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UVB Induces Apoptosis Via Downregulation of CALML3-Dependent JNK1/2 and ERK1/2 Pathways in Cataract

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UVB Induces Apoptosis Via Downregulation of CALML3-Dependent JNK1/2 and ERK1/2 Pathways in Cataract INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 41: 3041-3050, 2018 UVB induces apoptosis via downregulation of CALML3-dependent JNK1/2 and ERK1/2 pathways in cataract YIN JIA1*, QIN QIN1*, CHAO-PING FANG1, WEI SHEN2, TING-TING SUN1, YUAN-LAN HUANG3, WEN-JIE LI1 and AN-MEI DENG1 Departments of 1Laboratory Diagnosis and 2Ophthalmology, Changhai Hospital, The Second Military Medical University, Shanghai 200433; 3Medical Department, The 455th Hospital of PLA, Shanghai 200052, P.R. China Received November 13, 2015; Accepted January 19, 2018 DOI: 10.3892/ijmm.2018.3478 Abstract. The aim of the current study was to understand of CALML3 and involved oxidative stress, Ca2+, JNK1/2 and the mechanisms of apoptosis occurring in cultured human ERK1/2 signaling pathways, suggesting that investigating lens epithelial cells (HLECs) following ultraviolet B (UVB) CALML3 may useful for developing cataract treatment. irradiation. The investigations intended to confirm the pres- ence of apoptosis and to reveal the roles of oxidative stress, Introduction calcium (Ca2+), c-Jun NH2-terminal kinase (JNK)1/2, and extracellular signal-regulated kinase (ERK)1/2 signaling Cataracts are a cause major cause of blindness worldwide. It pathway in these progresses. Cell apoptosis, ROS genera- is reported that a number of factor lead to the development 2+ tion and intracellular Ca concentration was measured by and progression of cataracts, including H2O2, glucose and flow cytometry. The expression of CALML3, caspase-3, prolonged exposure to sunlight (1,2). Ultraviolet B (UVB) is Bax, Bcl-2, p-JNK1/2, JNK1/2, p-ERK1/2 and ERK1/2 associated with the formation of cataracts leading to cortical was measured by RT-qPCR and western blot analysis. lens opacity. The lens epithelium is a single layer of cells that Annexin V-fluorescein isothiocyanate/propidium iodide regulates lens homeostasis; however, the lens epithelium is staining demonstrated that UVB irradiation increased the exposed to UVB irradiation, as the top layer of cells in the apoptotic rate, reactive oxygen species (ROS) production eye (3). Even relatively low UVB irradiation damages the and intracellular Ca2+ concentration of HLECs in dose- and lens epithelium and causes a loss of ion homeostasis, which time-dependent manners. Overexpression of calmodulin like 3 contributes to the development of cataracts in the aging popu- (CALML3) reversed the effects of UVB irradiation on apop- lation (4). The precise mechanism of the association of UVB tosis, ROS production and Ca2+ concentration of HLECs, and irradiation with cataract development in humans unknown. decreased expressions of caspase-3 and Bax, with increased Apoptosis is a regulated cell death mechanism that main- expressions of Bcl-2. Notably, silencing of CALML3 had tains homeostasis, and has a role in cataract formation in similar effects to UVB irradiation and inhibited the activation humans (5). Loss of mitochondrial transmembrane potential JNK1/2 and ERK1/2 pathways. Nimodipine, a Ca2+-channel and the production of reactive oxygen species (ROS) cause antagonist, significantly attenuated the damages induced by damage to cells and induce apoptosis. UVB irradiation CALML3 downregulation. In conclusion, UVB irradiation induces apoptosis through a number of molecular processes, induced increase in apoptosis, ROS production and Ca2+ concen- such as targeting Bcl-2 family proteins, and activating caspase tration of HLECs, in part, by downregulating the expression and cell apoptosis signaling pathways (6). Bcl-2 has a negative role in the cellular apoptotic pathway, whereas Bax, a Bcl-2 homologue, can reverse the inhibitory effect of Bcl-2 in apop- tosis. A previous study demonstrated that Bax expression and caspase-3 activity were decreased in cataracts, suggesting a Correspondence to: Dr Wen-Jie Li or Dr An-Mei Deng, Department pivotal role in cataract formation (7). The mitogen-activated of Laboratory Diagnosis, Changhai Hospital, The Second Military protein kinases (MAPKs), including extracellular signal-regu- Medical University, 168 Changhai Road, Yangpu, Shanghai 200433, lated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and P. R. Ch i na E-mail: [email protected] p38 MAPK, are involved in the regulation of cell apoptosis E-mail: [email protected] and are activated during UVB-induced apoptosis of human retinal pigment epithelial cells (8,9). *Contributed equally Oxidative stress caused by ROS has been reported in numerous diseases, particularly aged-associated disorders, Key words: cataract, ultraviolet B irradiation, human lens epithelial such as cataracts (10). Previous studies demonstrated that the cells, calmodulin like 3, apoptosis, c-Jun NH2-terminal kinase 1/2, lens has an antioxidant system to counter the toxic damage extracellular signal-regulated kinase1/2 of ROS, including the activity of antioxidantive enzymes and antioxidants, such as superoxide dismutase (SOD), catalase 3042 JIA et al: UVB induces apoptosis via downreGulation of CALML3 (CAT), glutathione peroxidase (GSH-PX) and malondialde- Flow cytometry analysis of apoptosis, ROS production and hyde (MDA) (11-13). Chang et al (14) reported that the serum calcium concentration. Prior to analysis of analysis of apop- SOD, CAT and GSH-PX activities were decreased and the tosis, ROS production and calcium concentration, the HLECs level of MDA was increased in patients with cataracts. were trypsinized, centrifuged and resuspended twice with PBS. Cellular calcium (Ca2+) homeostasis in the lens epithelial The HLECs (1x105 cell/ml) were treated with UVB with 2 W/m2 cells is essential for maintaining lens clarity. Inappropriate for 60 min, and subsequently collected and incubated with 5 µl Ca2+ signaling and abnormal Ca2+ levels are associated with Annexin V buffer and 5 µl propidium iodide (PI) for 10 min various clinical disorders, including stroke, cardiac dysfunc- for apoptosis detection, incubated with medium containing tion, obesity, aging and Alzheimer's diseases (15-18). Ca2+ is 10 µM dichlorodihydrofluorescein diacetate (DCFH-DA) for also involved in cell growth, differentiation and motility during 30 min for ROS detection and incubated with Fluo-3 (10 µM; the transformation and progression of cancer. Calmodulin Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for like 3 (CALML3) is an epithelial‑specific calcium sensing 30 min for intracellular calcium concentration detection. protein that highly expressed in oral mucosa, skin, breast and prostate (19,20). A previous study reported that CALML3 Determination of SOD, MDA, CAT and GSH‑PX activities. is regulated by short-term blockade of the MaxiK channel The HLECs (1x105 cell/ml) were treated with UVB with 2 W/m2 associated with an increase in basal Ca2+ concentration (21). for 60 min. The SOD (WST-1 method), MDA (TBA method), However, the function of CALML3 in cataracts has not been CAT (visible light) and GSH-PHX assay kits (obtained from fully elucidated and requires further investigation. Nanjing Jiancheng Bioengineering Institute, Nanjing, China) The current study investigated the effect of UVB irradiation were used to determine the antioxidant activities. SOD, MDA, on apoptosis, and whether oxidative stress, Ca2+ homeostasis CAT and GSH-PHX activities were measured at the absor- and apoptosis-associated signaling pathways have a role in bance of 450, 532, 405 and 412 nm, respectively. UVB-induced apoptosis in human lens epithelium cells. Reverse transcription‑quantitative polymerase chain reac‑ Materials and methods tion (RT‑qPCR) assay. Total RNA was isolated with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according Cell culture. Human lens epithelial cells (HLEC; an immortal- to the manufacturer's protocols. Complementary DNA was ized cell line) used in this study obtained from Cell Bank of synthesized using a cDNA synthesis kit (Thermo Fisher Academia Sinica (Shanghai, China), were cultured in Dulbecco's Scientific, Inc.). The cDNA synthesis conditions were as modified Eagle's medium (DMEM)‑high glucose (HyClone; follows: 37˚C for 60 min, followed by 85˚C for 5 min and 4˚C GE Healthcare Life Sciences, Logan, UT, USA) supplemented for 5 min. qPCR was performed to detect the mRNA levels with 10% fetal bovine serum (FBS) and 1% penicillin-strepto- of the indicated genes. qPCR was performed on ABI 7300 mycin solution in an incubator (Thermo Fisher Scientific, Inc., (Applied Biosystems; Thermo Fisher Scientific, Inc.) thermal Waltham, MA, USA) at 37˚C with 5% CO2. Cells were treated cycler using a standard SYBR‑Green PCR kit (Thermo Fisher with 0.04 mM nimodipine for 24 h Scientific, Inc.) according to the manufacturer's instructions. The qPCR cycling conditions were as follows: 95˚C for 10 min, UVB treatment. The UVB treatment was performed by using followed by 40 cycles at 95˚C for 15 sec and 60˚C for 45 sec, and a UVB radiometer with a total output of 4 W/m2 (wavelengths a final extension step of 95˚C for 15 sec, 60˚C for 1 min, 95˚C of 297 nm). Prior to irradiation, the HLECs were washed twice for 15 sec and 60˚C for 15 sec. The relative mRNA expression with phosphate-buffered saline (PBS). The HLECs were then of target gene compared with GAPDH was calculated using irradiated at the desired intensity and time: 60 min at 0.5, 1, the 2-ΔΔCq (22) method. The primers used in this study listed in 2 and 4 W/m2; 2 W/m2 for 5, 15, 30, 60, 90, 120 and 180 min. Table I. Following UVB irradiation the HLECs were incubated in DMEM-high glucose (HyClone; GE Healthcare Life Sciences) Western blot assay. HLECs were seeded at a density of supplemented with 10% FBS and 1% penicillin-streptomycin 5x105 cells per well in 6-well plates, cultured overnight and solution in an incubator (Thermo Fisher Scientific, Inc.) at then treated with UVB 2 W/m2 for 60 and 180 min. Total 37˚C with 5% CO2. proteins were isolated from the HLECs and were subjected to 12% SDS-PAGE, electroblotted onto to a polyvinylidene Lentivirus and small interfering RNA transduction. The fluoride membrane (Roche Diagnostics, GmbH, Mannheim, CALML3 coding sequence (GENEWIZ lnc., Suzhou, Germany).
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