Downregulation of OPN3 Gene Induces Ferroptosis, Apoptosis, and Pyroptosis of Human Dermal Fibroblasts In Vitro

OPN3 is a G protein-coupled receptor (GPCR) that serves various light-dependent and light-independent functions in human skin cells. These functions include the regulation of ultraviolet A-induced photoaging in dermal fibroblasts and the pigmentation of epidermal melanocytes (Lan et al., 2020Lan Y. Wang Y. Lu H. Opsin 3 is a key regulator of ultraviolet A-induced photoageing in human dermal fibroblast cells.Br J Dermatol. 2020; 182: 1228-1244Google Scholar, Ozdeslik et al., 2019Ozdeslik R.N. Olinski L.E. Trieu M.M. Oprian D.D. Oancea E. Human nonvisual opsin 3 regulates pigmentation of epidermal melanocytes through functional interaction with melanocortin 1 receptor.Proc Natl Acad Sci U S A. 2019; 116: 11508-11517Google Scholar). GPCRs, which constitute the largest family of membrane proteins, are involved in nearly all physiological processes, including cell survival and death (Kamps and Coffman, 2005Kamps A.R. Coffman C.R. G protein-coupled receptor roles in cell migration and cell death decisions.Annals of the New York Academy of Sciences. 2005; 1049: 17-23Google Scholar). Our previous study demonstrated that OPN3 downregulation triggers apoptosis in melanocytes through the mitochondrial pathway of apoptosis, even in the absence of light stimulation (Wang et al., 2020Wang Y. Lan Y. Lu H. Opsin3 Downregulation Induces Apoptosis of Human Epidermal Melanocytes via Mitochondrial Pathway.Photochem Photobiol. 2020; 96: 83-93Google Scholar). This suggests that OPN3 plays a significant role in determining cell survival and death. In this study, we have also discovered that OPN3 is involved in ferroptosis, apoptosis, and pyroptosis of primary normal human dermal fibroblasts (NHDFs) in vitro. To investigate whether OPN3 is associated with cell survival and death in NHDFs, we evaluated the cell proliferation activity and transcriptomics of NHDFs. Following OPN3 knockdown, a significant reduction in cell proliferation activity was observed starting from 24 hours compared to the control group (Figure S1-2). To further explore the role of OPN3 in NHDFs' viability, RNA sequencing analysis of NHDFs in response to low expression of the OPN3 gene revealed the identification of both ferroptosis and apoptosis pathways, as depicted in Figure S3. Iron plays a pivotal role in initiating intracellular lipid peroxidation and ferroptosis. The intracellular labile iron pool and Fe2+ act as reservoirs of redox-active iron, directly promoting the generation of free radicals that initiate oxidative damage. Both iron overload and the accumulation of reactive oxygen species (ROS) are central factors in cell ferroptosis (Jiang et al., 2021Jiang X. Stockwell B.R. Conrad M. Ferroptosis: mechanisms, biology and role in disease.Nat Rev Mol Cell Biol. 2021; 22: 266-282Google Scholar). To further evaluate whether OPN3 mediates ferroptosis, we observed a significant increase in the expression of transferrin receptor (TFRC) and nuclear receptor coactivator 4 (NCOA4, termed ferritinophagy) in NHDFs successfully infected with LV-RNAi-OPN3, compared with the control group. Simultaneously, the expression of ferritin (ferritin heavy chain, FTH, and ferritin light chain, FTL) significantly decreased, along with lower levels of the iron export factor ferroportin (FPN) (Figure 1a), indicating an enhanced capacity for iron uptake. These results revealed that both iron storage and iron export capacities were compromised in LV-RNAi-OPN3 infected NHDFs, leading to cellular iron overload. Moreover, a significant increase in unstable Fe2+ levels was observed in OPN3-deficient NHDFs. Conversely, the viability of LV-control-RNAi cells was rescued, and Fe2+ levels were partially restored by adding liproxstatin-1 in vitro (Figure 1b-c), suggesting that intracellular iron homeostasis was altered, triggering ferroptosis in NHDFs. Glutathione peroxidase 4 (GPX4), a key mediator of ferroptosis, is the sole enzyme responsible for catalyzing glutathione (GSH) to reduce lipid peroxides in mammals (Rochette et al., 2022Rochette L. Dogon G. Rigal E. Zeller M. Cottin Y. Vergely C. Lipid Peroxidation and Iron Metabolism: Two Corner Stones in the Homeostasis Control of Ferroptosis.Int J Mol Sci. 2022; 24Google Scholar). Knockdown of the OPN3 gene resulted in decreased expression of GPX4 in NHDFs. Additionally, the expression of Acyl-CoA synthetase long-chain