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Effects of the P16/Cyclin D1/CDK4/Rb/E2F1 Pathway on Aberrant Lung Fibroblast Proliferation in Neonatal Rats Exposed to Hyperoxia

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Effects of the P16/Cyclin D1/CDK4/Rb/E2F1 Pathway on Aberrant Lung Fibroblast Proliferation in Neonatal Rats Exposed to Hyperoxia EXPERIMENTAL AND THERAPEUTIC MEDICINE 22: 1057, 2021 Effects of the p16/cyclin D1/CDK4/Rb/E2F1 pathway on aberrant lung fibroblast proliferation in neonatal rats exposed to hyperoxia SHIMENG ZHAO, ZHIGUANG CHEN, SHUANG HAN and HONGMIN WU Department of Neonatology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China Received August 10, 2020; Accepted March 11, 2021 DOI: 10.3892/etm.2021.10491 INK4a Abstract. p16 (p16) inhibits the vital G1 to S phase fibrosis without affecting tissue repair is the key to curing. To transition during cell cycle progression through the p16/cyclin address this problem, the mechanism that regulates the cell D1/CDK4/retinoblastoma(Rb)/E2F1 pathway. Hyperoxia can cycle during LF proliferation needs to be clarified. suppress the G1/S checkpoint and induce more lung fibroblasts A cell divides into two identical cells through the precisely (LFs) to transition from the G1 phase to the S phase and undergo controlled G1, S, G2 and M phases of the cell cycle. During cell proliferation. The present study investigated the rate of p16 cell cycle progression, cells that pass the G1/S checkpoint of gene promoter methylation and the protein expression levels of the cycle, characterized by the initiation of DNA synthesis, p16, cyclin D1, CDK4, Rb and E2F1 in LFs from the lungs of are committed to dividing into two complete cells (3). The rats exposed to hyperoxia and normoxia on postnatal days 3, 7 p16INK4a (p16)/cyclin D/CDK4/retinoblastoma‑associated and 14. In the hyperoxia‑exposed group, the methylation rate protein (Rb)/E2F1 pathway is recognized as a key pathway in was 50 and 80% on days 7 and 14, respectively. Cyclin D1 the regulation of cell proliferation, for which E2F1 is a notable and CDK4 overexpression was associated with p16 loss and Rb downstream effector. Deregulated E2F1 activity due to the inactivation by phosphorylation. Rb phosphorylation induced aberrance of the upstream components in this pathway, such as E2F1 release in the G1 phase, which promoted cell proliferation. inactivation of p16, confers a growth advantage to cells (4‑7). No methylation was observed in the normoxia‑exposed group. During the G1 phase of the normal cell cycle, Rb is inactivated by These observations suggested that p16 loss may stimulate sequential phosphorylation events that are mediated by various aberrant LF proliferation via the p16/cyclin D1/CDK4/Rb/E2F1 cyclin‑dependent kinases (CDKs), leading to the release of E2F pathway. transcription factors, the activation of numerous genes (such as p19ARF and p53 et al) and cell cycle progression (8). p16 is Introduction a cyclin‑dependent kinase inhibitor that positively regulates the expression of cyclin D1 at the post‑transcriptional level. Premature infants with respiratory disorders usually require p16 and cyclin D1 competitively bind to CDK4 to prevent cells inhalation of high levels of oxygen. Prolonged exposure to high from entering the corresponding phase of the cell cycle and, levels of oxygen may lead to lung injury, which often results in therefore, inhibit its protein kinase activity. This inhibition widespread lung fibrosis (1). The main manifestation of lung suppresses the CDK4‑mediated phosphorylation of the Rb fibrosis is the uncontrolled proliferation of lung fibroblasts protein. When phosphorylated (p)‑Rb binds to E2F1 to form (LFs) that are deposited in the pulmonary interstitial space or a complex, the transcriptional activation of E2F1 is inhibited, replace the normal lung epithelium (2). The hyperplasia and thereby preventing the cell from transitioning from the G1 to replacement of fibrous tissue after tissue injury are noteworthy the S phase and inhibiting cell proliferation (6,9). Therefore, pathophysiological processes of tissue repair. Discovering how together, p16, CDK4, Rb and E2F1 constitute a feedback loop to effectively prevent LF proliferation in order to alleviate lung that directly regulates the cell proliferation cycle. Once the p16 gene is mutated, transduction through the loop is unable to proceed. Our previous study found that LFs isolated from rats exposed to hyperoxia in vivo showed significantly greater Correspondence to: Dr Shimeng Zhao, Department of Neonatology, The First Affiliated Hospital of China Medical University, 155 Nan proliferation compared with those from rats exposed to Jing Northern Street, Shenyang, Liaoning 110001, P.R. China normoxia. In addition, using flow cytometry, it was revealed E‑mail: [email protected] that the percentage of LFs in the S and G2/M phases increased, and the proportion of LFs in the G0/G1 phase decreased Key words: hyperoxia, lung fibroblasts, p16/CDK4/Rb/E2F1, cell simultaneously (10). In another