INF2 regulates oxidative stress-induced apoptosis in epidermal HaCaT cells by modulating the HIF1 signaling pathway

Biomedicine & Pharmacotherapy 111 (2019) 151–161

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INF2 regulates oxidative stress-induced apoptosis in epidermal HaCaT cells by modulating the HIF1 signaling pathway

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Zhixiong Chen, Chenyu Wang, Nanze Yu, Loubin Si, Lin Zhu, Ang Zeng, Zhifei Liu, Xiaojun Wang Department of Plastic Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, No. 1 Shuaifuyuan Wangfujing Dongcheng District, Beijing, 100730, China

A R T I C LE I N FO

A B S T R A C T

Keywords: INF2 HaCaT cell Oxidative stress HIF1 Mitochondria

Promoting epidermal cell survival in an oxidative stress microenvironment is vital for skin regeneration after burns and/or wounds. However, few studies have explored the mediators related to epidermal cell apoptosis in an oxidative stress microenvironment. Cellular viability was determined using the MTT assay, TUNEL staining, western blot analysis and LDH release assay. Two independent siRNAs were transfected into HaCaT cell to repress INF2 and/or HIF1 in the presence of H2O2. Mitochondrial function was determined using JC-1 staining, mitochondrial ROS staining, immunofluorescence staining and western blotting. In the present study, our data demonstrated that the expression of inverted formin-2 (INF2) increased rapidly when the cells were exposed to H2O2. Interestingly, INF2 knockdown promoted HaCaT cell survival via reducing H2O2-mediated cell apoptosis. Molecular investigations demonstrated that INF2 deletion attenuated mitochondrial ROS overloading, restored the cellular redox balance, sustained the mitochondrial membrane potential, improved mitochondrial respiratory function and corrected the mitochondrial dynamics disorder in an H2O2-mimicking oxidative stress microenvironment. In addition, INF2 deletion upregulated the expression of HIF1. Interestingly, the inhibition of HIF1 increased cell death and caused mitochondrial stress despite the deletion of INF2, suggesting that the HIF1 signaling pathway is required for INF2 deletion-mediated HaCaT cell survival and mitochondrial protection. Altogether, our results identified INF2 as a novel apoptotic mediator for oxidative stress-mediated HaCaT cell death via modulating mitochondrial stress and repressing the HIF1 signaling pathway. This finding provides evidence to support the critical role played by the INF2-HIF1 axis in regulating mitochondrial stress and epidermal cell viability in an oxidative stress microenvironment.

1. Introduction Burn injuries lead to the excessive death of epidermal cells and affect 500,000 young people in the United States per year. Mechanistically, an inflammatory microenvironment, hypoxia stimulus and oxidative stress function together to induce cell death in epidermal cells [1]. However, epidermal cell survival and migration are vital for skin regeneration [2]. Interestingly, despite major advances in our molecular understanding of epidermis cell biology, no studies have identified the primary factors responsible for epidermal cell survival [3]. In this study, hydrogen peroxide (H2O2) was used to mimic the oxidative stress microenvironment induced by a burn injury [4]. Then, HaCaT cells were used to determine the key apoptotic trigger of epidermal cells in an oxidative stress microenvironment. Recently, mitochondrial homeostasis has been reported to be at the center of cell fate [5]. Properly functioning mitochondria produce ATP

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and modulate the cell redox balance to ensure epidermis cell metabolism [6,7]. However, damaged mitochondria participate in apoptotic signal activation and amplification via multiple effects [8]. For example, injured mitochondria with a lower mitochondrial potential cannot convert energy substrates into ATP [9,10]. In addition, abnormal mitochondria with a hyperpermeable membrane leak excessive calcium into the cytoplasm [11,12], and this process mediates cellular calcium overloading [13]. Moreover, damaged mitochondria are a source of ROS, and uncontrolled oxidative stress obligates a cell to undergo death via mitochondrial apoptosis [14]. However, the role of mitochondrial homeostasis in HaCaT cells has not been explored. We investigated whether mitochondrial stress is an upstream inducer of HaCaT cell apoptosis in an H2O2-mediated oxidative microenvironment. At the molecular level, inverted formin-2 (INF2), a novel mitochondrial dynamic regulator, is associated with cardiac ischemia

Corresponding author. E-mail address: [email protected] (X. Wang).

