==== Front
Biochem Soc Trans
Biochem Soc Trans
BST
Biochemical Society Transactions
0300-5127
1470-8752
Portland Press Ltd.

37248872
BST-51-1377
10.1042/BST20230121
Cell Death & Injury
Cell Membranes, Excitation & Transport
Cancer
Signaling
Review Articles
Caveolae and the oxidative stress response
Wu Yeping
Lim Ye-Wheen
http://orcid.org/0000-0002-7494-5248
Parton Robert G.
1 The University of Queensland, Institute for Molecular Bioscience, 4072 Brisbane, Australia
2 The University of Queensland, Centre for Microscopy and Microanalysis, 4072 Brisbane, Australia
Correspondence: Yeping Wu (yeping.wu@uq.edu.au) or Robert G. Parton (r.parton@imb.uq.edu.au)
28 6 2023
30 5 2023
51 3 13771385
18 3 2023
18 5 2023
19 5 2023
© 2023 The Author(s)
2023
https://creativecommons.org/licenses/by/4.0/ This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). Open access for this article was enabled by the participation of the University of Queensland in an all-inclusive Read & Publish agreement with Portland Press and the Biochemical Society under a transformative agreement with CAUL.

Oxidative stress is a feature of many disease conditions. Oxidative stress can activate a number of cellular pathways leading to cell death, including a distinct iron-dependent pathway involving lipid peroxidation, termed ferroptosis, but cells have evolved complex mechanisms to respond to these stresses. Here, we briefly summarise current evidence linking caveolae to the cellular oxidative stress response. We discuss recent studies in cultured cells and in an in vivo model suggesting that lipid peroxidation driven by oxidative stress causes disassembly of caveolae to release caveola proteins into the cell where they regulate the master transcriptional redox controller, nuclear factor erythroid 2-related factor 2. These studies suggest that caveolae maintain cellular susceptibility to oxidative stress-induced cell death and suggest a crucial role in cellular homeostasis and the response to wounding.

caveolae
Cavin1
cell death
lipid peroxidation
NRF2
oxidative stress
==== Body
pmcIntroduction; the cellular response to oxidative stress

Oxidative stress is attributable to an imbalance between the production of reactive oxygen species (ROS) and antioxidant defence. This phenomenon is associated with a wide range of diseases and physiological processes, but its complex effects are particularly well illustrated in cancer. The increased oxidative stress characteristic of highly metabolically active cancer cells can damage DNA and exacerbate inflammation, with a potential role in transformation, tumour cell growth, invasion, and metastasis [1]. However, oxidative stress can also trigger cell death, reducing the chance of transformation and tumorigenesis. Oxidative stress is the major cellular stress occurring upon external stimuli such as radiation [2] and is also a feature of tissue damage in response to wounding under normal physiological conditions [3].

ROS are a group of molecules derived from oxygen, including superoxide, hydrogen peroxide, hydroxyl radical, and singlet oxygen [4]. One of the cellular pathways sensitive to ROS is ferroptosis [5,6], a form of cell death, distinct from apoptosis, in which oxidised polyunsaturated phospholipids accumulate in an iron-dependent manner [7–11]. The pathways regulating ferroptosis are still being unravelled, but a number of features of the pathway are now well established. Suppression of ferroptosis through the clearance of lipid peroxides involves glutathione peroxidase 4 (GPX4), an antioxidant defence enzyme that repairs oxidative damage to lipids [12,13]. Both the availability of iron, as a labile iron pool [6,14] and specific membrane lipids are required for ferroptotic sensitivity. The lipid substrates for ferroptosis are surprisingly specific and a crucial enzyme in this process is long-chain acyl-CoA synthetase 4 (ACSL4) [15]. ACSL4 catalyses the incorporation of polyunsaturated fatty acids (PUFAs) into membrane phospholipids. Particularly important in this pathway are two omega-6 PUFAs, adrenic acid (AdA, 22 : 4) and arachidonic acid (AA, 20 : 4), which must be incorporated into phosphatidylethanolamine (PE) to render cells sensitive to ferroptosis, as shown by elegant inhibition and lipid rescue experiments [16]. The regulation of ACSL4 is therefore crucial for ferroptosis; key regulators include PKCbetaII [17] and Hilpda/HIG2 in clear cell carcinoma cells [18]. The latter is thought to link sources of cellular PUFAs stored in lipid droplets to surface ACSL4-dependent formation of PE-AdA and PE-AA. In contrast with the omega-6 PUFAs that are required for ferroptotic sensitivity, monounsaturated fatty acids, such as oleic acid, can inhibit ferroptosis, promoting the survival of metastasising cancer cell [19,20].

A crucial regulator of the oxidative stress response is nuclear factor erythroid 2-related factor 2 (NRF2), a master transcriptional regulator of cellular redox homeostasis that regulates the expression of a multitude of oxidative stress defence proteins [21]. NRF2 levels are kept low in resting cells through ubiquitylation by KEAP1 which targets NRF2 for degradation [22]. Increased NRF2 activity is associated with oncogenesis, promoting tumour progression, metastasis and resistance to anti-cancer therapies [21,23–25]. In the clinic, high NRF2 expression correlates with poor prognosis by helping cancer cells evade cell death [22]. These effects of NRF2, which inhibit ferroptosis [21,24,25], were assumed to be dependent only on the oxidative stress buffering capacity mediated by the transcriptional targets of NRF2. However, recent work suggests that the ability of NRF2 to regulate the labile iron pool in cells, through regulation of ferritinophagy, is a crucial aspect of its function [14].

