4‐octyl itaconate improves the viability of D66H cells by regulating the KEAP1‐NRF2‐GCLC/HO‐1 pathway

Abstract As a novel nuclear factor E2‐related factor 2 (NRF2) activator, the itaconate has shown significant therapeutic potential for oxidative stress diseases. However, its role in Vohwinkel syndrome in relation to the gap junction protein beta 2 (GJB2) mutation is still unclear. This study aimed at investigating the effect of 4‐octyl itaconate (OI) on HaCaT and D66H cells and clarify its potential mechanism in vitro. The optimal concentration and treatment time of OI on HaCaT cells and D66H cells were determined by CCK‐8 and LDH experiments. The effect of OI on cell proliferation was detected by EdU staining and FACS analysis of PI, while the apoptosis was evaluated by TUNEL staining and FACS analysis of Annexin V. The ROS staining was performed, and the levels of SOD, MDA, GSH and GSH/GSSG were detected to evaluate the effect of OI on oxidative damage induced by D66H‐type mutation. CO‐IP, Western blot, immunofluorescence and qPCR analyses were employed to detect the activation of KEAP1‐NRF2‐GCLC/HO‐1 pathway by OI. Finally, sh‐NRF2 was used to confirm the activation of this pathway by OI. Results showed that OI could improve the cell viability decreased by GJB2 gene mutation by regulating the balance between cell growth and apoptosis induced by oxidative damage. Furthermore, this alleviation process was regulated by the KEAP1‐NRF2‐HO‐1/GCLC pathway. In conclusion, OI could improve the viability of HaCaT and D66H cells via regulating the KEAP1‐NRF2‐GCLC/HO‐1 pathway, which provided a wide spectrum of potential targets for effective therapeutic treatments of Vohwinkel syndrome in the clinic.

is a deafness-related variant associated with Connexin-26 (Cx-26), which is the gap connection protein responsible for signal transmission and material exchange between adjacent cells and encoded by the gap junction protein beta 2 (GJB2) gene. [7][8][9][10][11] To date, a group of five GJB2 gene mutation sites have been reported to cause the classical VS, include p.Asp66 His (D66H), p.Gly130 Val (G130V), p.Gly59 Ser (G59S), p.Tyr65 His (Y65H) and p.Arg75 Gln (A75H), with the p.Asp66 His (D66H) as the first mutation type discovered and defined. [12][13][14][15] The human immortal keratinocyte line (HaCaT) cells are the well-established human immortalized keratinocyte in vitro model based on D66H-type mutation with a high survival rate and are commonly used for genetic and molecular investigations of skin diseases. 9 Studies have shown that the deletion or mutation in GJB2, which is also an oxidative stress-related gene, would activate cellular oxidative stress mainly by downregulating the nuclear factor E2-related factor 2 (NRF2) pathway. 16 As a transcription factor, the NRF2 plays a crucial role in the inflammatory responses and oxidative stress. [17][18][19] Under normal physiological conditions, the NRF2 is inactive by binding to Kelch-like ECH-associated protein l (KEAP1) in the cytoplasm. 20 With the presence of either endogenous or exogenous stimuli, the NRF2 is dissociated from KEAP1 and transferred into the nucleus to regulate the target genes, such as heme oxygenase 1 (HO-1), glutamate-cysteine ligase catalytic subunit (GCLC) and NAD(P) H quinone dehydrogenase 1 (NQO1), ultimately regulating the production of glutathione (GSH). 20,21 These findings imply that NRF2 is an indispensable signalling factor that regulates oxidative stress to affect cell fate with the GJB2 dysfunctions. Previous studies have shown that deletion or mutation of the GJB2 gene cause the oxidative stress to accelerate apoptosis in skin cells. 16,22,23 Therefore, the NRF2 could be a promising target in the treatment of hereditary skin disease caused by the GJB2 dysfunction.
As an intermediate metabolite of the tricarboxylic acid (TCA) cycle, the itaconate has been revealed with antioxidant effects by attenuating the intracellular succinate dehydrogenase (SDH) activity. 20 Recent studies have shown that the itaconate could promote the alkylation of KEAP1, thereby promoting the dissociation of NRF2 from KEAP1, leading to the activation of NRF2. 20 Itaconate belongs to the α, β unsaturated carboxylic acids with electrophilicity, which enables itaconate to interact with proteins containing sulphur groups at the cellular level, a process known as an electrophilic stress response (ESR). 24 Due to the ESR, the 4-Octyl itaconate (OI) contains an ester group, which makes the OI penetrate the cells at a high transformation rate without the assistance of transporting protein. 20 Because itaconate could activate NRF2, OI has also been shown to play an antioxidant role to improve the cell survival. 20 For example, OI could improve the LPS-induced chondrocyte inflammation and attenuate the H2O2-induced neuronal reactive oxygen species (ROS) generation and lipid oxidation. 25,26 Furthermore, OI could also improve the prognosis in prebrain/ liver ischemia-reperfusion injury and inhibit UVB-induced oxidative stress in melanocytes and keratinocytes. [27][28][29] These studies have revealed the sound antioxidant effects and protection of OI.
However, the role of OI in the destructive keratoderma has not been studied; in particular, its involvement in the NRF2 signalling pathway is still unknown.
We hypothesize that OI can prevent VS induced by the D66Htype mutation and may mediate the protection via activating the KEAP1-NRF2-GCLC/HO-1 signalling pathway based on study findings of OI in other disease models. This study aimed at investigating the effect and mechanism of OI on D66H-type mutation-induced VS in vitro to explore the potentially effective diagnosis and therapeutic treatment of VS in the clinical settings.

