Study On The Roles Of Metastasis Associated Protein 1(MTA1) In The Maturation Of Pulmonary Alveolar Capillary

Metastasis-associated protein(MTA)is a member of the nucleosome remodeling and decarboxylation complex(NuRD)family.The family is composed of three genes located in different chromosomes,namely,MTA1,MTA2 and MTA3.MTA is most well-known for its biological roles in controlling histone deacetylation and regulation of gene expression,and for promotion of cancer metastasis.MTA1 is one of the earliest and most studied molecules found in this family.Numerous studies have shown that the MTA proteins were highly expressed in various human malignant tumors,and participated in the development and metastasis of tumors,and regulated the signaling pathways in many cells.However,MTA1 expression is not restricted to tumors.It has also been shown that MTA1 was substantially expressed in normal mouse tissues and organs,especially in the brain,lung,and testes.These evidences suggest that MTA1 might function during both physiological and pathological processes.Our previous study showed that over one-third of homozygous MTA1-KO mice died within the first 7 days after birth.The lungs of died baby mice demonstrated marked pulmonary capillaries development abnormality.It suggested that MTA1 might play an important role in the development of lung,but the mechanism is still unknown.Hypoxia is an important situation in solid tumors,which can promote tumor angiogenesis,invasion and metastasis.Hypoxia-inducible factor 1(Hypoxia inducible factor 1,HIF-1)is a hypoxia response protein.There are two subunits,HIF-1? and HIF-1?,in the functioning HIF1 protein,which plays an important role in regulating vascular development,alveolar growth,preventing alveolar damage and promoting lung injury after alveolar regeneration.The content of HIF-1? is regulated by oxygen partial pressure and rapidly degraded under the condition of constant oxygen.The activity of HIF-1 mainly depends on the effective content of HIF-1?.Oxygen content and angiogenesis are key regulatory processes for lung development,and intracellular HIF-1? is the main transcription factor for effective oxygen reduction in angiogenesis.Notably,previous research has shown that specific histone deacetylase(HDAC)enzymes induce angiogenesis and influence the stability of HIF-1? in mice.Vascular development in the lungs is a complex,multistep process regulated by many factors.Notably,pulmonary vascular endothelial cells play a crucial role in the regulation of the pulmonary vascular system and in the maintenance of barrier function and the integrity of alveolar-capillary membranes.These cells as well as other cell types in the lungs express high levels of vascular endothelial growth factor(VEGF),which is one of the most important regulatory mediators of vascular regulation during angiogenesis and vascular maturation.In fact,VEGF has been shown to be essential in the fetal lung during development and administration has been shown to initiate vessel formation.It has been shown that oxygen tension and angiogenesis were critical regulators of embryonic lung growth.Transcription of VEGF is regulated by HIF-1?,and VEGF induced phosphorylation of endogenous MTA1 on tyrosine residues.These suggest that the expression of HIF-1?,as well as VEGF and its associated receptors,plays a key role in the process of angiogenesis.This is supported by a study showing a relationship between increased HIF-1? expression and its nuclear translocation with pronounced angiogenesis.Interestingly,recent evidence has shown that MTA1 protein expression is significantly correlated with tumor angiogenesis and appears to contribute to this process through the regulation of HIF-1?.However,few studies have reported the potential link between MTA1 and HIF-1? during normal physiological processes,and it is currently unclear if this possible angiogenesis-promoting relationship exists in the lungs.Objective:The present study was designed to examine the development of angiogenesis in the lung of MTA1-knockout(KO)mice in order to determine the roles of MTA1 during the various stages of pulmonary vascular development,and to clarify the molecular mechanisms involved.Methods:1.The immunohistochemistry(IHC),quantitative PCR and Western blot were used to determine the tissue distribution MTA1,and the analyze the dynamic changes of MTA1 during the development of mouse lung.By analyzing the postnatal survials and and body weight gains,we clarified the influences of MTA1 deficiency on the postnatal mortality and growth.And then,the HE staining was used to analyze the lung development in MTA deficient mice.2.The heart weight/body weight(HW/BW)ratio and the size of the right ventricular wall cardiomyocytes were also measured in 2-month old mice,and the expression of alpha-smooth muscle actin(?