Functional Roles Of CCAAT Enhancer Binding Protein A And Its SUMO-modification In Rat Lung Development

ObjectiveWe detect the dynamic expression of C/EBP?,SUMO-modified C/EBP?,the secretion of pulmonary surfactant and the amount of glycogen in rat lung tissue during different stages,to explore the functional roles of C/EBP? and its SUMO-modification in lung development.MethodsAccording to the histological stage of rat lung development,lung tissues were collected at 15.5 d(the late pseudoglandular period),17.5 d(the canalicular period),19.5 d(the early saccular period)of embryonic age and 0.5d(the middle saccular period),4 d(the late saccular period),7 d(the alveolar period,the alveolar stage),14 d(the alveolar period,the equilibrium stage)of postnatal age.The lung morphologic appearance was observed by HE staining.C/EBP? protein expression and Surfactant Proteins(SP-A,SP-B,SP-C,SP-D)secretion were detected by Western-blot assay.Phosphatidylcholine Assay Kit was utilized to analyze the secretion of PC.Periodic Acid-Shiff(PAS)staining was used to evaluate the amount of glycogen.The expression localization of sumoylated C/EBP? in lung was examed though the double immunofluorescence staining.The Co-immunoprecipitation assay was carried out to detect the mobility of sumo-modified C/EBP ?.Results(1)The expressions of C/EBP? and Surfactant Proteins examined by immunohistochemistry: C/EBP? and SP-A began to express at the late pseudoglandular period,while SP-B,SP-C and SP-D started to express at the canalicular period.All of them increased with the development of lung,C/EBP?,SP-A,SP-C were reached the highest level at postnatal age 0.5 d,while SP-D at the postnatal day 4,and then their expression gradually decreased.However,the expression of SP-B continued to rise.The level of C/EBP? and SPs maintained stable at the alveolar period.The C/EBP? protein level was positively correlated with SPs at embryonic and early postnatal stage(r=0.999,0.991,0.982,0.951,all P<0.05).(2)The secretion of PC analyzed by Phosphatidylcholine Assay Kit: The level of PC was very low at embryonic day 15.5,with the development of lung,the secretion of PC increased gradually,there was no significant correlation between the expression of PC and C/EBP?(P>0.05).(3)The amount of glycogen evaluated by PAS staining: The level of glycogen was high at the late pseudoglandular stage,the content of glycogen decreased with the development of lung,especially during the canalicular phase to preliminary saccular period,then it became stable at the alveolar period.The expression of C/EBP? was negative correlation with the content of glycogen in fetal lung(r=-1.000,P<0.01).(4)The localization of sumoylated C/EBP? detected by double immunofluorescence staining: C/EBP? was partially colocalized with SUMO-1 in the alveolar epithelial cell at the postnatal day 4,suggesting that sumoylation of C/EBP? occurred in the lung.(5)Lung tissues were lysed with mild lysis buffer and analyzed by immunoblotting with a specific antibody to C/EBP?,the mobility of sumo-modified C/EBP? detected by Co-IP: sumoylated C/EBP? exhibits a developmental expression pattern during lung development,which was scarcely detected at the late pseudoglandular stage at embryonic day 15.5,the expression increased and was maximal at embryonic day 17.5,following which,the sumoylated C/EBP? decreased until the saccular period.At the alveolar stage,it increased slightly and finally stabilized.The sumoylated C/EBP? protein level was positively correlated with the content of glycogen at embryonic and early postnatal stage(r=0.999,P<0.05).The pulmonary surfactant secretion was approximately contrary to sumoylated C/EBP? expression pattern,there have negatively correlation between them,except for SP-B and PC.Conclusion(1)C/EBP? protein showed a dynamic expression pattern during lung development,it has positive effect on the AEC II differentiation and pulmonary surfactant secretion.(2)SUMO post-translationally modifies C/EBP? occurred in the lung tissue;the expression of sumuylated C/EBP? corresponded to lung differentiation and several differentiation-dependent genes expression during lung development,these results clearly assert that sumoylation may plays a negative role in the C/EBP?-mediated pneumonocytes differentiation and pulmonary surfactant synthesis in the lung.