus indicates that various AuAbs to keratinocyte membrane proteins cause the development of epidermal vesicles by synergistic action with Dsg-IgG antibodies.4The calcium-transporting ATPase type 2C member 1 gene (ATP2C1) encodes the Ca2+/Mn2+transporter.Mutation of this gene is associated with intracellular calcium homeostasis and is considered an important cause of familial benign chronic pemphigus.5

Kalantari-Dehaghiet al.4reported that ATP2C1 antibody was found in the serum of patients with PV and that ATP2C1 may therefore be involved in the pathogenesis of PV.Plakophilin(PKP)3 is a desmoplakin that can form an intermediate fiber network with various cytokeratins.PVIgG-induced cytoskeletal disruption is associated with PKP3 and the formation of an intermediate fiber network by combination with various keratin proteins.Data regarding the effects ofATP2C1andPKP3gene mRNA transcription and protein expression on the presence of serum from patients with PV in cultivating medium are limited.We herein describe our investigation of the effects of the sera of patients with PV on ATP2C1 and PKP3 expression in HaCaT cells.

Materials and methods

Study design

Four experimental groups were established in this study:(i)the normal control group (Con), in which only 5% fetal bovine serum (FBS)-containing Dulbecco modified Eagle medium(DMEM)was used;(ii)the normal healthy serum group (NH), in which 5% healthy serum was used in DMEM;(iii) the PV serum group (PV), in which 5% PV serum was used in DMEM;and (iv) the anti-Dsg3 monoclonal antibody group (AK23, positive control group), in which 5% FBS DMEM with 2 μg/mL pathogenic AK23 was used.

Participants and serum collection

We enrolled seven patients with active PV at the Guangzhou Institute of Dermatology from July 2016 to December 2017.PV was diagnosed based on comprehensive clinical and histological examination results and immunological studies showing intraepidermal intercellular IgG and/or C3 deposits by direct immunofluorescence.The inclusion criteria for patients with PV were no use of immunosuppressive agents or glucocorticoids in the last 30 days, no other autoimmune diseases, and the absence of pregnancy in female patients.The study was approved by the Guangzhou Institute of Dermatology Research Ethics Committee (No.201802).

After obtaining informed consent, 10 mL of whole blood was collected from the seven patients with PV,and normal control serum was collected from three healthy volunteers.We mixed the serum specimens of the seven patients with PV to reduce experimental error.The anti-Dsg antibody concentrations in the mixed sera were 169 U/mL of Dsg1 and 117 U/mL of Dsg3;the anti-AK23, Dsg antibody titers in the healthy volunteers’ sera were negative.

Antibodies and reagents

HaCaT cells (immortalized human keratinocyte line) was from Guangzhou Yeshan Biological Technology Co.,Ltd.,Guangzhou,China,FBS,high-glucose DMEM,penicillin/streptomycin, and phosphate-buffered saline were supplied by HyClone(Logan,UT,USA).Antibodies included PKP3(Abcam,Cambridge,MA,USA),GAPDH(Abcam),ATP2C1 (ABclonal Biotechnology, Inc., Wuhan, China),and pathogenic pemphigus mouse monoclonal antibody AK23 (MBL, Tokyo, Japan).

Cell culture and treatments

HaCaT cells were used to establish anin vitromodel of PV according to a previous study.6Cells were cultured in highglucose DMEM supplemented with 1% penicillin/streptomycin and 10% FBS and maintained in a humidified atmosphere containing 5% CO2at 37 °C.The medium was changed every 24 hours until the cultures reached 70% to 80% confluency.HaCaT cells were treated with different mediums as described above according to the study design,and the cells were cultivated for another 24 hours.

Detection of mRNA transcription of ATP2C1 and PKP3 genes

After extracting total RNA by Trizol reagent,complementary DNA was synthesized using a reverse transcription kit and then subjected to qPCR with specific primers(Table 1).The qPCR was performed using SYBR?Premix Ex TaqTM(Tli RNase H Plus)on a QuantStudioTM6 Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA,USA).Briefly, 10 μL of mixed buffer (2×), 0.5 μL of forward primer,0.5 μL of reverse primer,4 μL of distilled water, and 5 μL of complementary DNA template were added to form a 20 μL reaction system;a two-step PCR amplification procedure was then conducted.The initial denaturation was performed at 95 °C for 30 seconds,followed by 40 cycles at 95 °C for 3 seconds and 60 °C for 34 seconds,and elongation at 60 °C for 1 minute.

