Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration.
How CD99 signals during this process remains unknown.We show that during TEM, endothelial cell (EC) CD99 activates protein kinase A (PKA) via a signaling complex formed with the lysine-rich juxtamembrane cytoplasmic tail of CD99, the A-kinase anchoring protein ezrin, and soluble adenylyl cyclase (sAC).PKA then stimulates membrane trafficking from the lateral border recycling compartment to sites of TEM, facilitating the passage of leukocytes across the endothelium.
Affiliation: Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60208.
- Adenylyl Cyclases/metabolism*
- Antigens, CD/immunology/metabolism*
- Cyclic AMP-Dependent Protein Kinases/metabolism*
- Endothelial Cells/metabolism*
- Signal Transduction/physiology*
- Transendothelial and Transepithelial Migration/physiology*
- Analysis of Variance
- Antibodies, Monoclonal/immunology
- Blotting, Western
- Flow Cytometry
- Genetic Vectors
- Human Umbilical Vein Endothelial Cells
- Mice, Knockout
- Microscopy, Confocal
- Microscopy, Fluorescence
- RNA, Small Interfering/genetics
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fig6: Genetic ablation of endothelial sAC inhibits leukocyte transmigration. (a) Quantitative PBMC TEM assays were performed on HUVECs expressing either scrambled (SCR) or sAC shRNA. Where indicated, the rescue construct (sACt) was also expressed. (b) Immunoblot analysis for sAC and β-actin was performed on samples treated in parallel. (c) Quantification of immunoblots. Total sAC expression was normalized to β-actin for each sample and then normalized to SCR control. (d and e) TEM-IF assays were performed on HUVECs expressing SCR, sAC, or CD99 shRNA. (f) Immunoblot analysis for CD99, sAC, and β-actin was performed on samples treated in parallel. (g) Quantification of immunoblots. Total sAC or CD99 expression was normalized to β-actin for each sample and then normalized to SCR control. (h) Quantitative PMN TEM-IF assays were performed on TNF-activated (4 h) HUVECs expressing SCR, sAC, or CD99 shRNA. (i) Quantitative PMBC TEM-IF assays were performed on TNF-activated (4 h) HUVECs expressing SCR, sAC, or CD99 shRNA. (j) Immunoblot analysis of samples treated in parallel were performed for sAC, ICAM-1, PECAM, JAM-A, and CD99 expression. Bars, 10 µm. Images are representative of three (b, e, and j) independent experiments. Numerical values are the mean of two (h and i) or three (a, c, d, and g) independent experiments. Error bars represent SD (c and g) or SEM (a, d, h, and i; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test [a and c] and ANOVA [d, g, h, and i]).
To confirm that EC sAC is critical for TEM, we used shRNA to knockdown sAC expression in HUVECs and tested for defects in TEM. Compared with scrambled shRNA (control), sAC shRNA resulted in significantly decreased transmigration (Fig. 6 a). Furthermore, this defect in TEM was rescued by the reexpression of a truncated isoform of sAC (sACt; Buck et al., 1999). Important to note, sACt was rendered resistant to shRNA-mediated knockdown by introducing multiple silent mutations in the target sequence of sACt. Samples for immunoblot analysis were collected in parallel to confirm knockdown and rescue of sAC (Fig. 5, b and c). These data demonstrate that genetic ablation of sAC in EC inhibits leukocyte transmigration.