Accumulation of annexin A2 and S100A10 prevents apoptosis of apically delaminated, transformed epithelial cells.

In various epithelial tissues, the epithelial monolayer acts as a barrier. To fulfill its function, the structural integrity of the epithelium is tightly controlled. When normal epithelial cells detach from the basal substratum and delaminate into the apical lumen, the apically extruded cells undergo apoptosis, which is termed anoikis. In contrast, transformed cells often become resistant to anoikis and able to survive and grow in the apical luminal space, leading to the formation of multilayered structures, which can be observed at the early stage of carcinogenesis. However, the underlying molecular mechanisms still remain elusive. In this study, we first demonstrate that S100A10 and ANXA2 (Annexin A2) accumulate in apically extruded, transformed cells in both various cell culture systems and murine epithelial tissues in vivo. ANXA2 acts upstream of S100A10 accumulation. Knockdown of ANXA2 promotes apoptosis of apically extruded RasV12-transformed cells and suppresses the formation of multilayered epithelia. In addition, the intracellular reactive oxygen species (ROS) are elevated in apically extruded RasV12 cells. Treatment with ROS scavenger Trolox reduces the occurrence of apoptosis of apically extruded ANXA2-knockdown RasV12 cells and restores the formation of multilayered epithelia. Furthermore, ROS-mediated p38MAPK activation is observed in apically delaminated RasV12 cells, and ANXA2 knockdown further enhances the p38MAPK activity. Moreover, the p38MAPK inhibitor promotes the formation of multilayered epithelia of ANXA2-knockdown RasV12 cells. These results indicate that accumulated ANXA2 diminishes the ROS-mediated p38MAPK activation in apically extruded transformed cells, thereby blocking the induction of apoptosis. Hence, ANXA2 can be a potential therapeutic target to prevent multilayered, precancerous lesions.

Mitochondria directly sense osmotic stress to trigger rapid metabolic remodeling via regulation of pyruvate dehydrogenase phosphorylation.

A high-salt diet significantly impacts various diseases, ilncluding cancer and immune diseases. Recent studies suggest that the high-salt/hyperosmotic environment in the body may alter the chronic properties of cancer and immune cells in the disease context. However, little is known about the acute metabolic changes in hyperosmotic stress. Here, we found that hyperosmotic stress for a few minutes induces Warburg-like metabolic remodeling in HeLa and Raw264.7 cells and suppresses fatty acid oxidation. Regarding Warburg-like remodeling, we determined that the pyruvate dehydrogenase phosphorylation status was altered bidirectionally (high in hyperosmolarity and low in hypoosmolarity) to osmotic stress in isolated mitochondria, suggesting that mitochondria themselves have an acute osmosensing mechanism. Additionally, we demonstrate that Warburg-like remodeling is required for HeLa cells to maintain ATP levels and survive under hyperosmotic conditions. Collectively, our findings suggest that cells exhibit acute metabolic remodeling under osmotic stress via the regulation of pyruvate dehydrogenase phosphorylation by direct osmosensing within mitochondria.

ContactBlot: Microfluidic Control and Measurement of the Cell-Cell Contact State to Assess Contact-Inhibited ERK Signaling.

Extracellular signal-regulated kinase (ERK) signaling is essential to regulated cell behaviors, including cell proliferation, differentiation, and apoptosis. The influence of cell-cell contacts on ERK signaling is central to epithelial cells, yet few studies have sought to understand the same in cancer cells, particularly with single-cell resolution. To acquire same-cell measurements of both phenotypic (cell-contact state) and targeted-protein (ERK phosphorylation) profiles, we prepend high-content, whole-cell imaging prior to end-point cellular-resolution Western blot analyses for each of hundreds of individual HeLa cancer cells cultured on that same chip, which we call contactBlot. By indexing the phosphorylation level of ERK in each cell or cell cluster to the imaged cell-contact state, we compare the ERK signaling between isolated and in-contact cells. We observe attenuated (∼2×) ERK signaling in HeLa cells that are in-contact versus isolated. Attenuation is sustained when the HeLa cells are challenged with hyperosmotic stress. Our findings show the impact of cell-cell contacts on ERK activation with isolated and in-contact cells while introducing a multi-omics tool for control and scrutiny of cell-cell interactions.