https://doi.org/10.1016/j.biopha.2018.12.046 Received 2 November 2018; Received in revised form 10 December 2018; Accepted 12 December 2018 0753-3322/ © 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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2.3. ELISA

reperfusion injury via activating mitochondrial division [15,16]. Excessive mitochondrial division causes the uneven distribution of mitochondrial DNA into daughter mitochondria [17,18]. Functionally, the consequence of INF2 activation is followed by mitochondrial membrane potential collapse, mitochondrial oxidative stress and caspase-9 activation [19,20]. In addition, INF2 activation affects ER stress and mitochondrial calcium homeostasis. The pro-apoptotic effects of INF2 have been demonstrated in prostate cancer [21], brain ischemic stress [22] and protamine-induced kidney injury [23]. This information illustrates that INF2 functions in cell survival via modulating mitochondrial function [24,25]. However, this concept has not been tested in HaCaT cells. Hypoxia-induced factor 1 (HIF1) is a critical player in the survival strategy of stressed cells, including lung cancer [26], pancreatic cancer [27], renal cell carcinomas [28], and mesenchymal stem cells [29]. Interestingly, the pro-survival influence of HIF1 in HaCaT cells has also been reported. Activated HIF1 attenuates inflammation-mediated HaCaT cell death via the PI3K-Akt signaling pathway [30]. In addition, ultraviolet B-induced HaCaT proliferation is associated with HIF1 upregulation through the EGFR-Akt axis [31,32]. However, the roles of HIF1 in mitochondrial homeostasis and HaCaT survival under an oxidative microenvironment have not been adequately explained [33]. In addition, OPA1 is a mitochondrial protector in response to several stress responses, including inflammation stimulus [34], ischemia attack [35], and oxidative injury [36]. Activated OPA1 attenuates mitochondrial oxidative stress, sustains the mitochondrial membrane potential, and blocks the activation of mitochondrial apoptosis, thus sending a prosurvival signal to mitochondria [37–39]. Based on this information, we asked whether HIF1 maintains HaCaT survival and mitochondrial homeostasis in a manner that is dependent on OPA1 activity in an H2O2 stress-mediated microenvironment. Altogether, the aim of our study is to explore whether INF2 alterations are associated with HaCaT viability and whether INF2 modulates HaCaT survival via sustaining mitochondrial function by activating HIF1 in the context of an H2O2mediated microenvironment.

To analyze changes in caspase-9, caspase-9 activity kits (Beyotime Institute of Biotechnology, China; Catalog No. C1158) were used according to the manufacturer’s protocol. In brief, to measure caspase-9 activity, 5 μl of LEHD-p-NA substrate (4 mM, 200 μM final concentration) was added to the samples for 1 h at 37 °C. Then, the absorbance at 400 nm was recorded via a microplate reader to reflect the caspase-3 and caspase-9 activities. To analyze caspase-3 activity, 5 μL of DEVD-pNA substrate (4 mM, 200 μM final concentration) was added to the samples for 2 h at 37 °C. The levels of antioxidant factors, including GPX, SOD, and GSH, were measured with ELISA kits purchased from the Beyotime Institute of Biotechnology [42]. The experiments were performed in triplicate and repeated three times with similar results. 2.4. Immunofluorescence Cells were washed twice with PBS, permeabilized in 0.1% Triton X100 overnight at 4 °C. After the fixation procedure, the sections were cryoprotected in a PBS solution supplemented with 0.9 mol/l of sucrose overnight at 4 °C. The primary antibodies used in the present study were as follows: Tom20 (1:1,000, Abcam, #ab186735), and Smad2 (1:1,000, Abcam, #ab33875). Subsequently, samples were incubated with Alexa Fluor 488 donkey anti‑rabbit secondary antibody (1:1,000; cat. no. A‑21206; Invitrogen; Thermo Fisher Scientific, Inc.) for ∼1 h at room temperature. DAPI was used to label the nuclei, and images were captured using an inverted microscope (magnification, x40; BX51; Olympus Corporation, Tokyo, Japan) 2.5. Quantitative real-time PCR For mRNA expression analysis, total RNA was isolated using Trizol (Invitrogen, Carlsbad, California, USA) according to a previous study. Then, cDNA was synthesized using 1 mg RNA and the First-Strand Synthesis Kit (Fermentas, Flamborough, Ontario, Canada) according to a previous study [43]. The cycling conditions were as follows: 92 °C for 7 min, 40 cycles of 95 °C for 20 s and 70 °C for 45 s. β-actin was amplified as an internal standard. All the primer sequences are listed below: Drp1 (forward primer 5′- CATGGACGAGCTGGCCTTC-3′, reverse primer 5′-ATCCTGTAGTGATGTATCAGG-3′), Fis1 (forward primer 5′-TGTCCAGTCCGTAACTGAC-3′, reverse primer 5′-TTCGATAC CTGACTTAC-3′), Mff (forward primer 5′-ATGCAGACAATTAAGTGTGT TGTTGTGGGCGA-3′, reverse primer 5′-reverse primer, TCAT AGCAG CACACACCTGCGGCTCTTCTT-3′), Mfn2 (forward primer 5′-CCTCTTG ATCCTGATCTTAACGT-3′, reverse primer 5′-GGACTACCTGATTGTCA TTC-3′), and OPA1 (forward primer 5′-GCTACTTGTGAGGTCGATTC-3′, reverse primer 5′-GCCGTATACCGTGGTATGTCTG-3′).