family member 4 (ACSL4), an essential component of fat metabolism involved in driving ferroptosis by affecting lipid peroxidation products, was upregulated after OPN3 knockdown in NHDFs (Figure 1a). OPN3 knockdown increased intracellular total ROS and lipid ROS levels in NHDFs, and these phenotypes were attenuated by liproxstatin-1 (Figure 1d-e). These results suggest that the accumulation of intracellular lipid peroxides ultimately leads to ferroptosis in NHDFs. Furthermore, changes in mitochondrial morphological characteristics were observed under transmission electron microscopy (TEM), including reduced mitochondrial volume, increased membrane density, and even the disappearance or reduction of mitochondrial cristae (Figure 1f). Taken together, these findings reveal that the knockdown of the OPN3 gene in NHDFs drives ferroptosis-induced cell death. Interestingly, we also observed two other programmed cell death processes, apoptosis and pyroptosis, in LV-RNAi-OPN3 NHDFs using transmission electron microscopy. Furthermore, the downregulation of the OPN3 gene in NHDFs induced apoptosis compared to the control group, as confirmed by flow cytometry analysis (Figure 2a). Additionally, it significantly decreased the ratio of BCL-2/BAX, as determined by western blot analysis, indicating that the endogenous apoptosis pathway is regulated by OPN3 in NHDF cells (Figure 2b). Scanning TEM revealed chromatin condensation, nuclear fragmentation, and the presence of apoptotic bodies in LV-RNAi-OPN3 cells (Figure 2c). Additionally, some cells exhibited a loss of cell membrane integrity, but the boundaries of the nucleus could still be distinguished, indicating early stages of cellular pyroptosis. The pore-forming protein known as gasdermin D (GSDMD) is cleaved by caspase-1, leading to the formation of pores that increase membrane permeability, ultimately causing pyroptosis and the release of IL-1β. Western blot results demonstrated a significant increase in the expression of GSDMD, cleaved GSDMD, caspase-1, and IL-1β (Figure 2d). Collectively, these findings suggest that the downregulation of the OPN3 gene simultaneously induces ferroptosis, apoptosis, and pyroptosis in NHDFs in vitro. It was previously believed that programmed cell death pathways operated independently in parallel without any overlap. However, recent research has demonstrated that pyroptosis, apoptosis, and necroptosis, collectively referred to as PANoptosis (P for pyroptosis, A for apoptosis, N for necroptosis), exhibit crosstalk and can mutually regulate each other (Sun et al., 2023Sun X. Yang Y. Meng X. Li J. Liu X. Liu H. PANoptosis: Mechanisms, biology, and role in disease.Immunol Rev. 2023; Google Scholar). Furthermore, prior studies have reported that ROS can induce multiple forms of cell death, including apoptosis, autophagy, and ferroptosis, due to the pleiotropic nature of redox signaling in cell biology (Sies and Jones, 2020Sies H. Jones D.P. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents.Nature reviews Molecular cell biology. 2020; 21: 363-383Google Scholar; Su et al., 2019Su L.-J. Zhang J.-H. Gomez H. Murugan R. Hong X. Xu D. et al.Reactive Oxygen Species-Induced Lipid Peroxidation in Apoptosis, Autophagy, and Ferroptosis.Oxidative Medicine and Cellular Longevity. 2019; 2019: 1-13Google Scholar). However, in our present study, we have observed a interesting phenomenon in which ferroptosis, apoptosis, and pyroptosis concurrently occur when the OPN3 gene is downregulated in NHDFs in vitro. Based on our research findings, we propose a concept named 'FAPtosis', which encompasses Ferroptosis, Apoptosis, and Pyroptosis. While further investigation is needed to identify the key molecules responsible for the interaction between ferroptosis, apoptosis, and pyroptosis in NHDFs, these findings underscore the existence of crosstalk among different signaling pathways involved in regulated cell death. In conclusion, this study has advanced our understanding of OPN3's role in regulating cell death in human skin cells, laying the groundwork for future investigations into the mechanisms through which OPN3 influences survival and death. MANUSCRIPT TYPE: Letters to the Editor TITLE: Downregulation of OPN3 gene induces ferroptosis, apoptosis, and pyroptosis of human dermal fibroblasts in vitro Ting Liu1# M.S, Wei Zhang1# Ph.D., Wen Zeng1# Ph.D., Yu Wang1 Ph.D., Huanhuan Luo1 Ph.D., Xian Dong1 M.D., Jianglong Feng1 M.D., Yulei Zhang1 M.S, Shuqi Jin1 