Caveolae and the oxidative stress response

A number of different cellular organelles have been linked to oxidative stress and to ferroptosis, including the peroxisome, the Golgi complex, the endoplasmic reticulum (ER), and mitochondria where ROS are generated (reviewed by Stockwell [7]). Of increasing interest to researchers is the potential role of caveolae, nanoscopic microdomains of the plasma membrane, in the cellular response to oxidative stress. Caveolae are bulb- or flask-shaped invaginations of the plasma membrane that can be readily recognised by electron microscopy (Figure 1A) [26]. The first identified marker of caveolae, caveolin 1 (CAV1), is a small integral membrane protein which forms an oligomeric disc in the cytoplasmic leaflet of the caveolar membrane [27]. Two other caveolin isoforms are expressed in mammalian cells with caveolin 2 (CAV2) coexpressed with, and oligomerising with, CAV1 and caveolin 3 (CAV3) being expressed predominantly in muscle cells [28–30]. CAV1 is not sufficient to form caveolae, but co-operates with peripheral membrane proteins termed cavins [31–36]. Early work on caveolae was focused solely on caveolins and their proposed interactions with many different proteins [37]. In recent years, a shift in focus has occurred with the increased mechanistic understanding that the characterisation of the cavins and other accessory proteins has brought. The current model dictates that caveola formation relies on multiple low-affinity interactions between caveolin proteins, cavin proteins, and membrane lipids to generate a metastable domain [38,39]. This domain can be disassembled in response to various stimuli including an increase in membrane tension, or treatment with UV light, which releases cavins from caveolae into the cytoplasm where they can interact with cellular targets [40–44].

Figure 1. The role of caveolae in cellular response to oxidative stress.

(A) Top inset: caveolae as viewed by electron microscopy. Scale bar = 100 nm. Bottom inset: An illustration of caveolar structure in a cell under steady state conditions. (B) A diagram illustrating caveola-mediated cellular pathway in response to oxidative stress. Exogenous H2O2 stimulation, or GPX4 inactivation by ferroptosis inducer RSL3 [13,87], promotes ROS accumulation in cells. Excessive ROS-driven lipid peroxidation causes membrane damage and caveolar disassembly, leading to the release of Cavin1. This allows Cavin1 to target NRF2 in the cytosol and promote NRF2 ubiquitination (i), and NRF2 proteasomal degradation (ii). The binding of Cavin1 to NRF2 also inhibits the nuclear import of NRF2 (iii) [78]. The inhibition of NRF2 by Cavin1 through multiple mechanisms reduce the transcriptional levels of NRF2 downstream genes involved in oxidant defence, ultimately promoting ROS-driven cell death including ferroptosis and apoptosis. (C) A model for the role of caveolae in cellular oxidative stress response and its bearing in a physiological context of wound healing in the zebrafish. Upon tissue injury (a.), a paracrinal wave of ROS (represented by red arrows) such as H2O2 signals occur at the local wounding site [78,88]. Cells at steady state near the wound front experience oxidative stress which may trigger ROS-driven cell death (represented by b. and c.; dotted red arrow represents conversion of the state of a cell undergoing oxidative stress to a cell experiencing cell death). Regeneration pathways are subsequently triggered by cell death [79] (blue arrow) or ROS (d.) [3,89,90].

Although a review of the extensive literature linking caveolae to oxidative stress is beyond the scope of this minireview, we will provide a brief overview of relevant and emerging studies. It has been known for some time that caveolae can be modified by oxidative stress, for example by phosphorylation of CAV1 on a key tyrosine, Y14 [45–48]. The tyrosine kinase c-Src, which could be oxidised and activated by ROS [46,49,50], has been shown to be responsible for CAV1 phosphorylation under oxidative stress [45,46,51]. CAV1 phosphorylation on Y14 could be abrogated by incubation with a Src family inhibitor [52]. CAV1 was also shown to be subjected to proteasomal degradation in response to oxidative stress [53,54] with resulting impairment of caveola-dependent cell processes, although caveolae were still detectable by electron microscopy [53]. A functional role for caveolae in the cellular oxidative stress response has also been proposed [55], although a unifying mechanism for how the entire caveolar system (caveolins, cavins, other accessory proteins and membrane lipids) contributes to the oxidative stress response both in cells and whole tissues has been lacking. Earlier studies mainly focussed on CAV1 and many built upon the earlier hypothesis that CAV1 can directly interact with target proteins, a model that is increasingly being questioned [27,48,56,57]. The reported roles of CAV1 are complex as CAV1 expression has been linked to both an increase in oxidative stress or, conversely, to an increase in oxidative buffering capacity. This can be attributed to distinct contexts such as cells with different levels of basal ROS content (e.g. normal cells versus cancer cells [1]), and different concentrations and duration of pro-oxidant treatments in these studies. Alterations in these factors may cause ROS accumulation at different levels and lead to the activation of distinct signalling pathways and biological processes [58,59], thereby affecting the modification and response of CAV1 to oxidative stress. In some studies, CAV1 exhibits an inhibitory effect on oxidative stress. For example, up-regulation of CAV1 was suggested to protect lung cancer cell against excessive ROS-induced cell death, allowing ROS-driven cancer progression [60]. Down-regulation of stromal CAV1 was linked to mitochondrial enzymatic dysfunction and increased ROS production, leading to more aggressive phenotypes of breast cancer cells [61]. CAV1 was also shown to be a negative regulator of ROS production in endothelial cells [62] and in podocytes [63], reducing podocyte cell death [63]. In other studies, however, CAV1 was shown to promote oxidative stress through inhibiting NRF2 (i.e. CAV1 loss increased antioxidant defence) [64,65]. These studies reported an association of a significant pool of NRF2 with CAV1 via the CAV1 scaffolding domain and a caveolin-binding motif in NRF2 [64,65] although the cellular sites of interaction were suggested to be either at the plasma membrane [64] or the nucleus and cytosol [65]. The NRF2-caveolin interaction was lost after 48 h of oxidative stress treatment [64].

