Mechanism Of Retinoic Acid And Mitogen-activated Protein Kinases Regulating Hyperoxia Lung Injury

Background:Acute and chronic lung injury are major causes of mortality and morbidity in both preterm and term neonates. Prolonged exposure to hyperoxia in the developing lung is believed to play critical roles in the development of acute and chronic lung injury. In animal models, we and others have demonstrated that exposure to hyperoxia during critical window of alveolarization impairs lung septation, decreases alveolar number and internal surface area, enlarges of alveolar ducts and results in emphysematous changes similar to those found in patients with bronchopulmonary dysplasia (BPD). Simultaneously, the pathological changes includes severe disruption of the alveolar-capillary barrier, parenchymal cell injury followed by an inflammatory response and later by fibroblast proliferation and collagen accumulation, with the consequent distortion of the lung architecture, and eventually to lung fibrosis.Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases with crucial roles in extracellular matrix remodeling, acting in concert with their tissue inhibitors (TIMPs). Some of their functions regulate processes associated with development, such as branching morphogenesis and angiogenesis as well as inflammatory processes and wound healing. MMP-2 and MMP-9, also called gelatinases-A and -B, respectively. A critical effect of MMP-2 and MMP-9 in prenatal and postnatal lung development is to induce alveolar epithylial cell migration and branching morphogenesis by proteolytically cleavage extracellular matrix components. We and others have demonstrated that hyperoxia exposure lead to acute lung injury and inhibited lung development, accompaniment with MMP-2 and MMP-9 level and MMPs/TIMPs ratio markedly increased, extracellular matrix degradation and extensive tissue remodeling.Alveolar epithelial cells include type I cells (AEC I) and type II cells (AECII). The type II epithelial cell (the stem cells of the alveolar epithelium) is essential for normal repair after lung injury because it repopulates dead AEC I through proliferation, migration and differentiation, subsequently recovery the alveolar-capillary barrier. Numerous factors delicately regulate alveolar epithelial cell proliferation and apoptosis. Evidences suggest that the response to DNA damage may be important because DNA fragmentation and increased expression of the tumor suppressor protein p53 and its downstream target genes have been observed in murine lungs exposed to hyperoxia. p53 responds to DNA damage by arresting the cell cycle in G1 /S phase to allow DNA repair to take place and may result in apoptosis if the cell is unable to repair the DNA damage.The processes of lung growth, development, injury and repair are extremely complex, involving a multitude of effectors. Many of these effectors activate signaling pathways that converge into mitogen-activated protein kinases (MAPKs). There are three major families of MAPKs: the extracellular signal-regulated kinases-1 and -2 (ERK-1/2), c-Jun NH2-terminal kinases (JNK), and p38 kinases. Both ERK-1 and -2 are thought to be involved primarily in proliferation and differentiation, whereas JNK and p38 are believed to be involved in stress responses and apoptosis. Once activated, MAPKs regulate gene expression through phosphorylation of downstream transcriptional factors. Recent studies suggest that MAPKs are involved in the regulation of MMPs/TIMPs expression. Furthermore, MAPKs activity contributes to growth arrest and apoptosis. But the roles of MAPKs in hyperoxia-mediated premature lung MMPs/TIMPs expression have not been explored.Vitamin A is to date the only intervention tested by randomized clinical trials demonstrated to produce a decrease in relative risk of death or BPD at 36 weeks of post-menstrual age and has no visible side effect, but the exact mechanism has not been elucidated. Retinoic acid (RA) is vitamin A derivatives that regulate important biological functions, including cell growth and differentiation, development, and carcinogenesis. Recent data suggests that exogenous RA can improve alveolar structure, decreases fibrosis and regulates MMPs/TIMPs expression in the newborn rat with oxygen-induced lung injury. In addition, it has been reported that RA was involved in cell cycle regulation. But whether the protection of RA was related to regulating hyperoxia-induced activation of MAPKs was yet unknown.Objective:Establishment of hyperoxia lung injury animal and cells model:1. To further explore the role of MMPs/TIMPs in hyperoxia lung injury;2. To investigate whether MAPKs are involved in regulation of MMPs/TIMPs expression;3. To prove whether the protective effect of RA on hyperoxia lung injury was related with regulation of MMPs/TIMPs expression