The mitochondrial Ca2+ uptake regulator, MICU1, is involved in cold stress-induced ferroptosis.

Ferroptosis has recently attracted much interest because of its relevance to human diseases such as cancer and ischemia-reperfusion injury. We have reported that prolonged severe cold stress induces lipid peroxidation-dependent ferroptosis, but the upstream mechanism remains unknown. Here, using genome-wide CRISPR screening, we found that a mitochondrial Ca2+ uptake regulator, mitochondrial calcium uptake 1 (MICU1), is required for generating lipid peroxide and subsequent ferroptosis under cold stress. Furthermore, the gatekeeping activity of MICU1 through mitochondrial calcium uniporter (MCU) is suggested to be indispensable for cold stress-induced ferroptosis. MICU1 is required for mitochondrial Ca2+ increase, hyperpolarization of the mitochondrial membrane potential (MMP), and subsequent lipid peroxidation under cold stress. Collectively, these findings suggest that the MICU1-dependent mitochondrial Ca2+ homeostasis-MMP hyperpolarization axis is involved in cold stress-induced lipid peroxidation and ferroptosis.

Osmotic gradients induce stable dome morphogenesis on extracellular matrix.

One of the fundamental processes in morphogenesis is dome formation, but many of the mechanisms involved are unexplored. Previous in vitro studies showed that an osmotic gradient is the driving factor of dome formation. However, these investigations were performed without extracellular matrix (ECM), which provides structural support to morphogenesis. With the use of ECM, we observed that basal hypertonic stress induced stable domes in vitro that have not been seen in previous studies. These domes developed as a result of ECM swelling via aquaporin water transport activity. Based on computer simulation, uneven swelling, with a positive feedback between cell stretching and enhanced water transport, was a cause of dome formation. These results indicate that osmotic gradients induce dome morphogenesis via both enhanced water transport activity and subsequent ECM swelling.

Genome-wide siRNA screening reveals that DCAF4-mediated ubiquitination of optineurin stimulates autophagic degradation of Cu,Zn-superoxide dismutase.

Cu, Zn superoxide dismutase (SOD1) is one of the genes implicated in the devastating neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Although the precise mechanisms of SOD1 mutant (SOD1mut)-induced motoneuron toxicity are still unclear, defects in SOD1 proteostasis are known to have a critical role in ALS pathogenesis. We previously reported that the SOD1mut adopts a conformation that exposes a Derlin-1-binding region (DBR) and that DBR-exposed SOD1 interacts with Derlin-1, leading to motoneuron death. We also found that an environmental change, i.e. zinc depletion, induces a conformational change in WT SOD1 (SOD1WT) to the DBR-exposed conformation, suggesting the presence of an equilibrium state between the DBR-masked and DBR-exposed states even with SOD1WT Here, we conducted a high-throughput screening based on time-resolved FRET to further investigate the SOD1WT conformational change, and we used a genome-wide siRNA screen to search for regulators of SOD1 proteostasis. This screen yielded 30 candidate genes that maintained an absence of the DBR-exposed SOD1WT conformation. Among these genes was one encoding DDB1- and CUL4-associated factor 4 (DCAF4), a substrate receptor of the E3 ubiquitin-protein ligase complex. Of note, we found that DCAF4 mediates the ubiquitination of an ALS-associated protein and autophagy receptor, optineurin (OPTN), and facilitates autophagic degradation of DBR-exposed SOD1. In summary, our screen identifies DCAF4 as being required for proper proteostasis of DBR-exposed SOD1, which may have potential relevance for the development of therapies for managing ALS.

ASK1 suppresses NK cell-mediated intravascular tumor cell clearance in lung metastasis.

