Ultraviolet Light, Unfolded Protein Response and Autophagy †

The endoplasmic reticulum (ER) plays an important role in the regulation of protein synthesis. Alterations in the folding capacity of the ER induce stress, which activates three ER sensors that mediate the unfolded protein response (UPR). Components of the pathways regulated by these sensors have been shown to regulate autophagy. The last corresponds to a mechanism of self‐eating and recycling important for proper cell maintenance. Ultraviolet radiation (UV) is an external damaging stimulus that is known for inducing oxidative stress, and DNA, lipid and protein damage. Many controversies exist regarding the role of UV‐inducing ER stress or autophagy. However, a connection between the three of them has not been addressed. In this review, we will discuss the contradictory theories regarding the relationships between UV radiation with the induction of ER stress and autophagy, as well as hypothetic connections between UV, ER stress and autophagy.


INTRODUCTION
The endoplasmic reticulum (ER) is the organelle in which many proteins are synthesized and folded into their three-dimensional (3D) structures. Each type of cell has a specific folding capability, and the ER operates very near the limit of that capacity. This folding capacity can be adapted to specific cell needs, but it is also a process that can be easily disturbed by both internal and external stimuli (1)(2)(3). When damaging stimuli affect the folding capacity and misfolded/unfolded proteins accumulate in the lumen of the ER, a condition known as "ER stress" is developed. Ultraviolet light (UV) has been shown to induce ER stress, which activates the unfolded protein response (UPR) (2,3). There are three known stress sensors located in the ER membrane that act as part of the UPR and are regulated by the chaperone protein BiP (binding-immunoglobulin protein). They are PKR-like ER kinase (PERK), inositol requiring enzyme 1a/b (IRE1a/b) and activating transcription factor 6a/b (ATF6a/b) (2)(3)(4). These ER sensors play critical roles in the mediation of ER stressactivated signaling pathways that maintain the physiological function of cells (2)(3)(4) (Fig. 1).
Besides being the compartment for the synthesis, folding and quality control of the nascent proteins, ER serves as one of the membrane donors required for the formation of autophagosomes, which are double-membrane vesicles involved in autophagy (also referred to as autophagocytosis or macroautophagy) (5)(6)(7)(8)(9). UV dynamically regulates the formation of autophagosomes (10)(11)(12)(13)(14)(15)(16)(17)(18) and thus autophagy, a mechanism of degradation and recycling of cytosolic constituents, such as misfolded proteins and damaged organelles (19,20). The cellular roles of autophagy are constitutive or adaptive because for example, it eliminates damaged or senescent cellular components (constitutive role), and provides energy in case of metabolism alteration and nutrient starvation (adaptive role) (19). In this review, we discuss the responses of the ER stress sensors and their roles in the regulation of the autophagic reaction in cells after exposure to different range wavelengths of the UV spectrum.

UV RADIATION AND THE ER STRESS SENSORS
Under ER stress, several signaling pathways are activated in the ER to reestablish its homeostasis. However, when cells undergo long periods of ER stress, these signals will eventually turn to be pro-apoptotic (2)(3)(4)(21)(22)(23). These cellular responses are extensively known and are referred to as UPR (2)(3)(4)(21)(22)(23). The three stress sensors, PERK, IRE1a/b and ATF6a/b, are ER transmembrane proteins that are involved in both transcriptional and translational regulation of gene expressions (Fig. 1). The ER lumen has an environment that is suitable for forming disulfide bonds in proteins and has a high Ca 2+ concentration as well (24,25). The high oxidizing environment of the ER (high oxidative potential) is responsible for the capacity of disulfide bonds formation (26), while Ca 2+ is required for the proper function of protein chaperones, which are enzymes that ensures the proper protein conformation of nascent proteins (27). Since oxidative species (reactive oxygen species [ROS] and reactive nitrogen species [RNS]) are capable of interacting with and modifying cellular molecules such as lipids, nucleic acid and proteins; it is then hypothesized that the increase in ROS and/or RNS is a causative factor of ER stress and UPR by altering the redox status, depleting the ER Ca 2+ stores, and inducing the malfunction of protein chaperones, leading to the accumulation of misfolded proteins (28)(29)(30). In the ER, ROS and RNS interaction with proteins within the ER membrane compartment results in structural alteration of these molecules, as well as their function, for example, the S-nitrosylation of a protein disulfide isomerase alters its folding capacity (31,32), while the S-nitrosylation of IRE1 and PERK, alter their ability to control the expression of genes involved in UPR (33)(34)(35). Indeed, and as described later, UVB irradiation has been shown in skin cells to induce ER stress and the UPR by altering the protein folding mechanism through ROS-mediated oxidative stress and the efflux of Ca 2+ from the ER (36,37). The effects of UV on the ER depend on the length, dose and wavelength (UVC, UVB or UVA) of UV exposure, which will be discussed in the following chapters.