by MAPKs;4. To further investigate the role of MAPKs as modulators of oxidant-mediated proliferation, differentiation and apoptosis and the protective effect of RA on hyperoxia lung injury.Methods:1. Gastation 21 d Sprague-Dawley (SD) fetuses (term = 22 d) were delivered by hysterotomy. Within 12 h～24 h of birth, premature rat pups were randomly divided into 4 groups: Group I, Air-exposed control group; GroupII, hyperoxia-exposed group; Group III, Air-exposed plus RA group, Group IV, hyperoxia-exposed plus RA group. Group I and III were remained in room air, and group IIand IV were placed in 85% oxygen. The pups in Group III and IV were injected with RA (500μg/kg, every day) intraperitoneally. All lung tissues of premature rat pups were collected at 4 d, 7 d and 14 d after birth. The levels of MMP-2,MMP-9,MT1-MMP,TIMP-1 and TIMP-2 mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). MMP-2 and MMP-9 activity was measured by zymography. The protein abundance of TIMP-1,TIMP-2,p-ERK1/2,ERK1/2,p-JNK1/2,JNK1/2,p-p38 and p38 was determined by western blot.2. The primary rat embryonic LFs and AECII (gestation 19 d-20 d) were cultured in vitro. Cells grew to subconfluence and then randomly divided into 4 groups: Group I, Air-exposed control group; GroupII, hyperoxia-exposed group; Group III, Air-exposed plus RA group, Group IV, hyperoxia-exposed plus RA group. For the study of RA effects, sub-confluence growing cells were cultured for 24 h in medium with or without 1μM RA. Cells were then exposed to hyperoxia in the presence or absence of RA for the indicated durations. Cells cultured without RA were cultured in medium containing the same amount of ethanol. The levels of MMP-2,MT1-MMP and TIMP-2 mRNA were detected by RT-PCR; MMP-2 and MMP-9 activity was measured by zymography; The abundance of p-ERK1/2,ERK1/2,p-JNK1/2,JNK1/2,p-p38 and p38 was determined by western blot. LFs nuclear proteins were prepared and p-c-Jun/c-Jun was detected by western blot.3. The LFs were treated by PD98059(10×10-6mol/L), a specific inhibitor of MKK1 and MKK2 (ERK upstream kinases), SP600125(10×10-6mol/L), a specific inhibitor of JNK, and SB203580(10×10-6mol/L), a specific inhibitor of p38. The levels of MMP-2,MT1-MMP and TIMP-2 mRNA were detected by RT-PCR; MMP-2 and MMP-9 activity was measured by zymography; The abundance of p-ERK1/2,ERK1/2,p-JNK1/2,JNK1/2,p-p38 and p38 was determined by western blot.4. All lung tissues of premature rat pups were collected at 4 d after birth. Terminal Transferase d-UTP nick end labeling (TUNEL) staining detected cell apoptosis. The expression of PCNA was detected by Immunohistochemistry. Western blot analyses for phosphorylated and total nonphosphorylated ERKs, JNKs or p38.5. AEC II and LFs Apoptosis were analyzed by Annexin V/Propidium Iodide double Staining and flow cytometry. the expression of p-ERK1/2,p-JNK1/2,p-p38,PCNA,P53 and Caspase-3 in AEC II were determined by western blot. Results:1. The effect of hyperoxia and RA on the expression of lung tissue MMP-2,MMP-9,MT1-MMP,TIMP-1 and TIMP-2 mRNA levels:In room air, from 4 d to 14 d, Expression of message for MMP-2 was decreased, MMP-9 and MT1-MMP mRNA expression levels did not change, TIMP-1 mRNA expression levels was increased, and TIMP-2 mRNA expression levels was declined in pups. Treatment with RA did not significantly change expression of message for MMP-2,MMP-9,MT1-MMP,TIMP-1 and TIMP-2 in air-exposure.Exposure to oxygen resulted in levels of MMP-2,MMP-9,MT1-MMP and TIMP-1 mRNA consistently greater than levels expressed from lungs of normoxic pups. but rat pups treatment with RA from the hyperoxic environment expressed significantly lower levels of mRNA for MMP-2,MMP-9,MT1-MMP and TIMP-1 than the hyperoxic control pups on each experimental day. But hyperoxia and RA had not changed the expression of TIMP-2 mRNA.Protein levels:In room air, levels of pro-MMP-2, active MMP-2 and pro-MMP-9 were declined from 4 d to 14 d, active MMP-9 in 14 d was decreased. There were not significantly different between animals exposed to room air in the presence or absence of RA.The mean levels of active MMP-2, pro-MMP-9 and active MMP-9 after exposure to O2 were higher than air groups on each experimental day, and Pro-MMP-2 activity levels did not change. The levels of active MMP-2, pro-MMP-9 and active MMP-9 were decreased markedly after RA treatment in hyperoxia exposure rat pups.Protein expression of TIMP-1 was increased from 4 d to 14 d in room air exposure pups. RA had no effect on the protein levels of TIMP-1 in the pups exposed to room air. Hyperoxic exposure, however, caused a rapid increase in TIMP-1 mean protein levels on each experimental day, and RA treatment lead to a further elevate. Hyperoxia and RA did not change the protein expression of TIMP-2. 