Early DNA damage detection and cellular autophagy as drivers of stress-adaptive H2S production: A paradox resolved
Appropriately responding and adapting to genotoxic, oxidative, and metabolic stress is essential for survival and is at the heart of maintaining homeostasis. In this issue of Cell Chemical Biology, Jiang et al. (2021) describe an autophagy-dependent mechanism for cytoprotective H2S generation initiated by DNA damage and other small molecule treatments.
<!>Genotoxic and nutrient stress trigger endogenous H2S production in vitro and in vivo<!>Autophagy as a central player in stress-induced H2S production
<p>Hydrogen sulfide (H2S) is a bioactive gasotransmitter and redox metabolite. While exposure from external sources is associated with occupational hazards, endogenous cellular H2S production plays beneficial roles. Supraphysiological production results in vasodilation, cardiac fitness, and resistance to oxidative and inflammatory stress. Likewise, the enhancement of endogenous H2S production is associated with and/or causative of an increased lifespan and is evolutionarily conserved (Hine et al., 2015; Hine et al., 2017). Increased H2S production and/or signaling is hypothesized to provide appropriate metabolic and environmental adaptation to stressors. Therefore, studying H2S regulation and optimizing its cellular production is fundamental to understanding basic biological mechanisms and for translating the findings into clinically actionable tools.</p><p>H2S exists in free forms that includes H2S and HS− as well as in protein-bound forms such as persulfides, polysulfides, and iron-sulfur clusters. Enzymatic generation of H2S is primarily mediated by three enzymes. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL, a.k.a. CSE, CTH) are involved in trans-sulfuration, where methionine is converted into cysteine via a homocysteine intermediate. Due to the flexibility of CBS and CGL for accepting a variety of substrates in a vitamin-B6-dependent fashion, these enzymes also consume homocysteine and cysteine to produce H2S (Kabil and Banerjee, 2014). Mercaptopyruvate sulfurtransferase (3MST) is a third enzyme that produces H2S in conjunction with cysteine aminotransferase (CAT). Expression and activity of these enzymes are predominantly tissue specific and regulated by a variety of transcriptional factors, such as activating transcription factor 4 (ATF4), that respond to intercellular and intracellular redox, nutritional, and endoplasmic reticulum (ER) stressors (Gao et al., 2015; Zivanovic et al., 2020). An unresolved issue in the field relates to why in conditions of stress, particularly pertaining to dietary sulfur amino acid restriction, cells catabolize cysteine for use in H2S production rather than conserving cysteine for protein or glutathione synthesis. Likewise, the source of cysteine as substrate, be it from autophagy or the proteasome, is not well understood.</p><p>It has previously been shown that H2S production is enhanced under growth- and thyroid-hormone restriction and is dependent on autophagy, a process whereby macromolecules and organelles are broken down and cellular building blocks recycled (Hine et al., 2017). However, this autophagy-dependent H2S production was only demonstrated under hormone depletion stress in vitro. In the current issue of Cell Chemical Biology, the Mitchell laboratory extends the observation that autophagy is necessary for providing the substrate for enhanced H2S production under a variety of cell intrinsic and cell extrinsic stressors and molecular triggers in vitro and in vivo (Jiang et al., 2021). Strikingly, the authors show that DNA damage and its early detection via Poly(ADP-ribose) polymerase-1 (PARP-1) triggers endogenous H2S production via the ATF4-dependent induction of CGL in parallel with ATG5/ATG7 genes and AMPK α1/α2-dependent autophagosome formation.</p><p>The key discovery of the current work demonstrates that DNA damage drives CGL-derived H2S without engaging cellular apoptosis. However, this was not the initial question that motivated the authors' inquiry. Rather, an H2S-activated P3 fluorescent probe high-content screen in HeLa cells with over 12,000 small molecules (Figure 1A) and the associated molecular mechanisms in vitro (Figure 1A) and in vivo (Figure 1B) were the driving force underlying the study. 