UV and PERK
When misfolded proteins build up in the ER, BiP releases the ER luminal domain of PERK, which trigger the activation and auto trans-phosphorylation of PERK, decreasing protein synthesis through the phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2a) at the serine 51 ( Fig. 1). Phosphorylated eIF2a inhibits the process of translation by limiting the number of polypeptide chains formed and enables the ER to potentially recover and restore a normal protein synthesis (38)(39)(40). The activation of PERK and the subsequent eIF2a phosphorylation has also been shown to prioritize the translation of certain mRNAs, like the Activating Transcription Factor 4 (ATF4) and C/EBP homologous protein (CHOP), which not only aid to restore the equilibrium in the ER, but also participate in the regulation of the autophagy response (41,42). UVC, at a dose ranging between 30 and 100 J m À2 , was the first to be shown to induce the eIF2a phosphorylation at 4-12 h after the irradiation in MCF-7 (breast cancer) and hamster insulinoma tumor cells (43). Overexpression of a dominant negative PERK inhibits UVC-induced eIF2a phosphorylation suggesting the involvement of PERK in the signaling pathway (43). UVC also induces the activation of GRP78/BiP promoter in COS-1 (monkey kidney fibroblast-like) cells (43), further confirming UVC induces ER stress in various cells. Prior to the finding, UVC was not identified as an inducer of eIF2a phosphorylation because the phosphorylation was not detected at 45 min post-UVC irradiation (44) suggesting UVC-induced ER stress is a relatively slow process. Following the initial discovery, many studies have been carried out using UVB, a more physiologically relevant UV wavelength. By comparing the cellular responses to UVB irradiation (at a dose of 10, 20 and 30 mJ cm À2 ) in HaCaT and Hs68 cells (human fibroblast), Farrukh et al. demonstrated that UVB irradiation results in the production of ROS, depletion of ER Ca 2+ , induction of ER stress and activation of UPR in a cell line-dependent manner. UVB exposure increases cytotoxicity in both Hs68 and HaCaT cells. However, GRP78/BiP expression and the phosphorylation of eIF2a only increased in Hs68 but not in HaCaT cells post-UVB irradiation (36). Similarly, Mera et al. (45) described that PERK was not observed to be phosphorylated post-UVB irradiation at a dose ranging between 10 and 40 mJ cm À2 . On the other hand, our studies showed an increased eIF2a phosphorylation in HaCaT cells at a dose range between 10 and 200 mJ cm À2 (46)(47)(48)(49), and the increased p-eIF2a correlates with a decrease, instead of an increase as previously reported with other ER stressors (41,50,51), of the protein levels of ATF4 and GADD34 post-UVB irradiation (48). UVB also induces an up migration of PERK during protein gel electrophoresis. However, this up-shift is less significant compared to the shift induced by the ER stressors tunicamycin or thapsigargin Figure 1. Schematic representation of the ER stress sensors and their mechanism of activation and action. PERK is auto-trans phosphorylated and activated when ER stress is developed. After active, its kinase domain phosphorylates the alpha subunit of the translation factor eIF2a, which inhibits the global synthesis of proteins favoring the translation of some mRNA such as ATF4. IRE1 is also activated by phosphorylation. Once active, its RNase function mediates the splicing of XBP1u, producing XBP1s, which is a transcription factor that regulates the expression of chaperones and proteins that participate in the UPR. The last sensor is ATF6, which after its activation, is translocated to Golgi where is cleaved by the proteases S1P and S2P. The cytosolic fraction of ATF6 (p50ATF6) is also a transcription factor that, as XBP1, regulates the expression of chaperones and proteins that participate in the UPR. BiP is the main chaperone protein in the ER, and it also regulates the activation of the three sensors of the UPR. ER, endoplasmic reticulum; UPR, unfolded protein response.