2. The effect of hyperoxia and RA on the expression of lung tissue p-ERK1/2,ERK1/2,p-JNK1/2,JNK1/2,p-p38,p38Western blot analyses showed that the amounts of JNK, p38 and ERK proteins in hyperoxia-exposure or RA-treated lung tissues were the same as in untreated lung tissues, whereas activation of these MAPKs was markedly altered by hyperoxia and RA. After hyperoxia exposure, p-ERK1/2, p-JNK1/2 and p-p38 were dramatically increased on each experimental day, p-JNK1/2 and p-p38 were markedly declined, but p-ERK1/2 was further elevated by RA treatment.3. The effect of hyperoxia and RA on the expression of AECII and LFs MMP-2,MT1-MMP and TIMP-2 mRNABoth MMP-2 and MT1-MMP mRNA expression levels were increased in cells exposed to hyperoxia for 2 h, 6 h, 12 h and 24 h, and decreased after RA treatment. The expression of TIMP-2 mRNA was not change by hyperoxia or RA treatment. Gelatinase zymography analyses showed that the levels of pro-MMP-2, active MMP-2, pro-MMP-9 and active MMP-9 were higher after exposure to O2 for 6 h and 12 h, and were lower by RA treatment.4. The effect of hyperoxia and RA on the protein levels of AECII and LFs p-ERK1/2,ERK1/2,p-JNK1/2,JNK1/2,p-p38,p38 expressionSimilar to premature lung tissues, hyperoxia-exposure or RA-treatment did not change the total of JNK, p38 and ERK proteins in AECII and LFs. After hyperoxia exposure, p-ERK1/2, p-JNK1/2 and p-p38 were significantly increased on each experimental hour, p-JNK1/2 and p-p38 were markedly decreased, but p-ERK1/2 was further increased by RA treatment.5. The effect of hyperoxia and RA on the LFs nuclear protein levels of p-c-Jun/c-Jun expressionp-c-Jun was elevated after exposure to O2 for 6 h and 12 h, and were decreased by RA treatment.6. The effect of hyperoxia, RA, PD98059, SP600125 and SB203580 on the expression of LFs MMP-2, MT1-MMP and TIMP-2 mRNATo inspect the roles of ERK, JNK and p38 signaling pathways in regulation the expression of MMP-2, MT1-MMP and TIMP-2 after hyperoxia exposure, LFs were exposed to hyperoxia or room air for 12 h in the presence of the kinase inhibitors PD98059, SP600125, SB203580, and RA respectively. The results showed that SP600125, SB203580, and RA inhibited p-JNK1/2 and p-p38, simultaneously decreased the expression of LFs MMP-2 and MT1-MMP mRNA, but PD98059 did not change their expression. In addition, PD98059, SP600125 and SB203580 had no effect on the the expression of TIMP-2 mRNA.7. The effect of hyperoxia and RA on the proliferation and apoptosis of prenatal lungLungs from pups exposed to hyperoxia for 4 d exhibited TUNEL-positive nuclei increased markedly throughout the parenchyma. TUNEL-positive nuclei in the parenchyma were mainly observed in alveolar type II cells, endothelial cells surrounding capillaries and type I cells. After RA treatment, TUNEL-positive nuclei decreased significantly in hyperoxia-exposed lung.In lung sections, after exposure to 85 % O2 for 4 d, the number of PCNA positive cells index was obviously decreased, and increased markedly by RA treatment. The air-space size was significantly enlarged and secondary crests were markedly decreased in hyperoxia-exposed animals. RA treatment improved lung air spaces and secondary crests in air-exposed pups, but had no effect on hyperoxia exposure pups. 8. The effect of hyperoxia and RA on the proliferation and apoptosis of AECII and LFs in vitroQuantitative data from flow cytometry analyses (PI/Annexin-V double staining) demonstrated that there was a significant increase in signs of both early apoptosis, as designated by quadrant II, and late apoptosis/necrosis (quadrant III) after AECII 12 h of hyperoxia. RA markedly decreased hyperoxia-induced AECII apoptosis and necrosis.Western blot analyses showed that the protein levels of PCNA was reduced, that of p53 and active fragment of Caspase-3 were increased after 12 h of hyperoxia in AECII; RA improved the expression of PCNA, and decreased the expression of p53 and active fragment of Caspase-3.The apoptosis and proliferation of LFs were not changed by hyperoxia exposure and/or RA treatment.Conclusions:1. MMP-2, MMP-9, MT1-MMP, TIMP-1 and TIMP-2 are all involved in alveolarization of premature rat lung development;2. the balance of MMPs/TIMPs was broken by hyperoxia during alveolarization of premature rat lung development, which lead to lung development inhibition and lung fibrosis;3. Hyperoxia exposure activated MAPKs (mainly JNK and p38), which played a role in broken the balance of MMPs/TIMPs;4. hyperoxia exposure lead to numerous AECII apoptosis and necrosis, but did not change LFs survival, both of which were involved in abnormal lung remodeling;5. RA had a protective effect on hyperoxia lung injury by which decrease active levels of JNK and p38, increase active levels of ERK, subsequently reduce the expression and activation of MMP-2, MMP-9 and MT1-MMP, and decline AECII apoptosis and necrosis.