Tumor metastasis is the leading cause of death worldwide and involves an extremely complex process composed of multiple steps. Our previous study demonstrated that apoptosis signal-regulating kinase 1 (ASK1) deficiency in mice attenuates tumor metastasis in an experimental lung metastasis model. However, the steps of tumor metastasis regulated by ASK1 remain unclear. Here, we showed that ASK1 deficiency in mice promotes natural killer (NK) cell-mediated intravascular tumor cell clearance in the initial hours of metastasis. In response to tumor inoculation, ASK1 deficiency upregulated immune response-related genes, including interferon-gamma (IFNγ). We also revealed that NK cells are required for these anti-metastatic phenotypes. ASK1 deficiency augmented cytokine production chemoattractive to NK cells possibly through induction of the ligand for NKG2D, a key activating receptor of NK cells, leading to further recruitment of NK cells into the lung. These results indicate that ASK1 negatively regulates NK cell-dependent anti-tumor immunity and that ASK1-targeted therapy can provide a new tool for cancer immunotherapy to overcome tumor metastasis.

SOD1 as a molecular switch for initiating the homeostatic ER stress response under zinc deficiency.

Zinc is an essential trace element, and impaired zinc homeostasis is implicated in the pathogenesis of various human diseases. However, the mechanisms cells use to respond to zinc deficiency are poorly understood. We previously reported that amyotrophic lateral sclerosis (ALS)-linked pathogenic mutants of SOD1 cause chronic endoplasmic reticulum (ER) stress through specific interactions with Derlin-1, which is a component of the ER-associated degradation machinery. Moreover, we recently demonstrated that this interaction is common to ALS-linked SOD1 mutants, and wild-type SOD1 (SOD1(WT)) comprises a masked Derlin-1 binding region (DBR). Here, we found that, under zinc-deficient conditions, SOD1(WT) adopts a mutant-like conformation that exposes the DBR and induces the homeostatic ER stress response, including the inhibition of protein synthesis and induction of a zinc transporter. We conclude that SOD1 has a function as a molecular switch that activates the ER stress response, which plays an important role in cellular homeostasis under zinc-deficient conditions.

ASK family kinases mediate cellular stress and redox signaling to circadian clock.

Daily rhythms of behaviors and physiologies are generated by the circadian clock, which is composed of clock genes and the encoded proteins forming transcriptional/translational feedback loops (TTFLs). The circadian clock is a self-sustained oscillator and flexibly responds to various time cues to synchronize with environmental 24-h cycles. However, the key molecule that transmits cellular stress to the circadian clockwork is unknown. Here we identified apoptosis signal-regulating kinase (ASK), a member of the MAPKKK family, as an essential mediator determining the circadian period and phase of cultured cells in response to osmotic changes of the medium. The physiological impact of ASK signaling was demonstrated by a response of the clock to changes in intracellular redox states. Intriguingly, the TTFLs drive rhythmic expression of Ask genes, indicating ASK-mediated association of the TTFLs with intracellular redox. In behavioral analysis, Ask1, Ask2, and Ask3 triple-KO mice exhibited compromised light responses of the circadian period and phase in their activity rhythms. LC-MS/MS-based proteomic analysis identified a series of ASK-dependent and osmotic stress-responsive phosphorylations of proteins, among which CLOCK, a key component of the molecular clockwork, was phosphorylated at Thr843 or Ser845 in the carboxyl-terminal region. These findings reveal the ASK-dependent stress response as an underlying mechanism of circadian clock flexibility.

Cold stress-induced ferroptosis involves the ASK1-p38 pathway.

A wide variety of cell death mechanisms, such as ferroptosis, have been proposed in mammalian cells, and the classification of cell death attracts global attention because each type of cell death has the potential to play causative roles in specific diseases. However, the precise molecular mechanisms leading to cell death are poorly understood, particularly in ferroptosis. Here, we show that continuous severe cold stress induces ferroptosis and the ASK1-p38 MAPK pathway in multiple cell lines. The activation of the ASK1-p38 pathway is mediated by critical determinants of ferroptosis: MEK activity, iron ions, and lipid peroxide. The chemical compound erastin, a potent ferroptosis inducer, also activates the ASK1-p38 axis downstream of lipid peroxide accumulation and leads to ASK1-dependent cell death in a cell type-specific manner. These lines of evidence provide mechanistic insight into ferroptosis, a type of regulated necrosis.

In Situ Single-Cell Western Blot on Adherent Cell Culture.