in HaCaT cells, suggesting that UVB could be inducing an unknown modification on PERK (34,47,48). These results further suggest that UVB induces posttranslational modification and activation of PERK (characteristic migration pattern) which might be via a non-canonical pathway. Besides UVC and UVB, UVA at a dose range of 0.2-10 J cm À2 has also been shown to trigger ER stress, PERK activation and eIF2a phosphorylation (52). In summary, there is mounting evidence that all three bands of UV could induce eIF2a phosphorylation (Table 1). However, the UV-induced activation of the PERK-eIF2a phosphorylation signaling pathway is highly dependent on UV wavelength, dose, time, as well as cell lines, and even cell culture conditions. For example, eIF2a phosphorylation is easily detected in HaCaT cells after UVB but not solar UV exposure when the cells were cultured in DMEM with 10% FBS. Instead, when cells were cultured in 1% FBS, eIF2a phosphorylation can be observed not only post-UVB but also post-solar UV irradiation (unpublished data).

UV and IRE1
IRE1a is an endonuclease and its role is to relay information about misfolded proteins from the ER to the nucleus (62). When sensing a build-up of misfolded proteins, IRE1a is dimerized and activated after the BiP protein is released from its luminal domain, which subsequentially results in an unconventional splicing of the X-box Binding Protein-1 (XBP1) mRNA ( Fig. 1). This splicing causes a shift in the reading frame, producing the transcription factor XBP1s as the final product. XBP1s transcriptionally regulates the expression of proteins involved in the process of protein folding, secretion, degradation associated with ER (ERAD), translocation of proteins across the ER and lipid synthesis, among others (4,63-66). IRE1a is also known for mediating the degradation of a specific subset of mRNAs, a mechanism known as RIDD (for Regulated IRE1-dependent decay) which is also involved in maintaining the cellular proteostasis (67)(68)(69)(70).
UVB, at a dose range between 10 and 40 mJ cm À2 , was the first to be shown to increase the expression of XBP1 mRNA and calnexin protein (a protein chaperone regulated by XBP1) in HaCaT cells (45). Then, the positive effect of UVA on the level of XBP1s at a dose range of 1-10 J cm À2 was reported (52). Both results are indicative of IRE1a pathway activation under the described conditions. In contrast, UVB 30 mJ cm À2 was also reported to reduce the levels of phosphorylated IRE1a and XBP1s in primary keratinocytes cells (37) and HaCaT cells after UVB 50 mJ cm À2 (54). Under our experimental conditions, the  effect of UVB (10 and 50 mJ cm À2 ) on IRE1a in HaCaT cells was not detected (47,48) (Table 1). Thus, the question regarding whether UV induces IRE1a activation remains contradictory.