2. Methods and materials 2.1. Cell culture and treatment HaCaT cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM supplemented with 10% FBS under 37 °C/5% CO2 conditions. To induce the oxidative stress models, different doses of H2O2 (0–1.0 mM) was added into the medium for 12 h [40]. To inhibit INF2 expression, two independent siRNAs against INF2 was transfected into HaCaT cells. In addition, HIF1 siRNAs were also transfected into INF2-deleted cells to represses HIF1 expression.

2.6. Mitochondrial potential analysis and detection of mPTP opening 2.2. Western blot analysis To observe the mitochondrial potential, JC-1 staining (Thermo Fisher Scientific Inc., Waltham, MA, USA; Catalog No. M34152) was used [44]. Then, 10 mg/ml JC-1 was added to the medium for 10 min at 37 °C in the dark to label the mitochondria. Normal mitochondrial potential showed red fluorescence, and damaged mitochondrial potential showed green fluorescence. The mPTP opening was measured via tetramethylrhodamine ethyl ester fluorescence according to a recent study [45].

The primary antibodies used in the present study were as follows [41]: Bcl2 (1:1000, Cell Signaling Technology, #3498), Bax (1:1000, Cell Signaling Technology, #2772), caspase9 (1:1000, Cell Signaling Technology, #9504), pro-caspase3 (1:1000, Abcam, #ab13847), cleaved caspase3 (1:1000, Abcam, #ab49822), survivin (1:1000, Cell Signaling Technology, #2808), complex III subunit core (CIII-core2, 1:1000, Invitrogen, #459220), complex II (CII-30, 1:1000, Abcam, #ab110410), complex IV subunit II (CIV-II, 1:1000, Abcam, #ab110268), Drp1 (1:1000, Abcam, #ab56788), Fis1 (1:1000, Abcam, #ab71498), Opa1 (1:1000, Abcam, #ab42364), Mfn2 (1:1000, Abcam, #ab56889), Mff (1:1000, Cell Signaling Technology, #86668), HIF1 (1:1,000, Abcam, #ab16066), INF2 (1:1000, Proteintech, catalog number 20466-1-AP) and Tom20 (1:1,000, Abcam, #ab186735). The experiments were performed in triplicate and repeated three times with similar results.

2.7. Mitochondrial ROS analysis Flow cytometry was applied as a quantitative method for evaluating mitochondrial ROS levels according to a previous study. Cells were seeded onto 6-well plates and then treated with erlotinib. Subsequently, the cells were isolated using 0.25% trypsin and then incubated with MitoSOX red mitochondrial superoxide indicator (Molecular Probes, 152

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following study. Meanwhile, two independent siRNAs against INF2 were transfected into HaCaT cells, and the knockdown efficiency was confirmed via western blotting shown in Fig. 1E and F. Then, cell viability was determined using an LDH release assay. Compared to the control group, H2O2 treatment increased the LDH levels in the medium (Fig. 1G), indicating cell membrane breakage and cell death. Interestingly, INF2 knockdown could repress the H2O2-mediated LDH release in HaCaT cells (Fig. 1G). These results were further supported by flow cytometry analysis using Annexin V/PI staining (Supplemental figure). Subsequently, cell death was observed using a TUNEL assay. Compared to the control group, the percentage of TUNEL-positive cells increased rapidly upon exposure to H2O2 (Fig. 1H and I). However, the loss of INF2 reduced the number of TUNEL-positive cells, suggesting that INF2 deletion preserves HaCaT cell survival in an oxidative microenvironment. This finding was further supported by analyzing the expression of cleaved caspase-3, caspase-8 and its substrate cleaved PARP. As shown in Fig. 1J–M, compared to the control group, the expression of cleaved caspase-3 and caspase-8 were significantly upregulated in response to H2O2 treatment; this effect was followed by an increase in cleaved PARP. However, the H2O2-mediated caspase-3/8 activation was abolished by INF2 silencing, reconfirming that INF2 deletion sustained HaCaT cell viability in the setting of oxidative injury.