Table 1Primer sequences included in the study.

Western blot analysis of ATP2C1 and PKP3 expression in HaCaT cells

Cell lysates for western blot analysis were prepared 24 hours after treatment.Cells were collected and subsequently lysed with lysis buffer from radioimmtmoprecipitation assay (Beyotime, Jiangsu, China) with phenylmethylsulfonyl fluoride (1 mmol/L) (Beyotime).The protein amount was determined using the bicinchoninic acid method(Thermo Fisher Scientific).Electrophoresis and western blotting were conducted according to standard procedures.Then,the membranes were incubated with primary antibodies specific for anti-ATP2C1(1:1,000 dilution), anti-PKP3 (1:20,000 dilution) at 4 °C overnight.The horseradish peroxidase-conjugated goat anti-rabbit antibody (ABclonal) was added for 1 hour at room temperature after washing the membranes with Trisbuffered saline +0.1% Tween 20.Membranes were developed using an enhanced chemiluminescence system(NCM Biotech, Suzhou, China).Western blots were analyzed by measuring the integrated density of bands after background subtraction using ImageJ.

Statistical analysis

The mRNA expression levels ofATP2C1andPKP3genes were compared with theGAPDHgene as a reference.Therelative qPCR detection of relative quantitation was calculated as 2-ΔΔCт.Comparison of the relative mRNA expression and protein expression among the different intervention groups was performed by one-way analysis of variance followed by Bonferroni correction using Graph-Pad Prism 5 (GraphPad Software, La Jolla, CA, USA).Differences were deemed significant when the calculatedPvalue was <0.05.The data are expressed as mean ±standard deviation.

Results

Characteristics of the patients with PV

Seven PV patients were included this study, including three males and four females with an average age of 50 ± 14.3 years (range from 32 to 72 years), and the course of disease was from 2 to 75 weeks,with an average of 10.4 ± 7.4 weeks.

APT2C1 and PKP3 mRNA expressions

Sera obtained from PV patients promoted mRNA transcription of theATP2C1andPKP3genes in HaCaT cells.When compared with the Con group,the relative mRNA transcription levels of the two genes increased by 384% and 404%,respectively.When compared with the NH group, the PV serum promotedATP2C1andPKP3mRNA expression by 218% and 241%,respectively(Fig.1 and Table 2).

Table 2ATP2C1 and PKP3 transcription levels in HaCaT cells under different medium conditions.

Figure 1. Comparison of mRNA levels of ATP2C1 and PKP in HaCaT cells after PV serum incubation.?P <0.05,??P <0.01, and???P <0.001.ATP2C1: ATPase type 2C member 1;PKP:plakophilin;Con: normal control group;NH: normal healthy serum group;PV: pemphigus vulgaris.

Effects of PV serum on protein expression of APT2C1 and PKP3

The Western blot results showed that the protein expression of PKP3 in the PV group was significantly increased after the addition of PV serum (P= 0.03vs.Con,P= 0.015vs.NH).The positive group showed similar results (P= 0.004vs.Con,P= 0.02vs.NH).There is no difference between PV group and positive group(1.39 ± 0.18vs.1.53 ± 0.16,P>0.05).However,ATP2C1 was significantly decreased in the NH group,PV group,and positive control group compared with the Con group (P<0.001) (Fig.2).

Discussion

This is the first study to exploreATP2C1andPKP3mRNA expression in HaCaT cells under the effects of serum from patients with PV.This preliminary study showed that 5% PV sera simultaneously enhanced mRNA level ofATP2C1andPKP3genes in HaCaT cells compared with normal healthy controls.Similarly, NH sera also increased transcription ofATP2C1andPKP3mRNA by 76% and 67%, respectively, when compared with the Con group, but without statistical significance;this may have been the result of cytotoxicity from cellular oxidative stress due to the presence of complement components in normal healthy sera.As mentioned above,the effect of complement cytotoxicity cannot be excluded and probably offset or partly contributed to the role of PV serum onATP2C1expression.However,the effect of anti-Dsg3 antibody on the expression of these two genes in HaCaT cells was also obvious.Conversely, anti-Dsg1 antibodies did not increase the expression of PKP3 in the present study(data not shown).The addition of sera from patients with PV increased PKP3 protein expression in HaCaT cells but decreased ATP2C1 protein expression(Fig.2).Many complicated and varied post-transcriptional mechanisms are involved in turning mRNA into protein.Transcription and protein expression are out of sync,and protein expression may lag behind mRNA expression;more in-depth research on this topic is required.