Cavin1 has been less extensively studied. Cavin1 deficiency was shown to impair ROS production induced by organ damage in mice [66], indicating a potential role for Cavin1 in regulating the redox state of cells in cancer. Oxidative stress can also activate senescence, where excessive ROS-induced DNA damage response (DDR) signals [67], such as p53, p16 and pRB [68] [69], may lead to a stable cell growth arrest, suppressing tumour development [70]. In this context, Cavin1 has been shown to be activators of the p53/p21 pathway, which places it as a regulator of oxidative stress-induced senescence [71,72].

Another link between caveolae and oxidative stress in cancer is their common interplay with an inflammatory response, a tumorigenesis driver. Inflammatory processes such as excessive ROS production, can be responsible for tumorigenesis through the induction of DNA mutations [73]. In the tumour environment, loss of mesenchymal stromal CAV1 could lead to oxidative stress and drive inflammation [61]. In addition to the direct effect on ROS production, CAV1 has been proposed as a negative regulator of prostaglandin-endoperoxide synthase (PTGS) 2 (also known as COX2) [74], which can be up-regulated by ROS and mediates inflammation [75]. Interestingly, differential regulation of cavin proteins by tumour necrosis factor (TNF), a PTGS2 and inflammation inducer [76], has been observed in mesenchymal stromal cells [77]. This study also revealed a role for Cavin2 as a suppressor of TNF signalling. However, the significance of up-regulated Cavin1 and Cavin3 in TNF-induced inflammation in cancer is yet to be defined. Taken together, these studies indicate that caveolae can act as key redox regulators modulating oxidative stress via multiple processes that are involved in cancer progression.

An integrated model for a role of caveolae in oxidative stress signalling

The discovery that disassembly of caveolae causes cavin proteins to be released into the cytoplasm [41] where they can interact with, and stabilise, target proteins [40] raised the possibility of examining the entire cellular protein complement linked to cavins to discover what pathways are regulated by caveola disassembly. This unbiased approach was made feasible by development of whole cell quantitative proteomic methods which could be applied to genome-edited cells lacking specific cavin proteins [78]. Unexpectedly, this unbiased approach led to new links between caveolae and oxidative stress.

A striking feature of HeLa cells lacking Cavin1 revealed by whole cell quantitative proteomics was the increase in protein levels of over 40 targets of NRF2 [78]. This corresponded to an increased oxidative stress buffering capacity of the Cavin1-null cells and importantly this was also the case for an in vivo model of Cavin1 deficiency; zebrafish lacking the two Cavin1 paralogs, Cavin1a and Cavin1b, similarly showed increased resistance to ROS accumulation after treatment with hydrogen peroxide [78]. In both systems, the increased oxidative stress buffering capacity could be attenuated by expression of Cavin1 but not by expression of CAV1, pointing to a primary role of Cavin1 (and presumably caveolae) in the oxidative stress response [78]. Note that this does not rule out a contributory role of CAV1 in the oxidative stress response.

Treatment with hydrogen peroxide represents a convenient but artificial test of the cellular ROS buffering capacity. However, ROS generation is a feature of tissue wounding in vivo. ROS generation triggers cell death and this process is required for efficient regeneration, a pathway dubbed the ‘Phoenix-Rising Pathway’ (see Figure 1C) [79]. The development of the zebrafish line lacking Cavin1 paralogs allowed the role of Cavin1 to be tested in an in vivo wounding assay [78]. This revealed that loss of Cavin1 caused a reduction in ROS accumulation at the wound site, reduced apoptosis, and significantly impaired epimorphic regeneration. Thus, the lack of the caveolar system impairs the endogenous ROS-induced apoptosis pathway required for efficient tissue regeneration in vivo. Rescue studies in the zebrafish showed that this process was dependent on Cavin-1 but not CAV1.

These experiments reveal an evolutionarily conserved role for caveolae in reacting to oxidative stress. However, it raised the question of the function of caveolae in wild-type cells subjected to oxidative stress. Specifically, why does a loss of Cavin1 (and caveolae) cause increased expression of NRF2 target proteins? And how does the caveolar system and Cavin1 specifically respond to oxidative stress? The results strongly suggested a link between Cavin1 and NRF2. In fact, oxidative stress was shown to cause partial caveola disassembly (as indicated by a reduction in caveolae by electron microscopy), release of Cavin1 into the cytosol, and interaction, direct or indirect, with NRF2 as shown by proximity ligation assays and by immunoprecipitation. This interaction was proposed to inhibit NRF2 activation through two mechanisms; firstly, it was shown that in this system Cavin1 was required for efficient ubiquitylation and degradation of NRF2; second, the Cavin1–NRF2 interaction in the cytosol inhibited nuclear import of NRF2 [78] (Figure 1B). It is also important to note the kinetics of this process. Rather than long-term treatment with oxidising agents over hours and days, the effect of oxidative stress in these studies occurred on the timescale of a few minutes showing a very rapid response to these stimuli. The combined effect of Cavin1 loss was therefore to rapidly increase NRF2 levels and activity.

In this model (Figure 1), Cavin1 released from caveolae in response to oxidative stress can prevent NRF2 activating its targets and so acts as a ‘suicide switch’ to cause cell death and remove the potentially DNA-damaged cells from the population, as observed in the zebrafish wounding assay. Cells lacking Cavin1 showed significantly increased resistance to ferroptosis triggered by inhibition of GPX4 [78] suggesting that caveolae may represent another branch of the ferroptotic regulatory network.

A final question relates to the signal that causes caveola disassembly and the release of cavins for interaction with NRF2. Specific induction of lipid peroxidation was shown to be sufficient to cause cavin release and association with NRF2 [78]. Consistent with this, inhibition of lipid peroxidation blocked cavin release and association of Cavin1 with NRF2. This raises the intriguing possibility that caveolae have evolved to respond to oxidative stress owing to their specialised lipid composition required for their formation and serving as sites for lipid peroxidation. This interesting model is speculative at present but can now be tested.