| Cell culture and treatments
The HaCaT cells were purchased from Sunncell (SNL-163, Wuhan, China

| CCK-8 assay
In the 96-well culture plates, 1.0 × 10 4 HaCaT or D66H cells were seeded per well and cultured for 12 h to cover 75% of the area of the bottom of the well. The cells were treated with OI for 24 h. Then, the cells were grown in 100 μL of complete medium for 1 h at 37°C and supplemented with 10 μL of CCK-8 working fluid (CKO4, DOJINDO Laboratory). The cytotoxicity rate was determined by measuring the absorption rate of the sample at 450 nm using a microplate reader (Multiskan GO, Thermo Scientific).

| Lactate dehydrogenase (LDH) assay
The absorbance of different groups of cells at 490 nm was measured by the LDH Cytotoxicity Assay Kit (C0016, Beyotime Biotechnology) with the relative content of intracellular lactate dehydrogenase (LDH) calculated. The data were normalized based on the HaCaT Control group (HC).

| Cell proliferation assay
In the 24-well culture plates, the cells covering 75% of the entire

| TUNEL staining
The One Step TUNEL Apoptosis Assay Kit (MA0224, Meilunbio) was used to measure the percentage of dead cells in HaCaT or D66H cultures by following the manufacturer's instructions. The cells were permeabilized with proteinase K at a concentration of 20 g/mL in order to enhance the efficacy of the staining, which was then followed by the application of DAPI and TUNEL staining. The confocal fluorescence microscope (TCS SP8, Leica) was used to capture the images, and ImageJ was used to determine the fluorescence counts on the data (Version: 2.0.0-rc-69/1.52p, http://imagej.net/Contr ibutors).

| Reactive oxygen species (ROS) assay
The cells were stained for 30 min with DCFH-DA dye in a 5% CO2 in-

| Intracellular superoxide dismutase (SOD) assay
An Enhanced BCA Protein Assay Kit was used to measure the protein content of cell lysates (P0009, Beyotime Biotechnology). The absorbance of each sample was measured at 520 nm, and the enzymatic activity of SOD was calculated according to the protocol provided by the Total Superoxide Dismutase Assay Kit with WST-8 (S0101S, Beyotime Biotechnology).

| Lipid peroxidation assay
The Enhanced BCA Protein Assay Kit was first used to measure the protein content of cell lysates (P0009, Beyotime Biotechnology).
After the treatment with Malondialdehyde (MDA) working solution, the sample was heated in a 100°C water bath for 10 min by following the instructions of the Lipid Peroxidation MDA Assay Kit (S0131S, Beyotime Biotechnology). The absorbance of each sample was then determined at 532 nm. Standard curve analysis was used to determine the MDA levels in the samples.

| GSH/GSSG measurements
In the 96-well culture plates, 1.0 × 10 4 HaCaT or D66H cells were seeded per well and cultured for 12 h to cover 75% of the bottom area of each well. Each sample was treated with 50 ul of GSH working solution for 5 min at room temperature. The GSH content and the GSH/GSSG ratio were determined based on the absorbance at 412 nm and a GSH and GSSG assay kit (S0053, Beyotime Biotechnology).

| Co-immunoprecipitation (CO-IP)
A total of 600 μg or more protein lysates per sample were collected. The lysates were precleared with control agarose resin in the Pierce Co-Immunoprecipitation (Co-IP) Kit (26,149, Thermo SCIENTIFIC). Then, the Keap1-binding protein was captured by either anti-Keap1 antibody (4678, CST, 1:50) or protein IgG beads, and the KEAP1-NRF2 immune complexes were detected by Western blot analysis.

| Western blot analysis
The HaCaT or D66H cells were cultured at a density of 2.0 × 10 6 cells per dish in 100 mm Cell Culture Dishes. The cellular proteins were extracted using RIPA buffer (R0020, Solarbio) containing 1% (v/v) PMSF (P0100, Solarbio). An Enhanced BCA Protein Assay Kit was used to measure the protein content (P0009, Beyotime Biotechnology). The instructions of the Nuclear Protein Extraction Kit (R0050, Solarbio) were strictly followed for the extraction of cytoplasmic and nuclear proteins. The protein sample as an equal amount was denatured and separated by SDS-PAGE electrophoresis and transfected into 0.2 μm PVDF (ISEQ 00010, Merck Millipore).
The primary and secondary antibody working fluids were prepared according to the instructions of the antibody manual and incubated at 4°C overnight and at room temperature for 2 h, respectively. The PVDF was exposed after staining with the ECL Kit (WBKLS0500, Merck Millipore). ImageJ software was used to quantify the grey