Integrating 2D culture of adherent mammalian cells with single-cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single-cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin-functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one-step copolymerization process that creates protein-decorated microwells. After single-cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell-based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab-gel western blot, while revealing cell-to-cell heterogeneity in stress responses.

The ASK1-specific inhibitors K811 and K812 prolong survival in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure. To develop effective treatments for this devastating disease, an appropriate strategy for targeting the molecule responsible for the pathogenesis of ALS is needed. We previously reported that mutant SOD1 protein causes motor neuron death through activation of ASK1, a mitogen-activated protein kinase kinase kinase. Additionally, we recently developed K811 and K812, which are selective inhibitors for ASK1. Here, we report the effect of K811 and K812 in a mouse model of ALS (SOD1(G93A) transgenic mice). Oral administration of K811 or K812 significantly extended the life span of SOD1(G93A) transgenic mice (1.06 and 1.08% improvement in survival). Moreover, ASK1 activation observed in the lumbar spinal cord of mice at the disease progression stage was markedly decreased in the K811- and K812-treated groups. In parallel, immunohistochemical analysis revealed that K811 and K812 treatment inhibited glial activation in the lumbar spinal cord of SOD1(G93A) transgenic mice. These results reinforce the importance of ASK1 as a therapeutic target for ALS treatment.

ß-TrCP-dependent degradation of ASK1 suppresses the induction of the apoptotic response by oxidative stress.

Apoptosis signal-regulating kinase 1 (ASK1) is a key player in the homeostatic response of many organisms. Of the many functions of ASK1, it is most well-known for its ability to induce canonical caspase 3-dependent apoptosis through the MAPK pathways in response to reactive oxygen species (ROS). As ASK1 is a regulator of apoptosis, its proper regulation is critical for the well-being of an organism. To date, several E3 ubiquitin ligases have been identified that are capable of degrading ASK1, signifying the importance of maintaining ASK1 expression levels during stress responses. ASK1 protein regulation under unstimulated conditions, however, is still largely unknown. Using tandem mass spectrometry, we have identified beta-transducin repeat containing protein (ß-TrCP), an E3 ubiquitin ligase, as a novel interacting partner of ASK1 that is capable of ubiquitinating and subsequently degrading ASK1 through the ubiquitin-proteasome system (UPS). This interaction requires the seven WD domains of ß-TrCP and the C-terminus of ASK1. By silencing the ß-TrCP genes, we observed a significant increase in caspase 3 activity in response to oxidative stress, which could subsequently be suppressed by silencing ASK1. These findings suggest that ß-TrCP is capable of suppressing oxidative stress-induced caspase 3-dependent apoptosis through suppression of ASK1, assisting in the organism's ability to maintain homeostasis in an unstable environment.

Mitogen-activated protein kinases as key players in osmotic stress signaling.

BACKGROUND: Osmotic stress arises from the difference between intracellular and extracellular osmolality. It induces cell swelling or shrinkage as a consequence of water influx or efflux, which threatens cellular activities. Mitogen-activated protein kinases (MAPKs) play central roles in signaling pathways in osmotic stress responses, including the regulation of intracellular levels of inorganic ions and organic osmolytes. SCOPE OF REVIEW: The present review summarizes the cellular osmotic stress response and the function and regulation of the vertebrate MAPK signaling pathways involved. We also describe recent findings regarding apoptosis signal-regulating kinase 3 (ASK3), a MAP3K member, to demonstrate its regulatory effects on signaling molecules beyond MAPKs. MAJOR CONCLUSIONS: MAPKs are rapidly activated by osmotic stress and have diverse roles, such as cell volume regulation, gene expression, and cell survival/death. There is significant cell type specificity in the function and regulation of MAPKs. Based on its activity change during osmotic stress and its regulation of the WNK1-SPAK/OSR1 pathway, ASK3 is expected to play important roles in osmosensing mechanisms and cellular functions related to osmoregulation. GENERAL SIGNIFICANCE: MAPKs are essential for various cellular responses to osmotic stress; thus, the identification of the upstream regulators of MAPK pathways will provide valuable clues regarding the cellular osmosensing mechanism, which remains elusive in mammals. The elucidation of in vivo MAPK functions is also important because osmotic stress in physiological and pathophysiological conditions often results from changes in the intracellular osmolality. These studies potentially contribute to the establishment of therapeutic strategies against diseases that accompany osmotic perturbation.