Conclusions and future directions

These studies delineate a novel pathway from cell surface caveolae to NRF2 and ferroptosis which involves lipid peroxidation. However, it opens up a number of new questions and avenues for investigation. Recent studies revealed that cell swelling caused by increased membrane permeability and sodium influx is an essential step leading to ferroptotic cell rupture [80,81]. It was also found that this cell swelling process increased tension on the plasma membrane [80]. Previous work showed that membrane stretch by osmotic swelling drives caveolar disassembly and the release of cavins [41,42,44]. This raises a possibility that in addition to lipid peroxidation, increased membrane tension upon cell swelling may also contribute to caveola disassembly and Cavin1 release during ferroptosis induction. There are clearly many different stimuli that cause caveola disassembly and yet it seems unlikely that all would lead to cavin-mediated inhibition of NRF2. One possibility is that a network of post-translational modifications may fine-tune cavin target recognition. It is also unclear how lipid peroxidation leads to disassembly of caveolae. Specific lipids required for caveola formation may be sites for peroxidation causing destabilisation of caveolae. Finally, there are considerable implications for understanding disease conditions associated with loss or dysfunction of caveolae. NRF2 has been extensively studied as a therapeutic target in oxidative stress-involved neurodegenerative diseases [82] and in radio-/chemo-resistant cancer [83,84]. There have been significant advances in drug development based on the mechanisms of NRF2 activation and inhibition [85,86]. The identification of Cavin1 as a binding protein and regulator of NRF2 expands the knowledge of NRF2 signalling and could potentially contribute to therapeutic strategies aimed at the pharmacologic regulation of NRF2 and redox status in cells.

Perspectives

Oxidative stress is ubiquitous in biological systems and is a key element in various physiopathological processes. Disruption of caveolae has been linked to an aberrant oxidative stress response, a contributing factor in disease progression.

Caveolae, composed of specific lipids and accessory proteins, are crucial in sensing and regulating oxidative stress. In the sensing stage, peroxidation of membrane lipids triggers the disassembly of caveolae to release the accessory protein Cavin1 into the cytosol. Redistributed Cavin1 in turn exerts a regulatory role in oxidative stress by inhibiting NRF2. This cellular pathway is essential for mediating oxidative stress-driven cell death including apoptosis and ferroptosis.

Future investigations into caveola signalling under oxidative stress should focus on (1) dissecting the mechanisms of caveola disassembly upon lipid peroxidation; (2) identifying post-translational modifications required for Cavin1 release and target recognition; (3) identifying the binding sites involved in the Cavin1–NRF2 interaction; (4) exploring the physiological relevance of the CAVIN1–NRF2–ROS axis in other oxidative stress-mediated biological processes.

Acknowledgements

R.G.P. is an Australian Research Council Laureate Fellow (FL210100107). This work was also supported by the National Health and Medical Research Council of Australia (grants APP1140064 and APP1150083) to R.G.P.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Open Access

Open access for this article was enabled by the participation of the University of Queensland in an all-inclusive Read & Publish agreement with Portland Press and the Biochemical Society under a transformative agreement with CAUL.