USA) for 30 min in the dark at 37 °C. Subsequently, PBS was used to wash cell two times, and then the cells were analyzed with a FACS Calibur Flow cytometer. Data were analyzed by FACS Diva software. The experiment was repeated three times to improve the accuracy. 2.8. TUNEL staining and MTT assay Apoptotic cells were detected with an In Situ Cell Death Detection Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA; Catalog No. C1024) according to the manufacturer’s protocol. Briefly, cells were fixed with 4% paraformaldehyde at 37 °C for 15 min. Blocking buffer (3% H2O2 in CH3OH) was added to the wells, and then cells were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. The cells were incubated with TUNEL reaction mixture for 1 h at 37 °C. DAPI (Sigma-Aldrich, St. Louis, MO, USA) was used to counterstain the nuclei, and the numbers of TUNEL-positive cells were recorded [46]. MTT was used to analyze the cellular viability. Cells (1 × 106 cells/well) were cultured on a 96-well plate at 37 °C with 5% CO2. Then, 40 μl of MTT solution (2 mg/ml; Sigma-Aldrich) was added to the medium for 4 h at 37 °C with 5% CO2. Subsequently, the cell medium was discarded, and 80 μl of DMSO was added to the wells for 1 h at 37 °C with 5% CO2 in the dark. The OD of each well was observed at A490 nm via a spectrophotometer (Epoch 2; BioTek Instruments, Inc., Winooski, VT, USA) [47].

3.2. INF2 regulates mitochondrial function

2.9. Transfections

Image intensity was quantified using Nikon NIS-Elements-AR software. ImageJ software (NIH, MD, USA) was used to quantify the protein levels on western blots. Data were analyzed using one-way ANOVA followed by Tukeyʼs test for post hoc analysis. Data are presented as the means ± S.E.M., and differences were deemed significant when P < 0.05.

Mitochondrial function is closely associated with cell homeostasis. Mitochondria are the source of cellular ROS. Interestingly, the levels of mitochondrial ROS were significantly increased in H2O2-treated cells, as assessed using a mitochondrial ROS probe (Fig. 2A and B). However, the superfluous mitochondrial ROS could be neutralized via silencing INF2 (Fig. 2A and B). This finding was supported by an analysis of the levels of cellular antioxidants. As shown in Fig. 2C–E, compared to the control group, the levels of GSH, GOD and GPX rapidly decreased in response to H2O2 treatment, indicating a redox imbalance. Interestingly, the loss of INF2 reversed the levels of GSH, GOD and GPX (Fig. 2C–E). In addition to mitochondrial ROS overloading, the mitochondrial pro-apoptotic factor cyt-c was liberated from the mitochondria into the cytoplasm/nucleus, as assessed via immunofluorescence (Fig. 2F and G). Interestingly, INF2 deletion repressed mitochondrial cyt-c translocation. After liberation into the cytoplasm, cyt-c can activate the mitochondrial apoptotic pathway in a manner dependent on caspase-9. Thereby, western blotting was used to analyze the expression of mitochondrial apoptosis-related proteins. As shown in Fig. 2H–L, compared to the control group, H2O2 treatment elevated the expression of caspase-9 and Bax. Interestingly, the levels of Bcl-2 and survivin correspondingly decreased in response to H2O2 treatment. Interestingly, INF2 deletion repressed the expression of caspase-9/Bax and upregulated the levels of Bcl-2/survivin. Altogether, this information suggested that mitochondrial apoptosis is positively regulated by INF2 in an oxidative stress microenvironment.

3. Results

3.3. INF2 induces mitochondrial dynamics disorder

3.1. INF2 deletion prevents H2O2-induced apoptosis in HaCaT cells

Recent studies have reported that mitochondrial apoptosis and function are highly modified by mitochondrial dynamics, including mitochondrial fission and fusion. Excessive mitochondrial fission and repressed fusion produce massive mitochondrial fragmentation, impairing mitochondrial metabolism and activating mitochondrial apoptosis pathway. Thus, we explored whether mitochondrial dynamics disorder was connected to INF2 activation in an H2O2-induced oxidative stress microenvironment. First, mitochondrial morphology was observed by immunofluorescence assay using the mitochondrial specific antibody Tom 20. Then, the average length of mitochondria was recorded. As shown in Fig. 3A and B, compared to the interconnective mitochondria in the control group, oxidative stress promoted the

