Role of the Endoplasmic Reticulum Stress Sensor IRE1 alpha in Ultraviolet B Radiation-Induced Damage Response
Open Access
- Author:
- Son, Jeongin
- Graduate Program:
- Molecular, Cellular, and Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 17, 2021
- Committee Members:
- Douglas Cavener, Major Field Member
Adam Glick, Chair & Dissertation Advisor
Girish Kirimanjeswara, Major Field Member
Daniel Hayes, Outside Unit & Field Member
Kumble Prabhu, Major Field Member
Melissa Rolls, Program Head/Chair - Keywords:
- unfolded protein response
UPR
ER stress
IRE1α
Calcium homeostasis
CIB1
UVB
Apoptosis
DNA Damage Repair
ROS
NRF2
Oxidative stress
Inflammation
Angiogenesis - Abstract:
- The endoplasmic reticulum (ER) is an important subcellular organelle for calcium storage and protein folding. Maintaining ER homeostasis is an essential process for cell survival. The ER can be challenged by numerous environmental insults, such as temperature, osmosis, toxic chemicals, and sunlight. To sense and respond properly to ER stress, there are three unfolded protein response (UPR) sensory molecules in metazoan cells: IRE1α, PERK, and ATF6. Although there are several reports about the association between the ER stress response and UVB irradiation, the role of activated UPR proteins in the UVB response has not been studied. In particular, the role of IRE1α under UVB-mediated stress is not clear. Here we showed for the first time that epidermal disruption of IRE1α can increase cell sensitivity to UVB irradiation with increased apoptosis and skin sloughing, and reduced γH2AX formation. As chemical inhibition of IRE1α’s RNase activity and knockdown of XBP1, an IRE1α downstream gene, through shRNA did not phenocopy the IRE1α deficiency, IRE1α dependent UVB sensitivity was independent of IRE1α’s RNase function. In addition, the increased UVB sensitivity in IRE1α deficient cells was associated with [Ca2+]i and ROS dysregulation. By using 2APB, an InsP3R inhibitor, we demonstrated that IRE1α deficiency-induced ER-to-cytosol calcium efflux through InsP3R, and basal [Ca2+]i increased. Pharmacologic and siRNA-mediated genetic approaches linked this [Ca2+]i dysregulation with UVB-mediated DNA damage repair (DDR) alteration. Also, the involvement of Calcium and Integrin binding protein 1 (CIB1) regulation through the IRE1α-TRAF2-ASK1 complex in UVB irradiated keratinocytes was revealed. These results indicate the essential role of IRE1α in [Ca2+]i homeostasis through CIB1 regulation by the formation of the IRE1α-TRAF2-ASK1 complex. This IRE1α-dependent calcium homeostasis is essential for UVB-mediated ER calcium efflux, which is essential for proper UVB-DDR. We further investigated the increased ROS signaling in IRE1α deficient cells. In chapter 4, we showed that in the absence of IRE1α, the stability and activation of PKR-like endoplasmic reticulum kinase (PERK) was compromised after UVB irradiation. Following UV irradiation, IRE1α deficiency not only caused PERK protein degradation but also suppressed nuclear factor erythroid-2-related factor 2 (NRF2) phosphorylation. This loss of NRF2 activation resulted in impairment of NRF2 dependent antioxidant gene expression, such as Ho-1, Nqo-1, Gclc, and Gclm. This PERK protein degradation in IRE1α deficient keratinocytes was not a UVB-specific response. Thapsigargin mediated PERK degradation in IRE1α knocked down keratinocytes indicated a general regulation of PERK stability by IRE1α. To answer the question if UVB-mediated skin inflammation was IRE1α dependent, the acute and chronic UVB response was tested in the skin of mice. To test this, we analyzed the recruited immune cell population and gene expression of the pro-inflammatory cytokines, IL-6 and TNFα. Notably, there were significantly fewer recruited neutrophils and leukocytes in UVB irradiated Ire1αΔep mice skin and decreased expression of pro-inflammatory cytokines. In chronic UVB exposure (6 weeks), there was significantly more Vegf, a pro-angiogenic gene, and proliferative cells in control mice skin than the Ire1αΔep mice. Therefore, we speculate that IRE1α is essential for the basal and UVB-induced oxidative stress, and UV skin immune responses. Also, IRE1α may be essential for proper UV-skin damage repair. Together, these studies establish a novel role for IRE1α in UVB-mediated stress as an immune and intracellular calcium regulator. These studies could highlight IRE1α as a potential therapeutic target of skin damage. Also, these data provide an important insight into the role of IRE1α as a cellular signaling modulator between UPR and DDR/ ROS signaling.