Apoptosis Signal-regulating Kinase 1 (ASK1)-p38 Pathway-dependent Cytoplasmic Translocation of the Orphan Nuclear Receptor NR4A2 Is Required for Oxidative Stress-induced Necrosis.

p38 mitogen-activated protein kinases (MAPKs) play important roles in various cellular stress responses, including cell death, which is roughly categorized into apoptosis and necrosis. Although p38 signaling has been extensively studied, the molecular mechanisms of p38-mediated cell death are unclear. ASK1 is a stress-responsive MAP3K that acts as an upstream kinase of p38 and is activated by various stresses, such as oxidative stress. Here, we show that NR4A2, a member of the NR4A nuclear receptor family, acts as a necrosis promoter downstream of ASK1-p38 pathway during oxidative stress. Although NR4A2 is well known as a nucleus-localized transcription factor, we found that it is translocated into the cytosol after phosphorylation by p38. Because the phosphorylation site mutants of NR4A2 cannot rescue the cell death-promoting activity, ASK1-p38 pathway-dependent phosphorylation and subsequent cytoplasmic translocation of NR4A2 may be required for oxidative stress-induced cell death. In addition, NR4A2-mediated cell death does not depend on caspases and receptor-interacting protein 1 (RIP1)-RIP3 complex, suggesting that NR4A2 promotes an RIP kinase-independent necrotic type of cell death. Our findings may enable a more precise understanding of molecular mechanisms that regulate oxidative stress-induced and p38-mediated necrosis.

A systematic immunoprecipitation approach reinforces the concept of common conformational alterations in amyotrophic lateral sclerosis-linked SOD1 mutants.

Mutations in the Cu, Zn superoxide dismutase (SOD1) gene are one of the causative agents of amyotrophic lateral sclerosis (ALS). Although more than 100 different mutations in SOD1 have been identified, it is unclear whether all the mutations are pathogenic or just single nucleotide polymorphisms (SNPs) unrelated to the disease. Our previous systematic analysis found that all pathogenic SOD1 mutants (SOD1(mut)) have a common property, namely, an association with Derlin-1, a component of the endoplasmic reticulum-associated degradation machinery. For the proposed mechanism, we found that most pathogenic SOD1(mut) have a constitutively exposed Derlin-1-binding region (DBR), which is concealed in wild-type SOD1 (SOD1(WT)). Moreover, we generated MS785, a monoclonal antibody against DBR. MS785 distinguished most ALS-causative SOD1(mut) from both SOD1(WT) and non-toxic SOD1(mut). However, MS785 could not recognize SOD1(mut) that has mutations in the MS785 epitope region. Here, we developed a new diagnostic antibody, which could compensate for this shortcoming of MS785. We hypothesized that in ALS-causative SOD1(mut), the DBR-neighboring region [SOD1(30-40)] may also be exposed. We then generated MS27, a monoclonal antibody against SOD1(30-40). We found that MS27 could distinguish SOD1(WT) from the pathogenic SOD1(mut), which has mutations in the MS785 epitope region. Moreover, all pathogenic SOD1(mut), without exception, were immunoprecipitated with a combination of MS785 and MS27. The MS785-MS27 combination could be developed as a novel mechanism-based biomarker for the diagnosis of ALS.

In vivo gene manipulation reveals the impact of stress-responsive MAPK pathways on tumor progression.

It has been widely accepted that tumor cells and normal stromal cells in the host environment coordinately modulate tumor progression. Mitogen-activated protein kinase pathways are the representative stress-responsive cascades that exert proper cellular responses to divergent environmental stimuli. Genetically engineered mouse models and chemically induced tumorigenesis models have revealed that components of the MAPK pathway not only regulate the behavior of tumor cells themselves but also that of surrounding normal stromal cells in the host environment during cancer pathogenesis. The individual functions of MAPK pathway components in tumor initiation and progression vary depending on the stimuli and the stromal cell types involved in tumor progression, in addition to the molecular isoforms of the components and the origins of the tumor. Recent studies have indicated that MAPK pathway components synergize with environmental factors (e.g. tobacco smoke and diet) to affect tumor initiation and progression. Moreover, some components play distinct roles in the course of tumor progression, such as before and after the establishment of tumors. Hence, a comprehensive understanding of the multifaceted functions of MAPK pathway components in tumor initiation and progression is essential for the improvement of cancer therapy. In this review, we focus on the reports that utilized knockout, conditional knockout, and transgenic mice of MAPK pathway components to investigate the effects of MAPK pathway components on tumor initiation and progression in the host environment.