| Immunofluorescence staining
After the treatment with OI, the HaCaT and D66H cells were fixed by 4% PFA (P1110, Solarbio) for 30 min. Prior to the blocking with 5% BSA or serum homologous to the secondary antibody, the 0.3% Triton X-100 (T8200, Solarbio) was permeabilized for

| Quantitative Real-time PCR (qPCR)
The total cellular RNA was extracted using TRIzol (15,

| Statistical analysis
Each experiment was repeated with three or more biological replicates. Data were presented as mean ± standard deviation (SD) with the one-way analysis of variance (anova) and independent t-test performed to determine the significant difference based on p < 0.05, using the Graph Pad Prism (Version: 9.4.0 (453), https://www.graph pad-prism.cn/).  Figure 1G). These results indicated that OI ameliorated the G1 phase arrest caused by the D66H-type mutation.

| OI inhibits apoptosis induced by GJB2 gene mutation
The

| OI prevents oxidative damage implemented by GJB2 gene mutation
The fluctuating levels of oxidative stress were detected in both HaCaT and D66H cells either with or without the presence of OI.
The D66H-type mutation induced excessive cell ROS production ( Figure 3A,B). The D66H cells exhibited the increased levels of both SOD and lipid peroxidation ( Figure 3C,D). Simultaneously, the relative content of GSH and the GSH/GSSG ratio was reduced by the D66H mutation ( Figure 3E,F)

| OI activates the KEAP1-NRF2-HO-1/GCLC signalling pathway in HaCaT cells
The Co-IP analysis was performed to verify the alternation of KEAP1-NRF2 interaction by OI treatment (Figure 4A,B). The whole input protein was detected by Western blot, showing that OI could stimulate the stable expression of protein NRF2 in HaCaT cells without affecting the expression of KEAP1 ( Figure 4A,C).
Following the OI administration, no discernible change of NRF2 expression was detected in cytoplasm, while a considerable increase in NRF2 expression was observed in the nucleus ( Figure 4D,E).
The immunofluorescence also revealed that NRF2 protein moved into the nucleus after the OI stimulation ( Figure 4G). Furthermore, the results of qPCR analysis revealed no discernible difference in mRNA levels of NRF2 between the control and the OI-treated samples ( Figure 4F).

| Knockdown of NRF2 attenuates the protective effect of OI on cell proliferation arrest, apoptosis and oxidative damage caused by GJB2 gene mutation
The sh-RNA was transfected into both wild-type and D66H-type cells to further verify the above findings. The NRF2 (−/−) was further evaluated based on sh-71 due to its promising results derived from the Western blotting and qPCR analyses ( Figure 6A-C). Blocking NRF2 prevented it from working with or without the presence of OI.
Most D66H mutant cells were detected in the G1 phase ( Figure 6D,E), whereas the protection of OI from apoptosis and oxidative damage caused by the D66H-type mutation disappeared ( Figure 6F-I). These results confirmed that the NRF2 signalling pathway was indispensable for the protective effect of OI on GJB2 mutation-induced cell necrosis, apoptosis and oxidative damage.

| DISCUSS ION
It has been reported that the GJB2 gene mutation (e.g. D66H, c.196G>C) could cause VS in many regions of the skin. 1,9,30 However, the explicit molecular mechanisms regulating the formation of the skin disease and their potential therapeutic treatments are still rarely reported, especially in the typical palmar keratoderma skin disease.
Our study is the natural extension based on our previous study to explore the possible relationship between the GJB2 gene mutation (i.e. D66H, c.196G>C) and palmar keratoderma skin disease, and the potential treatments of this skin disease. 31 Numerous studies have shown that the intercellular communication has a major impact on intracellular and extracellular signal transduction, ultimately determining the cell fate. This intercellular communication mainly relies on the junction protein, that is the CX26, which regulates the balance between apoptosis and proliferation to determine the survival of early cells. 32,33 This process would be possibly explained by oxidation, which was revealed in the hair cell damage caused by GJB2 gene mutations. 34 These results were consistent with the findings revealed in our study, showing that the D66H mutation could promote oxidative damage that disturbed the balance between apoptosis and proliferation in keratinized epithelial cells. These results indicated the promising therapeutic prospects of OI for the treatment of skin diseases caused by GJB2 mutations.
Moreover, the activation of the NRF2 signalling pathway could be a potential approach to protecting skin cells from damage. Previous studies have revealed that OI is a fat-soluble small-molecule chemical that can penetrate cells without transporters, and keratinocytes are positioned on the skin's surface and may directly touch foreign medications. 24 This gives OI the possibility to apply now to skin lesions to achieve therapeutic function, avoiding the discomfort Formal analysis (supporting); methodology (supporting). Li Zhang: Conceptualization (lead); project administration (lead); supervision (lead); validation (lead).

ACK N O WLE D G E M ENTS
This study was supported by Natural Science Foundation of Shandong Province (Grant Number: ZR2019MH029).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that there are no conflicts of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analyzed in this study can be obtained from the corresponding author on reasonable requirements.