Abbreviations

AA arachidonic acid

ACSL4 acyl-CoA synthetase 4

CAV1 caveolin 1

GPX4 glutathione peroxidase 4

NRF2 nuclear factor erythroid 2-related factor 2

PE phosphatidylethanolamine

PUFAs polyunsaturated fatty acids

ROS reactive oxygen species

TNF tumour necrosis factor
==== Refs
References

1 Hayes, J.D., Dinkova-Kostova, A.T. and Tew, K.D. (2020) Oxidative stress in cancer. Cancer Cell 38 , 167–197 10.1016/j.ccell.2020.06.001 32649885
2 Azzam, E.I., Jay-Gerin, J.P. and Pain, D. (2012) Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury. Cancer Lett. 327 , 48–60 10.1016/j.canlet.2011.12.012 22182453
3 Dunnill, C., Patton, T., Brennan, J., Barrett, J., Dryden, M., Cooke, J. et al. (2017) Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROS-modulating technologies for augmentation of the healing process. Int. Wound J. 14 , 89–96 10.1111/iwj.12557 26688157
4 Murphy, M.P., Bayir, H., Belousov, V., Chang, C.J., Davies, K.J.A., Davies, M.J., et al. (2022) Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo. Nat. Metab. 4 , 651–662 10.1038/s42255-022-00591-z 35760871
5 Conrad, M., Kagan, V.E., Bayir, H., Pagnussat, G.C., Head, B., Traber, M.G. et al. (2018) Regulation of lipid peroxidation and ferroptosis in diverse species. Genes Dev. 32 , 602–619 10.1101/gad.314674.118 29802123
6 Dixon, S.J., Lemberg, K.M., Lamprecht, M.R., Skouta, R., Zaitsev, E.M., Gleason, C.E., et al. (2012) Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149 , 1060–1072 10.1016/j.cell.2012.03.042 22632970
7 Stockwell, B.R. (2022) Ferroptosis turns 10: emerging mechanisms, physiological functions, and therapeutic applications. Cell 185 , 2401–2421 10.1016/j.cell.2022.06.003 35803244
8 Yagoda, N., von Rechenberg, M., Zaganjor, E., Bauer, A.J., Yang, W.S., Fridman, D.J., et al. (2007) RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels. Nature 447 , 864–868 10.1038/nature05859 17568748
9 Yang, W.S. and Stockwell, B.R. (2008) Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem. Biol. 15 , 234–245 10.1016/j.chembiol.2008.02.010 18355723
10 Yang, W.S., Kim, K.J., Gaschler, M.M., Patel, M., Shchepinov, M.S. and Stockwell, B.R. (2016) Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc. Natl Acad. Sci. U.S.A. 113 , E4966–E4975 10.1073/pnas.1603244113 27506793
11 Yang, W.S. and Stockwell, B.R. (2016) Ferroptosis: death by lipid peroxidation. Trends Cell Biol. 26 , 165–176 10.1016/j.tcb.2015.10.014 26653790
12 Seiler, A., Schneider, M., Forster, H., Roth, S., Wirth, E.K., Culmsee, C., et al. (2008) Glutathione peroxidase 4 senses and translates oxidative stress into 12/15-lipoxygenase dependent- and AIF-mediated cell death. Cell Metab. 8 , 237–248 10.1016/j.cmet.2008.07.005 18762024
13 Friedmann Angeli, J.P., Schneider, M., Proneth, B., Tyurina, Y.Y., Tyurin, V.A., Hammond, V.J., et al. (2014) Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice. Nat. Cell Biol. 16 , 1180–1191 10.1038/ncb3064 25402683
14 Anandhan, A., Dodson, M., Shakya, A., Chen, J., Liu, P., Wei, Y., et al. (2023) NRF2 controls iron homeostasis and ferroptosis through HERC2 and VAMP8. Sci. Adv. 9 , eade9585 10.1126/sciadv.ade9585 36724221
15 Doll, S., Proneth, B., Tyurina, Y.Y., Panzilius, E., Kobayashi, S., Ingold, I., et al. (2017) ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition. Nat. Chem. Biol. 13 , 91–98 10.1038/nchembio.2239 27842070
16 Kagan, V.E., Mao, G., Qu, F., Angeli, J.P., Doll, S., Croix, C.S., et al. (2017) Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis. Nat. Chem. Biol. 13 , 81–90 10.1038/nchembio.2238 27842066
17 Zhang, H.L., Hu, B.X., Li, Z.L., Du, T., Shan, J.L., Ye, Z.P., et al. (2022) PKCbetaII phosphorylates ACSL4 to amplify lipid peroxidation to induce ferroptosis. Nat. Cell Biol. 24 , 88–98 10.1038/s41556-021-00818-3 35027735
18 Zou, Y., Palte, M.J., Deik, A.A., Li, H., Eaton, J.K., Wang, W., et al. (2019) A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis. Nat. Commun. 10 , 1617 10.1038/s41467-019-09277-9 30962421
19 Magtanong, L., Ko, P.J., To, M., Cao, J.Y., Forcina, G.C., Tarangelo, A., et al. (2019) Exogenous monounsaturated fatty acids promote a ferroptosis-resistant cell state. Cell Chem. Biol. 26 , 420–432.e9 10.1016/j.chembiol.2018.11.016 30686757
20 Ubellacker, J.M., Tasdogan, A., Ramesh, V., Shen, B., Mitchell, E.C., Martin-Sandoval, M.S., et al. (2020) Lymph protects metastasizing melanoma cells from ferroptosis. Nature 585 , 113–118 10.1038/s41586-020-2623-z 32814895
21 Dodson, M., Castro-Portuguez, R. and Zhang, D.D. (2019) NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis. Redox Biol. 23 , 101107 10.1016/j.redox.2019.101107 30692038
22 Kansanen, E., Kuosmanen, S.M., Leinonen, H. and Levonen, A.-L. (2013) The Keap1-Nrf2 pathway: mechanisms of activation and dysregulation in cancer. Redox Biol. 1 , 45–49 10.1016/j.redox.2012.10.001 24024136
23 Homma, S., Ishii, Y., Morishima, Y., Yamadori, T., Matsuno, Y., Haraguchi, N., et al. (2009) Nrf2 enhances cell proliferation and resistance to anticancer drugs in human lung cancer. Clin. Cancer Res. 15 , 3423 10.1158/1078-0432.CCR-08-2822 19417020