CHIP-dependent termination of MEKK2 regulates temporal ERK activation required for proper hyperosmotic response.

The extracellular signal-regulated kinase (ERK) pathway is an important signalling pathway that regulates a large number of cellular processes, including proliferation, differentiation and gene expression. Hyperosmotic stress activates the ERK pathway, whereas little is known about the regulatory mechanisms and physiological functions of ERK activation in hyperosmotic response. Here, we show that MAPK/ERK kinase kinase 2 (MEKK2), a member of the MAPKKK family, mediated the specific and transient activation of ERK, which was required for the induction of aquaporin 1 (AQP1) and AQP5 gene expression in response to hyperosmotic stress. Moreover, we identified the E3 ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP) as a binding partner of MEKK2. Depletion of CHIP by small-interference RNA or gene targeting attenuated the degradation of MEKK2 and prolonged the ERK activity. Interestingly, hyperosmolality-induced gene expression of AQP1 and AQP5 was suppressed by CHIP depletion and was reversed by inhibition of the prolonged phase of ERK activity. These findings show that transient activation of the ERK pathway, which depends not only on MEKK2 activation, but also on CHIP-dependent MEKK2 degradation, is crucial for proper gene expression in hyperosmotic stress response.

ASK1 and ASK2 differentially regulate the counteracting roles of apoptosis and inflammation in tumorigenesis.

Apoptosis and inflammation generally exert opposite effects on tumorigenesis: apoptosis serves as a barrier to tumour initiation, whereas inflammation promotes tumorigenesis. Although both events are induced by various common stressors, relatively little is known about the stress-induced signalling pathways regulating these events in tumorigenesis. Here, we show that stress-activated MAP3Ks, ASK1 and ASK2, which are involved in cellular responses to various stressors such as reactive oxygen species, differentially regulate the initiation and promotion of tumorigenesis. ASK2 in cooperation with ASK1 functioned as a tumour suppressor by exerting proapoptotic activity in epithelial cells, which was consistent with the reduction in ASK2 expression in human cancer cells and tissues. In contrast, ASK1-dependent cytokine production in inflammatory cells promoted tumorigenesis. Our findings suggest that ASK1 and ASK2 are critically involved in tumorigenesis by differentially regulating apoptosis and inflammation.

p38 MAPKs regulate the expression of genes in the dopamine synthesis pathway through phosphorylation of NR4A nuclear receptors.

In Drosophila, the melanization reaction is an important defense mechanism against injury and invasion of microorganisms. Drosophila tyrosine hydroxylase (TH, also known as Pale) and dopa decarboxylase (Ddc), key enzymes in the dopamine synthesis pathway, underlie the melanin synthesis by providing the melanin precursors dopa and dopamine, respectively. It has been shown that expression of Drosophila TH and Ddc is induced in various physiological and pathological conditions, including bacterial challenge; however, the mechanism involved has not been fully elucidated. Here, we show that ectopic activation of p38 MAPK induces TH and Ddc expression, leading to upregulation of melanization in the Drosophila cuticle. This p38-dependent melanization was attenuated by knockdown of TH and Ddc, as well as by that of Drosophila HR38, a member of the NR4A family of nuclear receptors. In mammalian cells, p38 phosphorylated mammalian NR4As and Drosophila HR38 and potentiated these NR4As to transactivate a promoter containing NR4A-binding elements, with this transactivation being, at least in part, dependent on the phosphorylation. This suggests an evolutionarily conserved role for p38 MAPKs in the regulation of NR4As. Thus, p38-regulated gene induction through NR4As appears to function in the dopamine synthesis pathway and may be involved in immune and stress responses.