UV and ATF6
ATF6 is a type-II glycoprotein and a transcription factor. Under ER stress and after BiP releases it, ATF6 translocates from the ER to the Golgi apparatus where it is cleaved by the proteases S1P and S2P (71). ATF6 contains a basic-leucine zipper (bZIP) transcription factor domain at its cytosolic N-terminal domain, which is released after its cleavage in Golgi (72). The cleaved fragment of ATF6 (also called p50ATF6) is a transcription factor that likewise regulates the expression of various UPR genes (71)(72)(73). Cleaved ATF6 binds to its target DNA by recognizing the ER stress response element (ERSE) sequence present in many of the ER stress-responsive mammalian genes, such as the ERchaperones GRP78/BiP, calreticulin and GRP94, all involved in refolding proteins (4,72,74) (Fig. 1). A dose of 0.5 mJ cm À2 of UVC activates ER stress-ATF6a signaling cascade mediating ER stress-induced cellular senescence in normal and transformed cell lines (MCF-7, Caki-1 and MRC-5) (53). UVB, at a dose range between 10 and 40 mJ cm À2 , reduces the full-length ATF6 protein levels and increases the nuclear translocation of ATF6 suggesting the activation of ATF6 (45). UVA also triggers the activation of the ATF6a sensor (52,59). The full-length ATF6a was cleaved while the expression of nuclear ATF6a was upregulated in normal human dermal fibroblasts after exposing the cells to a dose of 500-2000 mJ cm 2 of UVA. Conversely, higher UVA doses also diminished the ATF6a protein (52). As with IRE1, under our experimental condition, UVB fails to activate ATF6a in HaCaT cells, but instead, it significantly decreases the total protein levels of ATF6a (47,48) ( Table 1). The studies focus on ATF6a since the isoform b has been described as a weak regulator of gene expressions and acts mostly as a repressor of ATF6a (75). However, on the heart tissue, ATF6b showed to have overlapping roles with ATF6a (76).

UV RADIATION, UPR AND AUTOPHAGY
UV induces an autophagic response, which participates in the elimination of damaged or senescent compounds, and provides energy in case of metabolism alteration through the degradation and reutilization of building blocks such as amino acids, nucleotides and fatty acids (19). Even though the mechanisms are still not fully understood, autophagy is often associated with the regulation of cell survival or death as being recently reviewed (77,78). In the skin, autophagy is also associated with photoaging prevention (79)(80)(81). ER stress and the activation of the UPR Radiation (0-6 Gy) Radiation-induced autophagy is PERK/eIF2a-dependent LNCaP Tunicamycin PERK and ATF4 are required for basal and ER stress-induced autophagy ATF4 was required for autophagosome formation, while PERK was required after this step (96) are strongly involved in autophagy regulation, either as a prosurvival or pro-death mechanism depending on the type of stimuli and the type of cells (82). Even though the relation between UV, ER stress-UPR components and autophagy has not been extensively studied, they are expected to be related since (1) UV activates ER stress and the UPR pathways (  (3) UV induces the autophagy response (10)(11)(12)81,115,116). One of the regulatory factors that connect UV with ER stress/UPR pathways and autophagy could be oxidative damage as UV is known for causing the production of ROS and RNS, which are associated with autophagy flux control (13)(14)(15)(16)(17). Indeed, ionizing radiation, which also induces ROS formation, was reported to induce autophagy through the activation of the PERK and IRE1 in a murine macrophage cell line (RAW 264.7), as well as in peritoneal macrophages from C57BL/6 female mice (83). Upon UV irradiation, autophagy response is a mechanism of cell survival that removes damaged cellular components such as oxidized lipids, aggregated proteins and organelles such as mitochondria; as well as regulates UV-induced DNA damage repair (79,80). However, its activity also showed to be important in mediating inflammatory and tumorigenesis processes (12,115,117,118). Autophagy plays contradictory roles regarding the regulation of tumorigenesis. It suppresses the early-stage tumorigenic process through the elimination of dysfunctional cellular compounds that are responsible for causing oxidative and genomic damage. In later stages of tumorigenesis, autophagy assists cancer cells to adapt to metabolic stress, promoting cell survival and progression (119)(120)(121). The known roles of autophagy after UV irradiation have been reviewed extensively (79,81,116), and are not going to be further explored here.