123 positive hits were discovered, including families of molecules associated with caloric restriction mimetics and genotoxic agents. The strongest H2S inducers from the genotoxic family were the cancer chemotherapeutic drugs irinotecan, teniposide, trifluridine, and bleomycin. The authors validated the dose-dependent increase of H2S generation after treatment with these drugs in HeLa cells while utilizing a second sulfide-selective fluorescent probe, SF7-AM.</p><p>To avoid cell- and/or cancer-specific bias in genotoxic-stress-induced H2S production, the authors shifted to utilizing fibroblasts and other cells types. In mouse embryonic fibroblasts (MEFs), teniposide and radiation both increased intracellular H2S and sulfane sulfur, indicating that this phenomenon is not solely due to a foreign chemical inducing an antioxidant/detoxification response. Likewise, ex vivo teniposide treatment of mouse dermal fibroblasts (MDFs) and circulating leukocytes increased H2S production.</p><p>To test if DNA damage induced H2S production and bioavailability in vivo, the authors employed chemically induced DNA damage as well as endogenous DNA lesion accumulation via DNA-repair deficiencies in mouse models. Intraperitoneal injection of etoposide boosted H2S levels within the bone marrow. The resulting buildup of DNA lesions and unrepaired endogenous DNA damage due to Csa and Xpa mutations in a Cockayne Syndrome progeroid mouse model increased H2S levels in leukocytes, lung endothelial cells, and liver. These initial results suggest that genotoxic stress from either exogenous or endogenous sources can trigger a physiological response to increase H2S production in a variety of cell and tissue types. However, the mechanism of action and whether this response is adaptive or maladaptive is yet to be discovered.</p><p>To determine the mechanism(s) leading to enhanced H2S under genotoxic stress, the authors perturbed cellular machinery involved in the DNA damage response, autophagy, metabolic stress response, and sulfur amino acid metabolism. The early DNA damage response is mediated by PARP-1. Knocking out (KO) PARP-1 in MEFs diminished H2S induction by both teniposide and radiation. Thus, the sensing and repair initiation of DNA damage via PARP-1 is necessary for the optimal increase of H2S. Since autophagy was previously shown to be important for cellular augmentation of endogenous H2S generation (Hine et al., 2017) and in the PARP-1 initiated DNA damage response (Muñoz-Gámez et al., 2009), the authors knocked out key proteins involved in autophagosome formation, including ATG5 and ATG7, as well as the metabolic stress sensor AMPK in MEFs for testing the necessity of autophagy for H2S induction during DNA damage. Concurrent with diminished cellular autophagy, all three MEF-KO lines displayed a decreased production of H2S induced by teniposide and an increased sensitivity and level of cell death.</p><p>Since the upstream PARP-1-initiated signaling and generation of sulfur amino acid substrate by autophagy were determined to be central for multiple forms of cell stress, one of the questions remaining to be answered was related to the identity of the enzymatic H2S generator. Cells treated with teniposide showed increased CGL mRNA and protein expression, but not CBS or 3MST expression. The authors next examined the transcriptional machinery linked to enhanced CGL as well as CGL itself. The transcription factor ATF4 is a central player in coordinating the adaptive stress response due to deficits in nutrients and ER stress as well as DNA damage. It elicits cellular survival and adaptation in part through the initiation of autophagy (B'chir et al., 2013) and the promotion of CGL expression (Gao et al., 2015). Here, ATF4-KO MEFs and CGL-KO MDFs displayed a significant decrease of H2S generation when exposed to teniposide. To determine the adaptive versus maladaptive consequences of increased H2S, the sensitivity to teniposide in WT and ATG5 KO MEFs ± H2S donor AP39 was analyzed. Fibroblasts lacking ATG5 having decreased H2S production were sensitized to teniposide-induced death, but were protected against teniposide when supplemented with AP39. Overall, autophagy-dependent H2S induction acts as an adaptive and protective response to DNA damage in the cells tested (Figure 2).