M.S, Hongguang Lu1* M.D., Ph.D. 1 Department of Dermatology, Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou, China # These authors contributed equally. *Corresponding author: Hongguang Lu Normal human dermal fibroblasts (NHDFs) were derived from discarded prepuce from pediatric patients aged 3-12 years (Affiliated Hospital of Guizhou Medical University, Guiyang, China) in an experiment with patient consent and the research protocol was approved by the ethics committee of the Affiliated Hospital of Guizhou Medical University. The foreskins underwent disinfection and cleaning procedures. The subcutaneous tissue was then eliminated and the remaining skin was cut into 2mm×2mm pieces. These skin pieces were digested with a 0.25% Dispase enzyme at 4°C for 14-16 hours. Following this, the epidermis was separated using eye tweezers and the dermis was placed in petri dishes, flipped over, and incubated in a 37°C environment with 5% CO2 for 2-3 hours. Eventually, the dermis was cultured in Dulbecco's Modified Eagle's Medium (DMEM) (0030034DJ, Gibco, USA) containing 10% FBS (Cat.No.FBSST-01033-500, Oricell, Guangzhou, China) and 1% antibiotic-antimycotic (Cat.No.P1400, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The petri dishes were maintained at 37°C with 5% CO2 for 4 days, during which the culture medium was changed. Approximately 3-7 days later, NHDFs (normal human dermal fibroblasts) started to emerge from the attached tissue. Once a significant number of NHDFs had emerged, the tissue was removed and the cells were digested using 0.25% pancreatic enzymes. The cells were then continued to be cultured in complete DMEM medium. This experiment was conducted using cells in the logarithmic growth stage. RSL-3 (HY-100218A, MCE) was dissolved in dimethyl sulfoxide (DMSO) (Cat#3871, Solarbio, Beijing, China) at a concentration of 10mM. The solution was stored at -80°C. NHDFs (2×103/well) were incubated in 96-well culture plates. Once the cells adhered, the medium was changed and different doses of RSL-3 (100μM, 10μM, 10μM, 1μM, 0.1μM, 0.01μM, 0.001μM) were added for 24 hours. After that, the medium was changed again and 10μL of cck8 solution was added to each well. The plates were then incubated at 37°C for 2 hours. The absorbance at 450nm was measured using a multimode microplate reader (VLBL0TD2, Thermo Scientific™, USA) to determine the range of cell survival and death concentrations. This experiment was repeated with RSL concentration gradients of 0.001μM, 0.002μM, 0.004μM, 0.008μM, 0.012μM, and 0.014μM. Five replicate wells were used for each concentration. The medium was changed and 10μL of cck8 solution was added to each well. After incubation at 37°C for 2 hours, the absorbance at 450nm was measured using a multimode microplate reader. The IC50 value of 0.006μM was calculated and used for subsequent experiments (figure S4). Liproxstatin-1 (HY-12726, MCE) was dissolved in DMSO at a final concentration of 10mM and stored at -80°C. LV-OPN3-RNAi cells were incubated in 96-well plates. Once the cells adhered, the medium was changed and 4μM liproxstatin-1 was added. After 24 hours, the medium was changed again and 10μL of cck8 solution was added to each well. The cells were then incubated at 37°C for 2 hours and the absorbance at 450nm was measured using a multimode microplate reader. The treatment with liproxstatin-1 rescued the viability of LV-control-RNAi cells after 72 hours. NHDFs were collected and total RNA was isolated and extracted using TRIzol (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. Reversed transcription was performed using the Fasting cDNA Dispelling RT SuperMix reverse transcriptase kit (KR116, TIANGEN BIOTECH (BEIJING), CO., LTD. China). Quantitative real-time reverse transcriptase PCR was carried out using a real-time PCR system (Bio-Rad, Laboratories, Inc. Hercules, CA, USA) and SYBR Green PCR Master Mix (FP209, Tiangen Biotechnology, Beijing, China). The relative RNA expression of OPN 3 was determined by calculating the 2-ΔΔCt value for each sample. The following human primers were used in this study: OPN3 forward, 5 ' - CAATCCAGTGATTTATGTCTTCATGATCAGAAAG-3', and OPN3 reverse, 5 '- GCATTTCACTTCCAGCTGCTGGTAGGT-3'; GAPDH forward, 5 '-GACATCCGCAAAGACCTG-3', and GAPDH reverse, 5 '-GGAAGGTGGACAGCGAG-3'. The cells were lysed on ice for 30 minutes using RIPA lysis buffer that contained 1 mM PMSF. The lysate was then fully oscillated on a vortex oscillator three times. After