A brief overview of the autophagy process
The mechanism of autophagic degradation of cellular components is done by the formation of structures called autophagosomes, which as the final step fuse with lysosomes (19). The formation of autophagosomes is divided into four steps: initiation, formation/nucleation, elongation/expansion and maturation/ degradation. Briefly, the autophagy mechanism starts with the formation of the phagophore assembly site, mediated by the ULK complex (ULK1-ATG13-FIP200-ATG101) located at the ER membrane. Then, phosphatidyl inositol 3-kinase (PI3K) is recruited to this site, forming domains called "omegasomes" due to their shape (Ω). PI3K complex (formed by VPS34, VPS15/ p150, and BECN1, plus ATG14L or UVRAG and regulatory proteins) phosphorylates phosphatidyl inositol (PI) to phosphatidyl inositol 3-phosphate (PIP3). Next, the exposure of PIP3 allows the recruitment of WIP12B, the latter recruiting the complex ATG12-ATG5-ATG16L. This last complex facilitates the lipidation of MAP1LC3 and GABARAP with phosphatidylethanolamine. Lipidated MAP1LC3 (LC3-II) is integrated into the growing phagophore (MAP1LC3 processing is also dependent on the activity of ATG7, ATG3 and ATG10) ( Fig. 1 from ref. (122)). Once the autophagosome is formed, its merges with a lysosome forming the autolysosome in a process dependent on tethering (SNARES), motor and Rab proteins (Rab7) (122)(123)(124)(125).
The roles of PERK, eIF2a phosphorylation and ATF4 in the regulation of autophagy post-UV exposure A time-dependent dynamic autophagic response was observed (10), which initially correlates positively (Pearson Corr. = 0.996, P = 0.0037) with the protein levels of phosphorylated eIF2a at 1 h post-UVB, but then there was a negative correlation between Autophagy was detected to be induced by the IRE1-JNK pathway (but not by XBP1) Jurkat and peripheral blood mitogen-activated T cells L-Arg depletion-induced ER stress IRE1 is required for autophagy activation after L-Arg depletion (102) them (Pearson Corr. = À0.98, P = 0.01) at 6 h post-UVB (126). Our analysis of autophagy flux in keratinocytes indicates that UVB (25 and 50 mJ cm À2 ) induces the autophagosome formation shortly after UVB (1 and 3 h after irradiation), but at longer times (6 h after irradiation), we also observed an inhibition on autophagosome formation (10). In addition, both eIF2a phosphorylation and the autophagy induction after UVB were cNOS dependent since the treatment of a cNOS inhibitor impaired this response (10). The result indicates that oxidative stress post-UVB could be a regulator of autophagy, as is also stated by others (13)(14)(15)(16)(17). Our results agree with the studies of Gu et al. on the impact of UVB on autophagy regulation which in parallel analyzes elements of the UPR pathway. The studies revealed that keratinocyte cells exposed to UVB downregulates the autophagy response by decreasing the expression of some autophagy-related genes (Atg) and reducing the protein levels and activation of PERK and IRE1, but not the phosphorylation of eIF2a (11,54). The observed inhibition of autophagy suggests a transient and quick induction of the autophagy response after short-wavelength UVB in keratinocytes which is later inhibited. The eIF2a phosphorylation has been reported as an essential factor for the autophagy response after stress stimuli such as starvation (via GCN2) or virus infection (via PKR) in yeast and MEF cells (106). Among the four known eIF2a kinases, PERK (EIF2AK3) and GCN2 (EIF2AK4) are activated by UV radiation (43,49,127,128). The eIF2a phosphorylation mediated by either GCN2 (by starvation) or PERK (by ER stress), participates in the regulation of autophagy via ATF4 on MEF cells (84). Indeed, numerous studies describe ATF4 as an important transcription factor involved in autophagy regulation (84,85,87,89,90,96). Interestingly, ATF4 protein levels after UVB only increase transiently, and long after UVB its levels become negatively correlated to the levels of phosphorylated eIF2a (48). This fact could be related to the late inhibition of the autophagy response observed by us and others on keratinocytes after UVB (10,11). Regarding the PERK-eIF2a-ATF4 signaling cascade, the importance of constituents of this arm of the UPR in regulating autophagy and cell fate has been described in different cell lines. In MEF cells, the transcription of SQSTM1 (cargo adaptor protein), ATG7 (autophagy-related protein) and NBR1 (cargo adaptor protein) were upregulated by ATF4 and CHOP simultaneously. However, in these same cells, the autophagy-related protein ATG16L1, MAP1LC3B, ATG12, ATG3, BECN1 and GABARAPL2 were ATF4-dependent but CHOP-independent; while ATG10, GABARP, and ATG5 were upregulated by CHOP (84). In C2C5 and MEF cells, autophagy was induced after the treatment with polyglutamine 72 (polyQ72). This