</p><p>The current work further reveals the intricate crosstalk between genomic damage, signal transduction, metabolism, and adaptive stress responses, which likely occur in a concomitant manner in pathophysiological conditions. It ultimately provides evidence for the mechanism and function of enhanced H2S in response to cellular stressors. It also introduces new questions and avenues of research. It's important to recognize that, while the authors focused on validating a few of the molecules that elicited an increase of cellular H2S, >120 were left unexplored. This provides abundant opportunities for the future study of safe methods for inducing endogenous H2S production. Drugs inhibiting H2S production have not been described and could be of significant scientific and clinical value.</p><p>While the interaction between H2S, stress, and autophagy has now been revised, it's still somewhat puzzling why cells evolved to produce H2S during genotoxic stress. In MDFs, the authors showed that H2S protects against teniposide-induced cell death. This aligns with work showing that H2S-induced persulfidation of MEK1 leads to PARP-1 activation and DNA damage repair (Zhao et al., 2014), suggesting a positive feedback regulation between PARP-1, CGL, and H2S. The mechanism has obvious benefits in promoting healthy, non-cancerous cell survival against genotoxic stress, and reciprocally suggests a targetable pathway to ensure that cancerous cells can't take advantage. This postulation is likely content-dependent due to H2S acting both as tumor suppressor and an enhancer reliant on cancer cell type (Hellmich and Szabo, 2015) and further complicated by distinct cellular interactions in the tumor microenvironment. Our laboratories recently showed that endogenous H2S production and persulfidation are strong suppressors against glioblastoma (GBM) (Silver et al., 2021). Since most GBM therapies elicit DNA damage, these genetic lesions should prompt H2S production with additional tumor suppression. Importantly, it's not known whether aging or pro-aging diets that suppress H2S production and signaling in the brain (Silver et al., 2021; Zivanovic et al., 2020) also dampen DNA-damage-induced H2S-tumor suppressor functions.</p><p>This cutting-edge work by the Mitchell laboratory has brought to end one chapter in the H2S field. Simultaneously, it opens many others, as these findings and methods will be of great interest, provide insight, and spark discoveries for investigators in cancer, aging, and metabolism fields.</p><!><p>(A) Overview of the in vitro cell systems used to screen for drugs and stressors that elicit endogenous H2S production and/or sulfide bioavailability. Initial high-throughput screening of a ~12,000 drug library in HeLa cells resulted in 123 positive hits, as detected by the sulfide-selective fluorescent probe P3. Subsequent validation of these positive hits focused on genotoxic agents in a variety of wild-type (WT) or gene-specific knockout (KO) cell types with additional sulfide-selective fluorescent probes SF7-AM, SSP4, and Hsip-1 DA. Cells deficient in PARP-1, ATG5, ATG7, AMPK, ATF4, and CGL failed to have robust induction of H2S when compared to their WT controls.</p><p>(B) Similar to cells in culture, the application of genotoxic stress in mice by etoposide treatment or through unrepaired DNA lesions caused by Csa and Xpa mutations in a Cockayne Syndrome progeroid model increased H2S in bone marrow, white blood cells (WBC), liver, lung, and endothelium.</p><!><p>Extracellular and Intracellular stressors, such as DNA damage and the deprivation of nutrients and endocrine growth factors, elicit an Integrated stress response. This response enables the cell to survive and show resiliency to these insults. Central to this response is activation of cellular autophagy through the formation of the autophagosome via concerted activities of PARP-1, AMPK, ATG5, ATG7, and ATF4. Additionally, a mechanistic transcriptional gene target of ATF4 is the trans-sulfuration and H2S generating enzyme cystathionine γ-lyase (CGL). Increased expression of CGL along with the triggering of cellular autophagy results in enhanced H2S production and sulfide bioavailability. Functionally, H2S acts as a gasotransmitterto modify cellular redox status and induce protein persulfidation (aka sulfhydration). Ultimately, in a dose- and cell-type-dependent manner, H2S controls mitochondrial bioenergetics and cell survival.</p>