-SMA)in the pulmonary arteries of 2-month old wildtype and MTA1 knockout mice.Heme staining and transmission electron microscopy were performed to observe changes in the formation and maturation of pulmonary alveolar capillary.Moreover,IHC,quantitative PCR and Western blot were used to detect CD31,CD34,VEGF,and HIF-1? in the lungs of MTA1-KO and wildtype mice at embryonic day 18.5 and 2 months.3.MTA1 was overexpressed or knockdown in the cultured 293 T cells and MLE-12 cells by transfecting with the recombindant plasmids with overexpressed MTA1 and MTA1-specific si RNA,respectively.The expression of MTA1,HIF-1? and VEGF were detected by Real-time PCR and Western blot to determine the regulatory role of MTA1 on HIF-1? and VEGF.Results: 1.MTA1 deficiency increases infant mortality and impedes growth.To determine the distribution of MTA1 protein and its role in mice,we examined the expression levels of MTA1 in different tissues by using Western blot analysis.The data suggest that MTA1 is expressed at high levels in lung tissue and may play an important role in various developmental processes.To investigate the function of MTA1,mice were studied at E18.5 and P7 as postnatal mortality was primarily observed in the first week after birth.In fact,our analysis shows that over 1/3 of homologous MTA1-KO mice died within this time.The MTA1-KO mice that did survive had retarded growth and their body weight was markedly decreased compared to that of wild type mice.These results suggest that MTA1 deficiency increases infant mortality and impedes growth.The results of histopathological analysis indicated that there were some defects in the lungs in MTA1 knockout mice,including the thickening alveolar walls and increased cell number.These results indicated that MTA1 could regulate the development and maturation of lung.2.MTA1 deficiency induces myocardial hypertrophy pulmonary arterial hypertension and impedes the genesis of capillary beds.In the adult mice that survived,MTA1 deficiency also appeared to induce myocardial hypertrophy,highlighted by an increased HW/BW ratio,and larger cardiomyocytes in the right ventricles(P<0.05).HE staining,Heme staining,IHC and transmission electron microscopy(TEM)were performed to observe changes in the formation of pulmonary alveolar capillary.The IHC results indicated that the percentages of CD31 and CD34 positive cells were similar in the alveolar walls of wildtype and MTA1 knockout mice.However,the results of Heme staining and TEM indicate that MTA1 deficiency reduces the numbers of open pulmonary alveolar capillaries and red blood cells(RBC)compared with that in wild type mice.These results indicated that MTA1 deficiency could reduce the mature capillaries in the pulmonary alveolus,which suggest that MTA1 is involved in the development and maturation of pulmonary alveolar capillaries.3.MTA1 modulates pulmonary alveolar capillary angiogenesis of mouse via stabilization of HIF-1?.In order to clarify the mechanism of MTA1 in regulating the development and maturation of pulmonary alveolar capillaries,we detected the expression of the expression of HIF-1? and VEGF in the lung tissue of MTA1-KO mice at E18.5.The results showed that the expression of HIF-1? and VEGF appeared to be decreased in the MTA1 deficient group compared to the wildtype.To further investigate the regulatory role of MTA1 on the generation of pulmonary capillaries via HIF-1? stabilization,we overexpressed MTA1 in 293 T cells and MLE-12 cells using the MTA1-pIRES2-EGFP vectors.In doing so,we found that the expression of HIF-1? and VEGF at both the protein and mRNA levels was significantly upregulated when MTA1 was overexpressed compared to the control group(transfected with the empty pIRES2-EGFP vector).And HIF-1? and VEGF levels were significantly decreased when MTA1 was down-expressed.These results suggested that MTA1 could stablize the cellular HIF-1?,and regulate the maturation of pulmonary alveolar capillaries.Conclusions:In conclusion,MTA1 participates in the formation of pulmonary capillaries via stabilization of HIF-1?.These data enhance our understanding of MTA1 function in development and may also provide insight into the diagnosis/treatment of related pulmonary diseases.