For siRNA transfection, cells were seeded onto 6-well plates and grown until they reached 50% confluent. Then, the medium was replaced with Opti-MEM medium according to a previous study. Subsequently, two independent siRNAs against HIF1 and two independent siRNAs against INF2 were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol [6]. After transfection for 48 h, the cells were isolated, and the transfection efficiency was determined by western blotting. The siRNAs sequences were as follows: siRNA1 against HIF1 (si1HIF1), 5′-CCTAUAGUUATUCAATUC-3′; siRNA2 against HIF1 (si2HIF1), 5′-CGTTAGTTCTGAATUATT-3′; The siRNAs sequences were as follows: siRNA1 against INF2 (si1-INF2), 5′- TTGTCCATTGCAAGGCC TCTGATT GAGTCTG -3′; siRNA2 against INF2 (si2-INF2), 5′- CTAATG CGTGCAATACGTGCGTCCTATATG-3′; siRNA control (si-ctrl), 5′-GCTT ACTTUTCTATACCTTUAT-3′. 2.10. Statistical analysis

First, different doses of H2O2 were added into the medium of HaCaT cells for 12 h to induce an oxidative microenvironment. Then, cell viability was measured via the MTT assay (Fig. 1A). Compared to the control group, cell viability was progressively reduced with an increase in H2O2 concentration. This alteration was closely followed by an increase in the expression of INF2, as assessed by qPCR (Fig. 1B) and western blotting (Fig. 1C and D). These results indicated that H2O2mediated oxidative injury is associated with INF2 upregulation. Notably, the minimum concentration of H2O2 to cause damage to HaCaT cells was 0.3 mM (Fig. 1A–C), and this concentration was used in the 153

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Fig. 1. INF2 deletion sustains HaCaT cell viability in an H2O2-induced oxidative stress microenvironment. A. HaCaT cells were incubated with different doses of H2O2, and then cell viability was determined via MTT assay. B. After treatment with different doses of H2O2, the transcription of INF2 was determined using a qPCR assay. C and D. Western blotting analysis of INF2 in response to different concentrations of H2O2. E and F. HaCaT cells were incubated in H2O2 (0.3 mM) for 12 h to induce oxidative stress. Two independent siRNAs against INF2 were transfected into HaCaT cells to prevent H2O2mediated INF2 upregulation. The knockdown efficiency was confirmed via western blotting. G. LDH release assay was used to evaluate cell apoptosis in response to INF2 knockdown. H–I. TUNEL staining of apoptotic cells. The number of TUNEL-positive cells was recorded. HaCaT cells were treated with 0.3 mM H2O2 and/or transfected with INF2 siRNA. J–L. Western blotting analysis for cleaved caspase-3 and cleaved PARP. HaCaT cells were treated with 0.3 mM H2O2 and/or transfected with INF2 siRNA. *p＜0.05.

proteins were rapidly downregulated in H2O2-treated cells (Fig. 3D–M), indicating blunted mitochondrial fusion in an oxidative stress microenvironment. Interestingly, the loss of INF2 reversed the expression of pro-fusion proteins and suppressed the activation of pro-fission factors in HaCaT cells (Fig. 3D–M). Altogether, the above data identified INF2 as a major regulator of mitochondrial dynamics via balancing mitochondrial fission and fusion.

formation of mitochondrial fragmentations, an effect that resulted in a decrease in mitochondrial length. Interestingly, the mitochondrial network was be sustained with INF2 deletion, which also restored the average length of mitochondria (Fig. 3A and B). This information indicated that H2O2 exposure resulted in damage to the mitochondrial structure. Subsequently, qPCR and western blotting were employed to analyze the expression of proteins related to mitochondrial dynamics, including mitochondrial fission and fusion. As shown in Fig. 3D–M, compared to the control group, H2O2 treatment elevated the levels of pro-fission proteins in HaCaT cells, suggesting that mitochondrial fission is activated in response to oxidative stress. In contrast, the levels of pro-fusion

3.4. INF2 affects mitochondrial energy metabolism The primary function of mitochondria is to produce ATP for cellular metabolism. Interestingly, ATP production was drastically reduced in 154

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Fig. 2. INF2 modulates mitochondrial function including mitochondrial oxidative stress and mitochondrial apoptosis. A and B. Mitochondrial ROS was evaluated via flow cytometry. HaCaT cells were treated with 0.3 mM H2O2 and/or transfected with INF2 siRNAs. C–E. The concentration of cellular antioxidants were measured via ELISA in HaCaT cells that were treated with 0.3 mM H2O2 and/or transfected with INF2 siRNAs. F and G. Immunofluorescence assay of cyt-c translocation into the cytoplasm/nucleus. H–L. HaCaT cells were treated with 0.3 mM H2O2 and/or transfected with INF2 siRNAs. Then, western blotting was used to observe the alterations in mitochondrial apoptosis, including caspase-9, Bax, survivin and Bcl-2. *p＜0.05.

