Effects And Signaling Pathways Of Focal Adhesion Kinase On Hyperoxia-induced Lung Injury In Premature Rats

[Objective] To explore the changes of expression of focal adhesion kinase (FAK) in different stages of the developing lungs and to explore the regulating role of FAK in lung development.[Methods] All lung tissues of fetal and neonatal rats were collected at the following ages: gestational ages (GA) 18, 20, 21 days, and 1, 4, 7, 10 and 21 day after birth. The expression of FAK protein was located and quantitated by immunohistochemistry and Western blot. The expression of FAK mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).[Results] It showed that FAK was mainly localized in the airway surface epithelium, alveolar epithelial cell and endothelial cells during the pseudoglandular stage. At the different stages of lung development , the expression of FAK were detectable at protein and gene levels. The expression was strongest on postnatal 4d . [Conclusion] FAK may play an important role in the developing lung. It not only modulates the process of development of airway surface epithelium and the morhogenesis of respiratory tube and capillary vessel, but also may be involved in alveolar epithelial proliferation and differentiation.Part 2 The effect of hyperoxia on the expression level of focal adhesion kinase of lung tissues of premature rat.[Objective] To establish 85% oxygen-exposed animal model and explore the effects of hyperoxia on the newborn rat lung tissues. To observe the expression of FAK in lungs of premature rats exposed to hyperoxia and explore the role of FAK in hyperoxia-induced lung injury.[Methods] The 1-day-old preterm rats were randomly assigned to two groups: air group and hyperoxia group. Hyperoxia groups were exposed continuously to about 85% oxygen, air groups were exposed continuously to room air. After 4, 7 and 14 days exposure, the expression of phosphor-FAK(FAK-Tyr397) and FAK protein was located and quantitated by immunohistochemistry and Western blot. The expression of FAK mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).[Results] Compared with air group, the expression of FAK peptide decreased markedly in hyperoxia group of 4d 7d and 14d respectively, especially in hyperoxia group of 14d (P<0.01) .The expression of FAK mRNA were similar to polypeptides.[Conclusion] Decreased expression of FAK induced by 85% oxygen is likely to contribute to the pathogenesis of hyperoxic lung injury and inhibition of premature lung development. Its mechanism may be related to inhibiting the process of alveolar epithelial proliferation and differentiation and the morphogenesis of blood vessel. [Objective]To establish a hyperoxia -exposed cell model to explore the effects of hyperoxia on levels of FAK in AEC II s isolated from fetal rat lungs , we also investigate the relationship of between FAK activated by Matrigel and proliferation or apoptosis of type II alveolar epithelial cells[Methods] AEC II were gained by primary culture from 19 d fetal rat lung. To establish a hyperoxia-exposed cell model, purified AECIIs were cultured for 6h,12h,24h,and 48h after culture bottles were filled with 95 % oxygen-5 % CO2 at 5 L/min for 10 min, and then sealed. Cells were harvested and total RNA was extracted by Trizol reagent and total protein was extracted by lysed liquor of cell. Levels of mRNA and protein of FAK and FAK-Tyr397 were measured by reverse transcription polymerase chain reaction (RT-PCR) and western blot. We observed the apoptosis of AEC II by flow cytometry assay using Annexin-V/PI double staining method.AEC II were randomly assigned to four groups and exposed to air or hyperoxia : air group (group I), hyperoxia/group (group II) , air plus Matrigel group (group HI), hyperoxia plus Matrigel group (group IV), Groups II and IV were cultured for 12 hour after their culture bottles were filled with 95 % oxygen-5 % CO2 at 5 L/min for 10 min and then sealed. Groups III and IV were cultured on Matrigel basement membrane matrix gel. All Groups were layed in CO2 culture chamber (37 ℃,5 % CO2) for 12 hours . Cells were harvested and total RNA was extracted by Trizol reagent and total protein was extracted by lysed liquor of cell. Levels of mRNA and protein of FAK were measured by reverse transcription polymerase chain reaction (RT-PCR) and western blot respectively.To investigate the relationship of between FAK activated or inactivated and proliferation or apoptosis of type II alveolar epithelial cells , AEC II were randomly assigned to six groups: air group (group I), hyperoxia group (group II), air + Matrigel group (group III), hyperoxia + Matrigel group (group IV), air + Matrigel +(RGD+YIGSR)group (group V), hyperoxia + Matrigel+(RGD+YIGSR) group (group VI). Levels of proliferation and apoptosis of AEC II were measured by immunohistochemical assay of PCNA and Tunnel method respectively.