Figure 2. ATP2C1 and PKP3 expression levels in HaCaT cells under different medium conditions.(A)The expression of ATP2C1,PKP3,and GAPDH was measured by Western blot.(B)Statistically significant relationships are indicated by?P <0.05,??P <0.01,and???P <0.001.ATP2C1: ATPase type 2C member 1;PKP3: Plakophilin 3;Con: normal control group;NH: normal healthy serum group;PV: pemphigus vulgaris.

The effect of PV serum on ATP2C1 and PKP3 expression in HaCaT cells have not been reported.This study preliminarily showed that the serum of patients with PV could promote the transcription ofATP2C1andPKP3genes at similar levels.However, the correlation between the expression level of ATP2C1 or PKP3 and the serum Dsg antibody titer is not completely clear, and further studies are needed.

PKPs belong to the p120ctn superfamily, containing a repetitive ARM domain7that is abundant in desmoadhesomes and interacts with PKPs.PKPs includes PKP1,PKP2,PKP3,PKP4,etc.,7and are essential for maintainingthe strength of desmosome connections,and PKP-deficient HaCaT cells show reduced membrane availability and binding frequency of Dsg1 and Dsg3 at their cell borders.8PKP3 is a catenin that links two adjacent porphyrin molecules, which are further linked to both Dsg and desmocollin to construct a desmosome unit.9PKP3 mediates both desmosome assembly and E-cadherin maturation through Rap1 GTPase, and PKP3 deficiency disrupts an E-cadherin/Rap1 complex required for adherent junction sealing.9Therefore, PKP3 may be closely related to the pathogenesis of pemphigus.10The present work confirmed that the transcription and protein expression of PKP3 in HaCaT cells was significantly upregulated when incubated with serum from patients with PV.

TheATP2C1gene is a pathogenic gene of chronic familial benign pemphigus (HHD).Proteomics studies have shown that patients with pemphigus have specific antibodies to ATP2C1.2The serum level of AuAbs to the secretory pathway Ca2+/Mn2+-ATPase isoform 1 is correlated with the disease stage of PV.1As non-Dsg antibodies,anti-ATP2C1 AuAbs are likely also involved in the pathogenesis of pemphigus.Although patients with HHD have also been reported to have AuAbs, such as desmocollin 1,desmocollin 2,11and Dsg antibody,12these AuAbs may be caused by the capture of Dsg protein by an antigen-presenting cell in the epidermis of patients with HHD.Therefore,the presence of these antibodies may be a result of HHD rather than a cause of comorbidity.

Taking the mouse anti-PKP3 antibody as a positive control,Lambertet al.10reported for the first time that the expression of PKP3 increased in non-basal membrane bullous skin disease by HEK293 cellsin vitrowhen incubated with serum from patients with PV, and anti-PKP3 antibodies were present in the serum of both patients with paraneoplastic pemphigus and those with pemphigus.Lianget al.13reported that sera from patients with PV decreased the transcription and protein expression level of Dsg3 in HaCaT cells.Until now,however,the effect of PV serum on the expression of ATP2C1 and PKP3 in HaCaT cells had not been studied.

In recent years,research has shown that AuAbs to other transmembrane protein components of keratinocytes(such as PKP3 and ATP2C1)are also present in the serum of patients with PV,except for Dsg AuAbs.14This further supports the multiple-hit theory of the pathogenesis underlying pemphigus,15which suggests that various AuAbs of keratinocytes cause blister formation by synergistic action with Dsg-IgG antibodies.The present study has indicated that PV serum or anti-Dsg antibody may positively regulate theATP2C1andPKP3genes and PKP3 protein expression.

Although the present work provides new insight into improving the pathogenesis of pemphigus, it has obvious limitations.First, because of concern regarding the negative effect of temperature on antibodies at the same time, serum complement was not inactivated before the study.Second,the titer of Dsg AuAbs in PV serum had not been tested individually before the experiment.Hopefully,this work represents the beginning of a new field of study regarding vital and life-threatening autoimmune bullous skin disease.

Source of funding

The study was supported by Scientific Research Plan from Guangzhou Science and Technology Bureau (No.201904010352).