24 Shin, D., Kim, E.H., Lee, J. and Roh, J.-L. (2018) Nrf2 inhibition reverses resistance to GPX4 inhibitor-induced ferroptosis in head and neck cancer. Free Radic. Biol. Med. 129 , 454–462 10.1016/j.freeradbiomed.2018.10.426 30339884
25 Roh, J.-L., Kim, E.H., Jang, H. and Shin, D. (2017) Nrf2 inhibition reverses the resistance of cisplatin-resistant head and neck cancer cells to artesunate-induced ferroptosis. Redox Biol. 11 , 254–262 10.1016/j.redox.2016.12.010 28012440
26 Parton, R.G. (2018) Caveolae: structure, function, and relationship to disease. Annu. Rev. Cell Dev. Biol. 34 , 111–136 10.1146/annurev-cellbio-100617-062737 30296391
27 Porta, J.C., Han, B., Gulsevin, A., Chung, J.M., Peskova, Y., Connolly, S. et al. (2022) Molecular architecture of the human caveolin-1 complex. Sci. Adv. 8 , eabn7232 10.1126/sciadv.abn7232 35544577
28 Lamaze, C., Tardif, N., Dewulf, M., Vassilopoulos, S. and Blouin, C.M. (2017) The caveolae dress code: structure and signaling. Curr. Opin. Cell Biol. 47 , 117–125 10.1016/j.ceb.2017.02.014 28641181
29 Parton, R.G. and Simons, K. (2007) The multiple faces of caveolae. Nat. Rev. Mol. Cell Biol. 8 , 185–194 10.1038/nrm2122 17318224
30 Parton, R.G., Way, M., Zorzi, N. and Stang, E. (1997) Caveolin-3 associates with developing T-tubules during muscle differentiation. J. Cell Biol. 136 , 137–154 10.1083/jcb.136.1.137 9008709
31 Bastiani, M., Liu, L., Hill, M.M., Jedrychowski, M.P., Nixon, S.J., Lo, H.P., et al. (2009) MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes. J. Cell Biol. 185 , 1259–1273 10.1083/jcb.200903053 19546242
32 Hill, M.M., Bastiani, M., Luetterforst, R., Kirkham, M., Kirkham, A., Nixon, S.J., et al. (2008) PTRF-Cavin, a conserved cytoplasmic protein required for caveola formation and function. Cell 132 , 113–124 10.1016/j.cell.2007.11.042 18191225
33 Jansa, P., Mason, S.W., Hoffmann-Rohrer, U. and Grummt, I. (1998) Cloning and functional characterization of PTRF, a novel protein which induces dissociation of paused ternary transcription complexes. EMBO J. 17 , 2855–2864 10.1093/emboj/17.10.2855 9582279
34 McMahon, K.A., Zajicek, H., Li, W.P., Peyton, M.J., Minna, J.D., Hernandez, V.J. et al. (2009) SRBC/cavin-3 is a caveolin adapter protein that regulates caveolae function. EMBO J. 28 , 1001–1015 10.1038/emboj.2009.46 19262564
35 Hansen, C.G., Bright, N.A., Howard, G. and Nichols, B.J. (2009) SDPR induces membrane curvature and functions in the formation of caveolae. Nat. Cell Biol. 11 , 807–814 10.1038/ncb1887 19525939
36 Hansen, C.G. and Nichols, B.J. (2010) Exploring the caves: cavins, caveolins and caveolae. Trends Cell Biol. 20 , 177–186 10.1016/j.tcb.2010.01.005 20153650
37 Couet, J., Li, S., Okamoto, T., Ikezu, T. and Lisanti, M.P. (1997) Identification of peptide and protein ligands for the caveolin- scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins. J. Biol. Chem. 272 , 6525–6533 10.1074/jbc.272.10.6525 9045678
38 Parton, R.G., Tillu, V., McMahon, K.A. and Collins, B.M. (2021) Key phases in the formation of caveolae. Curr. Opin. Cell Biol. 71 , 7–14 10.1016/j.ceb.2021.01.009 33677149
39 Tillu, V.A., Rae, J., Gao, Y., Ariotti, N., Floetenmeyer, M., Kovtun, O. et al. (2021) Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation. Nat. Commun. 12 , 931 10.1038/s41467-021-21035-4 33568658
40 McMahon, K.A., Stroud, D.A., Gambin, Y., Tillu, V., Bastiani, M., Sierecki, E., et al. (2021) Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response. eLife 10 , e61407 10.7554/eLife.61407 34142659
41 McMahon, K.A., Wu, Y., Gambin, Y., Sierecki, E., Tillu, V.A., Hall, T., et al. (2019) Identification of intracellular cavin target proteins reveals cavin-PP1alpha interactions regulate apoptosis. Nat. Commun. 10 , 3279 10.1038/s41467-019-11111-1 31332168
42 Sinha, B., Koster, D., Ruez, R., Gonnord, P., Bastiani, M., Abankwa, D., et al. (2011) Cells respond to mechanical stress by rapid disassembly of caveolae. Cell 144 , 402–413 10.1016/j.cell.2010.12.031 21295700
43 Lim, Y.W., Lo, H.P., Ferguson, C., Martel, N., Giacomotto, J., Gomez, G.A. et al. (2017) Caveolae protect notochord cells against catastrophic mechanical failure during development. Curr. Biol. 27 , 1968–1981.e67 10.1016/j.cub.2017.05.067 28648821
44 Tillu, V.A., Lim, Y.W., Kovtun, O., Mureev, S., Ferguson, C., Bastiani, M., et al. (2018) A variable undecad repeat domain in cavin1 regulates caveola formation and stability. EMBO Rep. 19 , e45775 10.15252/embr.201845775 30021837
45 Volonte, D., Galbiati, F., Pestell, R.G. and Lisanti, M.P. (2001) Cellular stress induces the tyrosine phosphorylation of caveolin-1 (Tyr(14)) via activation of p38 mitogen-activated protein kinase and c-Src kinase. evidence for caveolae, the actin cytoskeleton, and focal adhesions as mechanical sensors of osmotic stress. J. Biol. Chem. 276 , 8094–8103 10.1074/jbc.M009245200 11094059
46 Cao, H., Sanguinetti, A.R. and Mastick, C.C. (2004) Oxidative stress activates both Src-kinases and their negative regulator Csk and induces phosphorylation of two targeting proteins for Csk: caveolin-1 and paxillin. Exp. Cell Res. 294 , 159–171 10.1016/j.yexcr.2003.11.010 14980511