assessed using JC-1 staining. This result was accompanied with a decrease in the expression of the mitochondrial respiratory complex, suggesting that oxidative stress impairs mitochondrial respiratory function. Interestingly, the loss of INF2 maintained the mitochondrial membrane potential and reversed the activity of the mitochondrial respiratory complex (Fig. 4D–G). These results indicated that INF2

HaCaT cells after exposure to H2O2 (Fig. 4A). However, the loss of INF2 maintained the ATP content in HaCaT cells despite treatment with H2O2. At the molecular level, mitochondria convert the mitochondrial membrane potential into ATP with the help of the mitochondrial respiratory complex. Interestingly, the mitochondrial membrane potential was rapidly dissipated under H2O2 stimulus (Fig. 4B and C), as 155

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Fig. 3. Mitochondrial dynamics are controlled by INF2 in the setting of H2O2-induced oxidative injury. A and B. Immunofluorescence assay for mitochondrial morphology using the mitochondria-specific antibody Tom 20. The average length of mitochondria in HaCaT cells that were treated with 0.3 mM HaCaT and/or transfected with INF2 siRNAs was recorded. C–H. After treatment, RNA was isolated from HaCaT cells, and then qPCR was used to analyze the transcription of proteins related to mitochondrial fission/fusion. I–M. HaCaT cells were treated with 0.3 mM H2O2 and/or transfected with INF2 siRNA. Then, western blotting was used to determine the expression of mitochondrial fission proteins and mitochondrial fusion factors. *p＜0.05.

in an H2O2-induced oxidative stress microenvironment.

deletion permits mitochondrial energy metabolism in an H2O2-induced oxidative stress microenvironment. Subsequently, we observed mitochondrial glucose metabolism via measuring the remaining glucose content and lactic acid production in the medium. As shown in Fig. 4H and I, compared to the control group, glucose uptake was downregulated in H2O2-treated cells, an effect that was followed by a decrease in lactic acid production. Interestingly, INF2 deletion promoted glucose uptake and therefore enhanced lactic acid generation, suggesting that INF2 deletion sustained mitochondrial energy metabolism

3.5. INF2 represses the HIF1-OPA1 signaling pathway HIF1 is a pro-survival mediator for HaCaT cells under different stress conditions, and OPA1 is a novel mitochondrial defender that modulates mitochondrial dynamics and mitochondrial oxidative stress. In the present study, we asked whether HIF1 and OPA1 are involved in H2O2-mediated HaCaT apoptosis and mitochondrial dysfunction. 156

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Fig. 4. INF2 governs mitochondrial respiratory function and energy metabolism. A. Cellular ATP production was determined in HaCaT cells that were treated with 0.3 mM H2O2 and/or transfected with INF2 siRNA. B and C. Mitochondrial membrane potential was observed using a JC-1 probe. The red-to-green fluorescence intensity was used to quantify the mitochondrial membrane potential. D–G. After treatment, proteins were isolated from HaCaT cells, and then western blotting was used to analyze the expression of the mitochondrial respiratory complex. H–I. ELISA assay was used to quantify glucose uptake and lactic acid production in HaCaT cells that were treated with 0.3 mM H2O2 and/or transfected with INF2 siRNAs. *p＜0.05 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

INF2-deleted cells to repress HIF1 expression, and the knockdown efficiency was confirmed via western blotting (Fig. 5A–C). In response to HIF1 downregulation, the expression of OPA1 was inhibited, as evaluated via western blotting (Fig. 5A–C) and immunofluorescence (Fig. 5D–F). Altogether, these data established the regulatory effect of INF2 on the HIF1-OPA1 signaling pathway under oxidative stress injury.

Western blotting analysis demonstrated that HIF1 and OPA1 expression were downregulated in response to H2O2 treatment (Fig. 5A–C), and this effect could be inhibited by INF2 deletion, suggesting that oxidative stress repressed HIF1 and OPA1 via INF2. These results were confirmed via immunofluorescence, which demonstrated a parallel decrease in HIF1 and OPA1 upon H2O2 2 stimulus (Fig. 5D–F). However, INF2 deletion reversed the fluorescence intensity of HIF1 and OPA1. Subsequently, we investigated whether HIF1 is the upstream regulator of OPA1. Two independent siRNAs against HIF1 were transfected into

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Fig. 5. The HIF1-OPA1 signaling pathway is regulated by INF2 in an H2O2-induced oxidative stress model. A–C. Western blotting was used to establish the regulatory effects of INF2 on HIF1 and OPA1. HaCaT cells were treated with 0.3 mM H2O2 and transfected with INF2 siRNA and/or HIF1 siRNA. Then, OPA1 expression was measured by western blotting. D–F. Immunofluorescence assay of HIF1 and OPA1. HaCaT cells were transfected with two independent siRNAs against HIF1 to repress INF2-mediated HIF1 activation. The knockdown efficiency was verified via immunofluorescence assay. *p＜0.05.

in a manner that is dependent on the HIF1 signaling pathway in an oxidative stress microenvironment.

