Abstract
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 -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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | L650-L662 |
| Journal | American Journal of Physiology - Lung Cellular and Molecular Physiology |
| Volume | 284 |
| Issue number | 4 28-4 |
| State | Published - Apr 1 2003 |
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Keywords
- Activator protein-1
- Fetal sheep
- Gene expression
- Maturational differences
- Nitric oxide
- Protein translocation
- Pulmonary arterial endothelial cells
ASJC Scopus subject areas
- Physiology
- Pulmonary and Respiratory Medicine
- Physiology (medical)
- Cell Biology
Cite this
Developmental differences in the shear stress-induced expression of endothelial NO synthase : Changing role of AP-1. / Wedgwood, Stephen; Mitchell, Calista J.; Fineman, Jeffrey R.; Black, Stephen M.
In: American Journal of Physiology - Lung Cellular and Molecular Physiology, Vol. 284, No. 4 28-4, 01.04.2003, p. L650-L662.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Developmental differences in the shear stress-induced expression of endothelial NO synthase
T2 - Changing role of AP-1
AU - Wedgwood, Stephen
AU - Mitchell, Calista J.
AU - Fineman, Jeffrey R.
AU - Black, Stephen M.
PY - 2003/4/1
Y1 - 2003/4/1
N2 - 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 -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.
AB - 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 -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.
KW - Activator protein-1
KW - Fetal sheep
KW - Gene expression
KW - Maturational differences
KW - Nitric oxide
KW - Protein translocation
KW - Pulmonary arterial endothelial cells
UR - http://www.scopus.com/inward/record.url?scp=0037377477&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037377477&partnerID=8YFLogxK
M3 - Article
C2 - 12533439
AN - SCOPUS:0037377477
VL - 284
SP - L650-L662
JO - American Journal of Physiology - Heart and Circulatory Physiology
JF - American Journal of Physiology - Heart and Circulatory Physiology
SN - 0363-6135
IS - 4 28-4
ER -