47 Parton, R.G., Kozlov, M.M. and Ariotti, N. (2020) Caveolae and lipid sorting: shaping the cellular response to stress. J. Cell Biol. 219 , e201905071 10.1083/jcb.201905071 32328645
48 Jung, W., Sierecki, E., Bastiani, M., O'Carroll, A., Alexandrov, K., Rae, J., et al. (2018) Cell-free formation and interactome analysis of caveolae. J. Cell Biol. 217 , 2141–2165 10.1083/jcb.201707004 29716956
49 Giannoni, E., Buricchi, F., Raugei, G., Ramponi, G. and Chiarugi, P. (2005) Intracellular reactive oxygen species activate Src tyrosine kinase during cell adhesion and anchorage-dependent cell growth. Mol. Cell. Biol 25 , 6391–6403 10.1128/mcb.25.15.6391-6403.2005 16024778
50 Corcoran, A. and Cotter, T.G. (2013) Redox regulation of protein kinases. FEBS J. 280 , 1944–1965 10.1111/febs.12224 23461806
51 Chen, D.B., Li, S.M., Qian, X.X., Moon, C. and Zheng, J. (2005) Tyrosine phosphorylation of caveolin 1 by oxidative stress is reversible and dependent on the c-src tyrosine kinase but not mitogen-activated protein kinase pathways in placental artery endothelial cells1. Biol. Reprod. 73 , 761–772 10.1095/biolreprod.105.040881 15958730
52 Wehinger, S., Ortiz, R., Díaz, M.I., Aguirre, A., Valenzuela, M., Llanos, P. et al. (2015) Phosphorylation of caveolin-1 on tyrosine-14 induced by ROS enhances palmitate-induced death of beta-pancreatic cells. Biochim. Biophys. Acta Mol. Basis Dis. 1852 , 693–708 10.1016/j.bbadis.2014.12.021
53 Mougeolle, A., Poussard, S., Decossas, M., Lamaze, C., Lambert, O. and Dargelos, E. (2015) Oxidative stress induces caveolin 1 degradation and impairs caveolae functions in skeletal muscle cells. PLoS ONE 10 , e0122654 10.1371/journal.pone.0122654 25799323
54 Luanpitpong, S., Talbott, S.J., Rojanasakul, Y., Nimmannit, U., Pongrakhananon, V., Wang, L. et al. (2010) Regulation of lung cancer cell migration and invasion by reactive oxygen species and caveolin-1*. J. Biol. Chem. 285 , 38832–38840 10.1074/jbc.M110.124958 20923773
55 Wang, S., Wang, N., Zheng, Y., Zhang, J., Zhang, F. and Wang, Z. (2017) Caveolin-1: an oxidative stress-related target for cancer prevention. Oxid. Med. Cell. Longev. 2017 , 7454031 10.1155/2017/7454031 28546853
56 Byrne, D.P., Dart, C. and Rigden, D.J. (2012) Evaluating caveolin interactions: do proteins interact with the caveolin scaffolding domain through a widespread aromatic residue-rich motif? PLoS ONE 7 , e44879 10.1371/journal.pone.0044879 23028656
57 Collins, B.M., Davis, M.J., Hancock, J.F. and Parton, R.G. (2012) Structure-based reassessment of the caveolin signaling model: do caveolae regulate signaling through caveolin-protein interactions? Dev. Cell 23 , 11–20 10.1016/j.devcel.2012.06.012 22814599
58 Schieber, M. and Chandel, N.S. (2014) ROS function in redox signaling and oxidative stress. Curr. Biol. 24 , R453–R462 10.1016/j.cub.2014.03.034 24845678
59 Marinho, H.S., Real, C., Cyrne, L., Soares, H. and Antunes, F. (2014) Hydrogen peroxide sensing, signaling and regulation of transcription factors. Redox Biol. 2 , 535–562 10.1016/j.redox.2014.02.006 24634836
60 Suchaoin, W. and Chanvorachote, P. (2012) Caveolin-1 attenuates hydrogen peroxide-induced oxidative damage to lung carcinoma cells. Anticancer Res. 32 , 483–490 https://ar.iiarjournals.org PMID: 22287735
61 Pavlides, S., Tsirigos, A., Vera, I., Flomenberg, N., Frank, P.G., Casimiro, M.C., et al. (2010) Loss of stromal caveolin-1 leads to oxidative stress, mimics hypoxia and drives inflammation in the tumor microenvironment, conferring the “reverse Warburg effect”: a transcriptional informatics analysis with validation. Cell Cycle 9 , 2201–2219 10.4161/cc.9.11.11848 20519932
62 Ju, H., Zou, R., Venema, V.J. and Venema, R.C. (1997) Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. J. Biol. Chem. 272 , 18522–18525 10.1074/jbc.272.30.18522 9228013
63 Sun, L.N., Liu, X.C., Chen, X.J., Guan, G.J. and Liu, G. (2016) Curcumin attenuates high glucose-induced podocyte apoptosis by regulating functional connections between caveolin-1 phosphorylation and ROS. Acta Pharmacol. Sin. 37 , 645–655 10.1038/aps.2015.159 26838071
64 Volonte, D., Liu, Z., Musille, P.M., Stoppani, E., Wakabayashi, N., Di, Y.P. et al. (2013) Inhibition of nuclear factor-erythroid 2-related factor (Nrf2) by caveolin-1 promotes stress-induced premature senescence. Mol. Biol. Cell 24 , 1852–1862 10.1091/mbc.E12-09-0666 23637463
65 Li, W., Liu, H., Zhou, J.S., Cao, J.F., Zhou, X.B., Chen, Z.H. et al. (2012) Caveolin-1 inhibits expression of antioxidant enzymes through direct interaction with nuclear erythroid 2 p45-related factor-2 (Nrf2). J. Biol. Chem. 287 , 20922–20930 10.1074/jbc.M112.352336 22547061
66 Zheng, Y., Lee, S., Liang, X., Wei, S., Moon, H.G. and Jin, Y. (2013) Suppression of PTRF alleviates the polymicrobial sepsis induced by cecal ligation and puncture in mice. J. Infect. Dis. 208 , 1803–1812 10.1093/infdis/jit364 23908488
67 Borodkina, A., Shatrova, A., Abushik, P., Nikolsky, N. and Burova, E. (2014) Interaction between ROS dependent DNA damage, mitochondria and p38 MAPK underlies senescence of human adult stem cells. Aging (Albany NY) 6 , 481–495 10.18632/aging.100673 24934860