[Results] Compared with air group,the expression of FAK peptide decreased markedly in hyperoxia groups of 12h 24h and 48h, especially in hyperoxia groups of 24h and 48h (P<0.01) , however, the phosphorylation lever of FAK (FAK-Tyr³⁹⁷) decreased markedly in hyperoxia group of 6h (P<0.05) , with time the expression of FAK-Tyr³⁹⁷ decreased more markedly. The expression of FAKmRNA were similar to FAK-Tyr³⁹⁷. At the same time, apoptosis and necrosis of AEC II were detectable by flow cytometry assay in all groups,but the maximal apoptosis rateof AEC II was in hyperoxia group of 12h, with time apoptosis rate decreased and necrosis rate increased.Treated with Matrigel, FAK activity of AEC II increased, which was abolished by YIGSR pentapeptides add RGD tripeptides. Compared with air group of 12h, the expression of PCNA decreased and apoptotic index increased markedly in hyperoxia group of 12h, air group of 12hour treated with Matrigel had no effect on apoptosis index of AEC II, but incr-eased the expression of PCNA significantly (p<0.01) . The expression of PCNA increased and apoptotic index decreased markedly in hyperoxia group of 12h treated with Matrigel as compared with hyperoxia group of 12h (p<0.05) .After the treatment with YIGSR pentapeptides (50ug/ml) and RGD tripeptides(50ug/ml)together for12h,the expression of PCNA decreased and apoptosis index increasedmarkedly as compared with air +Matrigel group and hyperoxia +Matrigel group respectively- (p<0.01) .[Conclusion] Hyperoxia decreased levels of mRNA and protein of FAK and FAK-Tyr³⁹⁷ in AEC II from fetal rat lung,which may be a contributory mechanism of impaired proliferation, apoptosis and necrosis of AECII in hyperoxia induced lung injury in premature rat. Matrigel could inhibit apoptosis and promote proliferation of AECII resulted from hyperoxia in vitro.It may play a protective role in hyperoxia-induced lung injury partly due to activated FAKPart 4 Anti-apoptotic effect of focal adhesion kinase on hyperoxia -exposed AECII and its Signaling pathway[Objective] To investigated whether the signaling pathway of PI-3K/Akt is involved in the FAK- elicited anti-apoptotic effect on hyperoxia-exposed AEC II[Methods] AEC II were randomly assigned to four groups: (1) air + Matrigel group , (2) hyperoxia + Matrigel group, (3) air+Matrigel+( RGD+YIGSR) group , (4) hyperoxia + Matrigel+(RGD+YIGSR)group . All Groups were layed in CO₂ culture chamber (37 ℃,5 % CO2) for 12 hours, cells were harvested and total protein was extracted by lysed liquor of cell. The level of PI-3K was measured by western blot. Another two groups: (5) air + Matrigel +LY294002 (25umol/L) group and (6) hyperoxia +Matrigel+LY294002 (25umol/L) group were added in order to detect the levels of downstream proteins of PI-3K. Western blotting was performed to assess the levels of tota- and phosphor-Akt, and flow cytometry assay was used to evalue the levels of phosphor-Bad and GSK3[Results ] Hyperoxia significantly inhibited the expression of PI-3K. Compared with (1) group, the expression level of PI-3Kof AEC II treated with YIGSR and RGD together for 12h markedly decreased (p<0.01) . Compared with (1)group, the PI-3K inhibitor, ly294002 (25umol/L), could not decrease the level of tota- Akt, but decrease the level of phosphor- Akt significantly (p<0.01) ,while, (3)group which treated with YIGSR and RGD together had lower levels of tota- and phosphor-Akt than (1)group (p<0.01) . Compared with (2)group, the level of phosphor-Akt decreased markedly in both (4)group and (6)group (p<0.01) .The levels of phosphor-Bad and GSK3 was measured by flow cytometry assay. It was found that hyperoxia could significantly inhibit the expressions of. phosphor-Bad and promote the expressions of GSK3. Both ?group and ?group had lower levels of phosphor-Bad and higher levels of GSK3 than those in (1) group (p<0.01) . Compared with (2)group, the expressions of phosphor-Bad decreased markedly and GSK3 increased in both (4)group and(6)group (p<0.01)[Conclusion] These results confirm that FAK is upstream of phosphati-dylinositol 3-kinase/Akt in regulating AECII survival. Hyperoxia-induced AECII apoptosis and impaired proliferation were mediated through inhibition of PI-3K/Akt signaling pathway which was elicited by FAK. The decreased expression levels of PI-3K and Akt of AECII resulted from hyperoxia may contribute to hyperoxia-induced premature lung injury..Matrigel could inhibit apoptosis and promote proliferation of hyperoxia-exposed AECII in vitro and plays a protective role in hyperoxia-induced AECII injury partly due to FAK-PI-3K/Akt signaling pathway.