treatment induces ER stress which, through the activation of the PERK/eIF2a pathway, converts LC3-I to LC3-II via upregulation of the Atg12 mRNA, suggesting the activation of the ATG5-ATG12-ATG16 complex (85). In the human P493-6 B cell line, as well as in MEF cells, and murine models, it was also observed a relation between UPR and autophagy. In these cells, c-Myc (which increases protein synthesis inducing ER stress) was responsible for activating the PERK arm of the UPR, helping to induce a cytoprotective autophagy (86). Moreover, a study comparing the response of the tumor cell lines HT29, MCF-7, U373 and HCT116 indicates that hypoxia induces the conversion of LC3-I to LC3-II and the autophagy flux as a mechanism of survival also by a PERK-dependent pathway (87). Hypoxia causes the upregulation of MAP1LC3B, ATG5 and ULK1 mediated by the transcription factors ATF4 and CHOP (87)(88)(89) (Table 2).
Interestingly, Milani et al. (90) described in the tumor cell line MCF-7, an important role of ATF4 regulating autophagy by a PERK-independent mechanism. Indeed, they observed that the induction of autophagy depends on the proteasomal stabilization of ATF4 which upregulates MAP1LC3B. This role of ATF4 in autophagy induction independent of PERK was again reported later in a prostate cancer cell line (LNCaP) by the group of Engedal N (96). This group observed that tunicamycin induces autophagy mediated by PERK and ATF4. However, PERK and ATF4 could serve independent roles in regulating autophagy through regulating different steps of the process. For example, ATF4 was required for autophagosome formation, while PERK was required after this step (96). More recently, a PERKindependent role of ATF4 and CHOP in stressed LNCaP and HCT116 cell lines was reported to regulate cell death by upregulating DR5 and MAP1LC3B (110) ( Table 2).
It is necessary to highlight that many studies (Tables 2-4) relate the ER stress induced by thapsigargin with autophagy; however, they show controversial results regarding (1) the relation between ER stress and the autophagy response and (2) the pro-dead and pro-survival role of autophagy (or of autophagy components). However, these differences could be related to the dose of thapsigargin, the cell type studied or even the methods used to evaluate the autophagy (82,91,93,(107)(108)(109)(110). In conclusion, eIF2a could directly or indirectly, as well as positively or negatively, regulate the expression of components and/or regulators involved in the autophagy pathway at transcriptional and/or translational levels (84)(85)(86)(87)90,96,110,129).
The roles of IRE1a and XPB1 in the regulation of autophagy post-UV exposure The IRE1 pathway has been involved in the negative and positive regulation of autophagy (Table 3). In U2OS and LNCaP cells, the IRE1 knockdown induced an enhanced autophagy and Caco-2 Tunicamycin and thapsigargin Knockdown of ATF6a has little effect on the upregulation of LC3-II (93) response under basal conditions (96). Also, in Huntington's disease mice and cell culture models, the XBP1 deficiency induced protective autophagy that mediates the clearance of the mutant Huntingtin protein by the augmented expression of Forkhead box O1 (FoxO1) (92), while IRE1 overexpression and its activation inhibited autophagy via the IRE1-TRAF2 pathway (98). Contrarily, in three human colon cancer cell lines (HT29, SW480 and Caco-2), autophagy showed to be dependent on the IRE1 pathway since the knockdown of IRE1a (and CHOP) inhibits the ER stress-induced autophagy response (93). In endothelial cells, XBP1s regulate autophagy by inducing BECLIN1 expression (97). Another mechanism was described in the neuroblastoma cell line SK-N-SH and MEF cells, which involves IRE1 and the c-Jun N-terminal kinase (JNK). This work shows autophagosome formation was accelerated when the cells were treated with the ER stressors tunicamycin and thapsigargin. The authors concluded not only that autophagy plays pivotal role in protecting the cells against death induced by ER stress, but also that the IRE1-JNK pathway was required for autophagy activation after ER stress (91). However, in HCT116 and MEF cells in which the proteasome was inhibited causing ER stress, autophagy was detected to be induced as a mechanism of cell survival regulated by the IRE1-JNK pathway but not by XBP1 (101). It is postulated as a possible mechanism of this IRE1-JNK-dependent induction of autophagy, that could be through the regulation of BECLIN1's dissociation from members of the anti-apoptotic Bcl-2 family proteins (102), and/or autophagy-genes regulation via FoxO and c-Jun/c-Fos (111,112,114). Moreover, and as well as with IRE1 and XBP1, JNK has been described to be a negative regulator for autophagy (113,114). Suggesting that autophagy regulation via IRE and XBP1 is context/stimuli-and cell-type specific ( Table 3).