68 Beausejour, C.M., Krtolica, A., Galimi, F., Narita, M., Lowe, S.W., Yaswen, P. et al. (2003) Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J. 22 , 4212–4222 10.1093/emboj/cdg417 12912919
69 Takahashi, A., Ohtani, N. and Hara, E. (2007) Irreversibility of cellular senescence: dual roles of p16INK4a/Rb-pathway in cell cycle control. Cell Div. 2 , 10 10.1186/1747-1028-2-10 17343761
70 Bertram, C. and Hass, R. (2008) Cellular responses to reactive oxygen species-induced DNA damage and aging. Biol. Chem. 389 , 211–220 10.1515/BC.2008.031 18208352
71 Bitar, M.S., Abdel-Halim, S.M. and Al-Mulla, F. (2013) Caveolin-1/PTRF upregulation constitutes a mechanism for mediating p53-induced cellular senescence: implications for evidence-based therapy of delayed wound healing in diabetes. Am. J. Physiol. Endocrinol. Metab. 305 , E951–E963 10.1152/ajpendo.00189.2013 23941874
72 Volonte, D. and Galbiati, F. (2011) Polymerase I and transcript release factor (PTRF)/cavin-1 is a novel regulator of stress-induced premature senescence. J. Biol. Chem. 286 , 28657–28661 10.1074/jbc.C111.235119 21705337
73 Federico, A., Morgillo, F., Tuccillo, C., Ciardiello, F. and Loguercio, C. (2007) Chronic inflammation and oxidative stress in human carcinogenesis. Int. J. Cancer 121 , 2381–2386 10.1002/ijc.23192 17893868
74 Chen, S.F., Liou, J.Y., Huang, T.Y., Lin, Y.S., Yeh, A.L., Tam, K. et al. (2010) Caveolin-1 facilitates cyclooxygenase-2 protein degradation. J. Cell Biochem. 109 , 356–362 10.1002/jcb.22407 19960513
75 Asting, A.G., Caren, H., Andersson, M., Lonnroth, C., Lagerstedt, K. and Lundholm, K. (2011) COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status. BMC Cancer 11 , 238 10.1186/1471-2407-11-238 21668942
76 Chen, C.C., Sun, Y.T., Chen, J.J. and Chiu, K.T. (2000) TNF-alpha-induced cyclooxygenase-2 expression in human lung epithelial cells: involvement of the phospholipase C-gamma 2, protein kinase C-alpha, tyrosine kinase, NF-kappa B-inducing kinase, and I-kappa B kinase 1/2 pathway. J. Immunol. 165 , 2719–2728 10.4049/jimmunol.165.5.2719 10946303
77 Annabi, B., Zgheib, A. and Annabi, B. (2017) Cavin-2 functions as a suppressive regulator in TNF-induced mesenchymal stromal cell inflammation and angiogenic phenotypes. Int. J. Stem Cells 10 , 103–113 10.15283/ijsc16032 28024316
78 Wu, Y., Lim, Y.W., Stroud, D.A., Martel, N., Hall, T.E., Lo, H.P. et al. (2023) Caveolae sense oxidative stress through membrane lipid peroxidation and cytosolic release of CAVIN1 to regulate NRF2. Dev. Cell 58 , 376–397.e4 10.1016/j.devcel.2023.02.004 36858041
79 Li, F., Huang, Q., Chen, J., Peng, Y., Roop, D.R., Bedford, J.S. et al. (2010) Apoptotic cells activate the “phoenix rising” pathway to promote wound healing and tissue regeneration. Sci. Signal. 3 , ra13 10.1126/scisignal.2000634 20179271
80 Hirata, Y., Cai, R., Volchuk, A., Steinberg, B.E., Saito, Y., Matsuzawa, A. et al. (2023) Lipid peroxidation increases membrane tension, Piezo1 gating, and cation permeability to execute ferroptosis. Curr. Biol. 33 , 1282–1294.e5 10.1016/j.cub.2023.02.060 36898371
81 Riegman, M., Sagie, L., Galed, C., Levin, T., Steinberg, N., Dixon, S.J. et al. (2020) Ferroptosis occurs through an osmotic mechanism and propagates independently of cell rupture. Nat. Cell Biol. 22 , 1042–1048 10.1038/s41556-020-0565-1 32868903
82 Brandes, M.S. and Gray, N.E. (2020) NRF2 as a therapeutic target in neurodegenerative diseases. ASN Neuro 12 , 1759091419899782 10.1177/1759091419899782 31964153
83 No, J.H., Kim, Y.-B. and Song, Y.S. (2014) Targeting Nrf2 signaling to combat chemoresistance. J. Cancer Prev. 19 , 111–117 10.15430/JCP.2014.19.2.111 25337579
84 Zhou, S., Ye, W., Shao, Q., Zhang, M. and Liang, J. (2013) Nrf2 is a potential therapeutic target in radioresistance in human cancer. Crit. Rev. Oncol. Hematol. 88 , 706–715 10.1016/j.critrevonc.2013.09.001 24126138
85 Forman, H.J. and Zhang, H. (2021) Targeting oxidative stress in disease: promise and limitations of antioxidant therapy. Nat. Rev. Drug Discov 20 , 689–709 10.1038/s41573-021-00233-1 34194012
86 Robledinos-Antón, N., Fernández-Ginés, R., Manda, G. and Cuadrado, A. (2019) Activators and inhibitors of NRF2: a review of their potential for clinical development. Oxid. Med. Cell. Longev. 2019 , 9372182 10.1155/2019/9372182 31396308
87 Yang, W.S., SriRamaratnam, R., Welsch, M.E., Shimada, K., Skouta, R., Viswanathan, V.S., et al. (2014) Regulation of ferroptotic cancer cell death by GPX4. Cell 156 , 317–331 10.1016/j.cell.2013.12.010 24439385
88 Niethammer, P., Grabher, C., Look, A.T. and Mitchison, T.J. (2009) A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature 459 , 996–999 10.1038/nature08119 19494811
89 Yoo, S.K., Freisinger, C.M., LeBert, D.C. and Huttenlocher, A. (2012) Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish. J. Cell Biol. 199 , 225–234 10.1083/jcb.201203154 23045550
90 Romero, M.M.G., McCathie, G., Jankun, P. and Roehl, H.H. (2018) Damage-induced reactive oxygen species enable zebrafish tail regeneration by repositioning of Hedgehog expressing cells. Nat. Commun. 9 , 4010 10.1038/s41467-018-06460-2 30275454