3.6. Loss of the HIF1 signaling pathway abolishes the INF2 deletionmediated cell protection in an H2O2 oxidative microenvironment Although we confirmed the inhibitory influence of INF2 on the HIF1-OPA1 singling pathway, whether the HIF1 signaling pathway is associated with HaCaT cell survival and mitochondrial protection is unknown. First, an MTT assay and LDH release assay were used to detect cell viability after silencing HIF1. Compared to the control group, H2O2-mediated cell apoptosis could be reversed by INF2 deletion (Fig. 6A and B). Interestingly, the loss of HIF1 re-activated cell death despite the knockdown of INF2, suggesting that HIF1 is required for INF2 deletion-mediated cell survival (Fig. 6A and B). Mitochondrial function was analyzed via detecting mitochondrial ROS production and ATP levels. Compared to the control group, mitochondrial ROS was significantly increased in response to H2O2 treatment (Fig. 6C), and this effect was inhibited by INF2 deletion. Interestingly, the loss of HIF1 caused mitochondrial ROS overloading in INF2-deleted cells (Fig. 6C), suggesting that HIF1 is necessary for maintaining the cell redox balance. Moreover, ATP production was repressed by H2O2 (Fig. 6D), while INF2 deletion reversed ATP generation via the upregulation of HIF1 expression. Caspase-9 activity was measured to reflect mitochondrial apoptosis. As shown in Fig. 6E, H2O2-mediated caspase-9 activation could be abolished by INF2 deletion, and this effect was highly dependent on HIF1 expression because the loss of HIF1 resulted in caspase-9 activation in HaCaT cells. Altogether, our results indicated that INF2 modulates HaCaT cell apoptosis and mitochondrial function

4. Discussion Enhancing epidermal cell survival is key to treating burns and wounds. After burn and skin tissue damage, uncontrolled inflammation stress, abnormal oxidative injury and chronic hypoxia stimulus impair epidermal cell viability and subsequently blunt cell migration, reducing the ability of the skin to repair itself. Although several studies have used stem cell transplantation and/or biomaterials to promote wound healing, little attention has been paid to identifying the critical mediator that regulates epidermal cell apoptosis in the harsh oxidative stress microenvironment. In the present study, we used a loss-of-function assay to verify the pro-apoptotic effect of INF2 on HaCaT cells in an H2O2-induced oxidative stress microenvironment. We demonstrated that the expression of INF2 was rapidly upregulated with an increase in the concentration of H2O2. However, the loss of INF2 attenuated H2O2mediated HaCaT cell apoptosis, and this process was modulated via maintaining mitochondrial function, correcting mitochondrial dynamics disorder and promoting mitochondrial energy metabolism. At the molecular level, the mitochondrial stress was regulated by INF2 via the HIF1-OPA1 signaling pathways. The inhibition of HIF1 abolished the beneficial effect of INF2 deletion on HaCaT cell survival and mitochondrial function. Overall, our research provides evidence to demonstrate the pro-apoptotic mechanism exerted by INF2 via repressing 158

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Fig. 6. The HIF1-OPA1 signaling pathway is involved in HaCaT cell apoptosis and mitochondrial damage. A. The MTT assay was used to evaluate the viability of HaCaT cells that were treated with 0.3 mM H2O2 and transfected with INF2 siRNA and/or HIF1 siRNAs. B. LDH release assay for HaCaT cell death. HaCat cells were treated with 0.3 mM H2O2 and transfected with INF2 siRNA and/or HIF1 siRNAs. C. Flow cytometry analysis of mitochondrial ROS production in HaCaT cells that were treated with 0.3 mM H2O2 and transfected with INF2 siRNA and/or HIF1 siRNAs. D. ATP production was measured in HaCaT cells in response to HIF1 deletion. E. ELISA analysis of caspase-9 activity in HaCat cells that were treated with 0.3 mM H2O2 and transfected with INF2 siRNA and/or HIF1 siRNAs. *p＜0.05.