centrifugation at 4°C, 12000rpm for 15 minutes, the supernatant was transferred to a 1.5ml EP tube. The protein concentration was measured using the NanoDrop™ 2000/2000c spectrophotometer (Thermo Fisher Scientific., USA). Next, the protein sample was added to 5× denatured loading buffer (Cat. No. ZS306, Zoman Biotechnology Co.Ltd. Beijing, China) and denatured at 100°C for 10 minutes. Then, the protein samples were subjected to SDS-PAGE gel electrophoresis and transferred to a PVDF membrane (Bio-Rad Inc.). A 5% skimmed milk powder TBST buffer was used to block the membrane at room temperature for 1 hour. The membrane was then incubated with the primary antibody overnight at 4°C in a shaker , followed by incubation with the corresponding secondary antibody at room temperature for 1 hour. After rinsing the membranes with 1×TBST washing buffer, the target bands on the membranes were detected using the Bio-Rad exposure imaging system (Bio-Rad Laboratories, Inc.).T he gray values of each target protein were analyzed usingimage J software for statistical analysis, and the representative blots were shown in the figures. The primary antibodies and secondary antibodies were used in this research as follows: anti-OPN3 (Cat.No.ab228748, 1:1,000; Abcam), and the mouse monoclonal antibody to Tublin alpha (Cat.No. T0033, 1:1000; Affinity Biosciences LTD.), anti-ACSL-4 (Cat.No. sc-365230, 1:1000; Santa), anti-GPX4 (Cat.No. T56959; 1:1000; Abmart), anti-TFRC (Cat.No. T56618; 1:1000; Abmart), anti-FTH (Cat.No. T55648; 1:1000; Abmart), anti-FTL (Cat.No. T56955; 1:1000; Abmart), anti-FPN (Cat.No. TD13561; 1:1000; Abmart), anti-BCL-2 (Cat.No. T40056F; 1:1000; Abmart), anti-BAX (Cat.No. YT0455; 1:1000; Immunoway), anti-GSDMD (Cat.No. TA4012; 1:1000; Abmart), anti-Caspase 1 (Cat.No. WL02996; 1:500; Wanleibio, china), anti-IL-1β (Cat.No. WL00891; 1:500; Wanleibio, china), secondary anti-rabbit (Cat.No. ab6721; 1:1,0000; Abcam) and anti-mouse (Cat.No. ab6789; 1:1,0000; Abcam) According to the lentivirus user manual (Shanghai Genechem Co., Ltd, China), the 2-4 passages of cells were incubated in 12-well plates at a density of 1.5×104 cells/well with lentivirus vector of LV-OPN3-RNAi and negative control for 3 days at a multiplicity of infection (MOI) of 20 to knockdown endogenous expression. Following this, stably expressing NHDFs were selected using 10ug/mL puromycin (P8230, Solarbio, China). The transfection efficiency of the lentivirus infection was then assessed using Western blotting and RT-qPCR. Cell viability was assessed using the Cell Counting Kit (CCK)-8 Cell Proliferation (Cell Counting Kit, C0037, Beyotime Institute of Biotechnology, Shanghai, China). NHDFs (2×103/well) were incubated in 96-well plates with five wells per replicate. The medium was replaced and 10μL of cck8 solution was added to each well every 24 hours. Following a 2 hours incubation at 37°C, the absorbance at 450nm was measured using a multimode microplate reader. The cell viability was determined by comparing the absolute absorbance of each sample to that of the control group. To evaluate cell proliferation, the EdU cell proliferation kit (C0085S Beyotime, Suzhou, China) was utilized. Following the knockdown of OPN3, cells were seeded in a 24-well plateat a density of 2×103 cells per well . Each well was supplemented with 10 mmol/L EdU and incubated at 37°C for 2 hours. The cells were then fixed with 4% polymethyl at room temperature for 15 minutes. Afterwards, permeabilization was achieved by incubating the cells in a solution containing 3% TritonX for 15 minutes. Subsequently, a Click reaction solution was added and allowed to react for 30 minutes at room temperature. To visualize the EdU-positive cells, the cells were incubated with Hocheset33342 at 37°C, 5% for 10 minutes, while being protected from light. The presence of red fluorescence indicated the presence of EdU-positive cells. Finally, under a fluorescence microscope, 5 visual fields were randomly selected for the analysis of the ratio of proliferating cells to total cells. The FerroOrange fluorescent probes (F374, Dojindo, Kumamoto, Japan) were utilized for the detection of intracellular Fe2+ as per the instructions provided by the manufacturer. In brief, NHDFs were seeded in DMEM medium in 96-well black plates and treated with 4μM liproxstatin-1 for a duration of 96 hours. Subsequently, the cells were harvested and enzymatically digested into single cells using pancreatic enzymes. After washing