study, we confirmed that proliferation 5‑aza‑2'‑deoxycytidine, a DNA methylation inhibitor, inhibited hyperoxia‑induced LF proliferation by restoring p16 expression (11). However, the underlying mechanism has not been investigated and the results from previous studies are 2 ZHAO et al: LOSS OF p16 STIMULATES LF PROLIFERATION VIA THE CDK4/RB/E2F1 PATHWAY incomplete. In the present study, the protein expression of p16, cultured in air and 5% CO2 at 37˚C in MEM with 10% FBS, cyclin D1, CDK4, Rb and E2F1 was measured in LFs from rats 10,000 U/ml penicillin and 10,000 µg/ml streptomycin for 72 h. exposed to hyperoxia or normoxia to further understand the The LF purity was confirmed at >95% by immunocytochemical occurrence and development of the aberrant LF proliferation staining for vimentin. Briefly, cultured LFs were fixed in induced by hyperoxia. 4% paraformaldehyde for 30 min at 37˚C. Next, a primary antibody against vimentin (1:100; cat. no. sc‑5565; Santa Cruz Materials and methods Biotechnology, Inc.) was added to the cells and incubated overnight at 4˚C. Then, cells were stained with goat anti‑rat Animals and oxygen exposure. The present study was secondary antibody (1:100; Beijing Zhongshan Golden Bridge approved by The China Medical University Animal Research Biotechnology Co., Ltd.) for 30 min at 37˚C. When passaged, Committee (Shenyang, China; approval no. 2017PS140K). the cells were seeded into 150‑cm2 culture flasks at a density Healthy Sprague‑Dawley rats (n=40; female:male, 4:1) of 2.5x105 or 5x105 cells/flask. All the experiments were obtained from The Center of Animal Experiments, China performed using fibroblasts from passage number two. Medical University, were used for this experiment. The higher the concentration of oxygen inhaled, the more obvious MTT assay. A 3‑(4,5‑dimethy‑lthiazol‑2‑yl)‑2,5‑diphenyltet‑ the changes in lung fibrosis. A fraction of inspired oxygen razolium (MTT) assay was performed for cell proliferation. 4 (FiO2) >85% (the lethal oxygen concentration) is frequently Cells were seeded into 96‑well plates at a density of 5x10 cells employed in experimental studies (12‑14). In the present study, per well and cultured at 37˚C in air and 5% CO2 for 24 h. the newborn rats (<12‑h old) were randomly divided into the Next, 20 µl of 5 mg/ml MTT (Sigma‑Aldrich; Merck KGaA) normoxia (FiO2, 21%) or hyperoxia (FiO2, 90±2%) groups. was added into each well and the cells were cultured for an The hyperoxia group was monitored twice daily (MT‑01 additional 4 h at 37˚C. After culture, the supernatant was Gas Monitor). CO2 was absorbed by soda lime to maintain a discarded and the resulting formazan crystals were solubilized concentration of <0.5%. The average birth weight of the rats by adding 150 µl of DMSO to each well. The optical density was 5.12±0.26 g in the hyperoxia group and 4.98±0.19 g in level was measured at a wavelength of 570 nm. Experiments the normoxia group. No statistically significant difference were performed in quintuplicate. was observed. Within 12 h of birth, the newborn rats (with the mother rats) were housed in standard cages placed inside Methylation‑specific polymerase chain reaction (MSP). LFs 425‑l capacity plexiglass isolation chambers that received from the hyperoxia and normoxia groups were collected humidified 2O or room air (30l/min) at ambient pressure for at 120 h post‑incubation in MEM with 10% FBS. A ZR‑96 up to 14 days. Each rat was fed at 20‑26˚C and 12 h light/dark Quick‑gDNA™ kit (Zymo Research Corp.) was used to cycle. The cages were opened for 0.5 h every day to add extract the DNA as recommended by the manufacturer. standard laboratory food and water and change the padding. An EZ DNA Methylation‑Gold™ kit (Zymo Research To avoid oxygen toxicity, the mother rats were rotated between Corp.) was used to perform the bisulfite modification of the the normoxia and hyperoxia environments every day. genomic DNA according to the manufacturer's instructions. PCR amplification was performed using p16 promoter gene Histological examination. On postnatal days 3, 7 and 14, 5 pup fragment‑specific primers for methylated or unmethylated rats in each group were sacrificed with an intraperitoneal DNA (Sangon Biotech Co., Ltd.). The primers used to amplify injection of pentobarbital sodium (100 mg/kg) and were unmethylated p16 were as follows: Sense, 5'‑TTT TTG GTG exsanguinated by aortic transection. Right lung tissue samples TTA AAG GGT GGT GTA CT‑3' and antisense, 5'‑CAC AAA were washed with PBS, placed in 4% paraformaldehyde at AAC CCT CAC TCA CAA CAA‑3', which yielded a 132‑bp room temperature for 24 h, then serially dehydrated with fragment. The primers used to amplify methylated p16 were increasing concentrations of ethanol before being embedded in as follows: Sense, 5'‑GTG TTA AAG GGC GGC GTA GC‑3' paraffin. The paraffin‑embedded lung tissue samples were cut and antisense, 5'‑AAA ACC CTC ACT CGC GAC GA‑3', which into 4‑µm sections, stained with hematoxylin and eosin (H&E; yielded a PCR product of 122 bp.
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