HIF1 attenuates ER stress in type II alveolar epithelial cells, promotes the cell cycle transition in colorectal cancer, enhances glycolysis and ATP production, inhibits mitochondrial fission in pancreatic cancer [27,58], affects the response of gastric cancer to radiation therapy, attenuates intestinal reperfusion stress and suppresses inflammation injury in cancer stem cells. In HaCaT cells, HIF1 is associated with skin repair via attenuating inflammation injury, enhancing epidermal cell response to hypoxia stress and inhibiting the sensitivity of skin to ultraviolet stress [59]. In the present study, we found that HIF1 is downregulated in an oxidative stress microenvironment. However, the re-introduction of HIF1 via silencing INF2 promoted HaCaT cells survival and maintained mitochondrial function. This finding was similar to those of previous studies in which HIF1 attenuated mitochondrial stress via repressing mitochondrial fission [27], improving mitochondrial oxygen metabolism, neutralizing ROS, and regulating the inflammation response. Furthermore, we found that increased HIF1 led to the upregulation of OPA1, a novel mitochondrial protector. Structurally, OPA1 inhibits mitochondrial network fragmentation and promotes mitochondrial fusion, contributing to the removal of damaged mitochondria and communication between mitochondria [60]. Higher expression of OPA1 protects mitochondrial function and metabolism in several types of cells [61]. In the present study, we observed that OPA1 expression was positively regulated by HIF1. However, the detailed role of OPA1 in oxidative stress-induced HaCaT cell survival and mitochondrial protection have not been elucidated in the current study [62]. Additional studies using an OPA1 overexpression assay are required to uncover its beneficial effects on HaCaT cell mitochondrial homeostasis in the setting of an oxidative stress microenvironment. Taken together, our results identified INF2 as a novel pro-apoptotic inducer of oxidative injury-challenged epidermal HaCaT cells. Increased INF2 amplified the oxidative injury signal to mitochondria via closing the HIF1-OPA signaling pathway, finally leading to mitochondrial stress and cell apoptosis. Strategies to repress INF2 expression and reverse the HIF1-OPA1 axis are vital for maintaining

the HIF1-OPA1 axis in HaCaT cells in an oxidative stress microenvironment. This finding lays a foundation to identify potential mediators of epidermal cell viability during oxidative injury. INF2 belongs to the formin family that regulates actin and microtubule dynamics [48]. INF2 interacts with filamentous actin [49]. The expression of INF2 is relatively high in immune cells, neurons, and many epithelial cells, highlighting that INF2 might be particularly important in those cell types. Previous studies have suggested the regulatory effects of INF2 on the cellular actin structure. Increased INF2 promotes actin filament assembly [50]. In HeLa cells, the overexpression of INF2 induces the formation of actin stress fibers, and excessive fiber production is closely associated with cell death and impaired cell migration [51]. In addition, INF2 interacts with the endoplasmic reticulum (ER) and consequently promotes actin filament accumulation around the ER [52], an effect that is accompanied by ER stress and cellular calcium imbalance. The role of INF2 in activating fatal mitochondrial fission has been confirmed by several careful studies [22]. In the present study, we found that INF2 expression was upregulated after exposure to H2O2, suggesting that oxidative stress is an upstream regulator of INF2 [53]. This finding was similar to a recent study in which cerebral ischemia reperfusion elevates the expression of INF2 in vivo and in vitro. Subsequently, we confirmed that H2O2-activated INF2 primarily induced mitochondrial stress, as evidenced by mitochondrial dysfunction, mitochondrial dynamics disorder and mitochondrial metabolism collapse. Notably, previous studies focused on the regulatory effects of INF2 on mitochondrial dynamics, especially mitochondrial fission [54,55]. Our study provides ample evidence to validate the influence of INF2 on mitochondrial apoptosis, mitochondrial oxidative stress and mitochondrial metabolism [56,57]. However, whether mitochondrial fission is required for INF2-mediated mitochondrial stress remains unknown, and more research is required to explore this question. At the molecular level, HIF1 is a hypoxia-related protective factor. Several biological stress processes are regulated by HIF1. Increased 159

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epidermal cell viability in an oxidative injury microenvironment. However, there are several limitations in the present study. First, we only used siRNA to perform the loss-of-function assay for INF2. More researches are required to conduct the gain-of-function assay for INF2 using adenovirus transfection. The recuse experiments would provide more evidences for the role of INF2 in cell death. Besides, only cell experiments were carried out in the current study. Additional studies using animal models are necessary to support our finding.

[11]

[12]

[13]

Funding

[14]

Not applicable. [15]

Availability of data and materials [16]

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

[17]

Ethics approval and consent to participate Not applicable.

[18]

Consent for publication

[19]

Not applicable. [20]

Competing interests [21]

The authors declare that they have no competing interests. Acknowledgments

[22]

Not applicable. [23]

Appendix A. Supplementary data [24]

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2018.12.046.

[25]

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