three times with Hanks' Balanced Salt Solution, the cells were incubated with 1μM FerroOrange for a period of 30 minutes. The fluorescence intensity of each sample was then promptly measured using a multimode microplate reader, with an excitation spectrum of 543nm and an emission spectrum of 580nm. Apoptosis was detected by BD FACSCalibur flow cytometry with annexin V-FITC/PI double staining. Following OPN3 knockdown for 24 hours, cells were inoculated in 6-well plates at a density of 2×105 /well. After 24 hours, two wells were collected from each group and resuspended in 400μL annexin buffer containing 5μL annexin V-FITC (7sea Biotechnology, Shanghai, China, A005-2). Cells were incubated at room temperature for 15 minutes away from light. And then, after adding 10μL PI to the buffer, cells were incubated in an ice bath under dark conditions for 5 minutes. Flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) analysis was performed within 30 minutes. Cells were seeded in DMEM medium in 6-well black plates(100×103/well). LV-OPN3-RNAi cells were treated with 4μM liproxstatin-1, while LV-control-RNAi cells and LV-OPN3-RNAi cells were treated with DMSO. After incubating for 72 hours, the cells were harvested and digested into single cells using pancreatic enzymes. The cells were then washed with 1×PBS and resuspended in DMEM medium containing 10μM DCFH-DA probe (S003S, Beyotime, Suzhou, China). All samples were incubated protected from light at 37°C and 5% CO2 for 30 minutes, followed by three washes with 1×PBS. Finally, flow cytometry was used to analyze more than 10,000 cells with a PE channel to determine the total level of reactive oxygen species (ROS). For the measurement of intracellular lipid ROS level, cells were collected and trypsinized into single cells. The cells were then washed with 1×PBS and resuspended in DMED medium containing 10% FBS. A 10μM C11 BODIPY (Thermo Fisher Scientific; cat#D3861) probe was added to the samples, which were incubated in the dark at 37°C and 5% CO2 for 30 minutes. After three washes with 1×PBS, flow cytometry was performed to analyze more than 10,000 cells with the fluorescein isothiocyanate green channel (FITC) to determine the amount of intracellular lipid ROS level. The cells were collected and washed with 1×PBS. After discarding the supernatant, 1ml electron microscope fixative was added to the mung bean-sized cell stem, which was then fixed at 4°C. Subsequently, the cells were embedded, sectioned, and stained. Image acquisition was performed using transmission electron microscopy. The distribution of cell death types in NHDFs was observed through transmission electron microscopy at low-magnification (scale: 10 μm). Five fields were randomly selected for the quantification of cell death types, each identified according to their distinct characteristics. Data analyses were conducted using the GraphPad Prism 9.3 software package and SPSS software. The data is presented as mean ± SD from multiple experiments. The mean values of the two groups were compared using a T-test, while the one-way ANOVA was utilized for comparisons among three or more groups, followed by a Tukey post hoc test. Statistical significance was defined when the p value <0.05.Figure S2OPN3 knockdown inhibits NHDFs proliferation. (a) CCK-8 assay showed that cell viability decreased in LV-OPN3-RNAi group after 24 hours, compared with the LV-control-RNAi group (n = 3). (b-c) Cell proliferation was inhibited in LV-OPN3-RNAi group after 24 hours by EdU assay (n = 5, scale bar = 50μm). CCK, cell counting kit; EdU, 2′-deoxy-5-ethynyluridine. *P < 0.05, **P < 0.01, and ***P < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure S3The Bulb map of highly represented pathways via KEGG pathway analysis. RNA sequencing analysis of the NHDFs response to low-expressed OPN3 gene revealed that the top 40 pathways were significantly enriched by the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis of differentially expressed genes. (p-adjust<0.05)View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure S4Cell suivival and IC50 curve in NHDFs treated with different dose RSL-3. (a) Cell survival rate with the incubator of different doses of RSL-3 for 24 hours. (n = 3). (b) The IC50 value of 0.006μM was calculated. RSL-3, ferroptosis activator. ns, no significant, *P < 0.05, **P < 0.01, and ***P < 0.001. (RSL-3 groups V.S. control group)View Large Image Figure ViewerDownload Hi-res image Download (PPT)