Developmental differences in the shear stress-induced expression of endothelial NO synthase: changing role of AP-1.
Endothelial nitric oxide synthase (eNOS) mRNA and protein levels increase during late gestation and then decrease postnatally in sheep lung parenchyma. The increase in fluid shear stress at birth, resulting from increased pulmonary blood flow, is an important mediator of postnatal eNOS gene expression. Our objective was to identify factors stimulating eNOS expression in pulmonary arterial endothelial cells (PAEC) in response to shear stress and to determine if these factors are developmentally regulated. PAEC were isolated from fetal lambs and adult sheep. Transcriptional activity from a 1,600-bp eNOS promoter fragment increased in both fetal and adult PAEC exposed to 8 h of shear stress. Conversely, activity driven from an 840-bp promoter fragment containing a putative activator protein (AP)-1 binding site was increased only in fetal PAEC. This increase was completely abolished in an identical construct containing a mutant AP-1 sequence. The AP-1 protein c-Jun was localized to the cytosol in static adult PAEC and to the nucleus in static fetal PAEC. After shear, c-Jun was nuclear localized in both cell types. However, transcriptionally active phosphorylated c-Jun was elevated only in the nuclei of sheared fetal PAEC. Resting levels of eNOS and NO were 2- and 20-fold higher, respectively, in fetal cells. Shear increased eNOS and NO in both cell types: levels were approximately 2.5-fold higher in fetal PAEC. Phosphorylation of Akt and eNOS was evident in sheared fetal but not adult PAEC. We have therefore identified mechanisms of eNOS regulation at the transcriptional level and to be enzyme activation specific to the fetal pulmonary arterial circulation.
Wedgwood
Stephen
S
Department of Pediatrics, Northwestern University Medical School, Chicago, Illinois 60611-3008, USA.
Mitchell
Calista J
CJ
Fineman
Jeffrey R
JR
Black
Stephen M
SM
D000818:Animals; D015153:Blotting, Western; D004730:Endothelium, Vascular / Q000196:embryology / Q000201:enzymology; D005260:Female; D019793:Fluorescein; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D008168:Lung / Q000098:blood supply*; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D011247:Pregnancy; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D011651:Pulmonary Artery / Q000196:embryology* / Q000201:enzymology*; D013314:Stress, Mechanical; D018808:Transcription Factor AP-1 / Q000378:metabolism*
C111205:4,5-diaminofluorescein; D007202:Indicators and Reagents; D011518:Proto-Oncogene Proteins; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019793:Fluorescein