The results of Son et al., Gu et al., and our analysis of IRE1a and XBP1 in keratinocytes exposed to UVB, suggest no activation of this arm of the UPR (37,48,54,130). These antecedents indicate that in keratinocytes exposed to UVB, this pathway may not be involved in autophagy induction. However, we cannot exclude that the lack of activation of these components after UVB could be related to the dynamic of the autophagy response observed in keratinocytes, as described under other cellular stressors (131). In this context, it is important to mention that no significant changes in BECLIN1 protein levels, which expression is regulated among others by XBP1s as discussed before (97), were observed at 1 h post-UVB by us, while a significant decrease was only observed at 6 h after UVB 50 mJ cm À2 in HaCaT cells (unpublished results).
The roles of ATF6 in the regulation of autophagy post-UV exposure Finally, there is little evidence available that links the ATF6 arm of the UPR with the regulation of the autophagy mechanism ( Table 4). The only direct evidence relating ATF6 and autophagy has been observed under virus infection (94,104,105), and in the context of IFN-c stimulation (103). In these works, ATF6 activation was necessary for autophagy induction. Moreover, it is known that ATF6 is one of the transcription factors that regulate the expression of GRP78/BiP as well as XBP1s and CHOP after UPR activation (63,71,74,132,133). As previously reported, these proteins are all involved in the autophagy regulation (84,87,92,93,97,109,(134)(135)(136).
Our results also indicate that after UVB the protein levels of the full-length ATF6a decrease, as stated also by Mera et al. (45), but instead, no changes in p50ATF6a nor GRP78/BiP protein levels were observed by us (48), suggesting that after UVB these components may not be involved in the induction of autophagy. Again, we cannot rule out the fact that the lack of induction of these components is related to the lack of or transient autophagy response observed after UVB in keratinocytes.

CONCLUSION
The antecedents available in the literature show significant contradictions regarding the activation of the UPR and autophagy post-UV exposure. The differences could be due, for example, to the cell models, time of analysis, culture condition and UV wavelength used. Autophagy seems to be regulated by UPR components after some stress stimulus. However, after UV irradiation other signaling pathways come into play, positively and negatively regulating the dynamic autophagy process (Fig. 2).

FUNDING
This work was partially supported by NIH ES030425 (to S. Wu).

CONFLICTS OF INTEREST
There are no conflicts to declare. Figure 2. Schematic representation of the hypothetical connections discussed in this review between UV radiation, autophagy, and ER stress. The literature strongly suggest that ER stress positively regulate autophagy via PERK/eIF2a/ATF4 pathway. However, the protein levels of ATF4 are also negatively regulated by UV via an unknown pathway, which can be a factor counteracting the induction of autophagy. UV irradiation also shows to induce ER stress and autophagy. However, this induction appears to be highly dependent on the cell type and experimental conditions. The roles of IRE1a and ATF6 pathways in regulating autophagy are unclear. ER, endoplasmic reticulum; UV, ultraviolet.