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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fig2: Engagement of EC CD99 activates PKA. (a) Quantitative TEM assays were performed using HUVECs pretreated with nonblocking anti–VE-cadherin, anti-PECAM, or anti-CD99 mAb. After 50 min, histamine (10 µM) was added to samples for 10 min at 37°C. (b) TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin (nonblocking control) or anti-CD99 mAb. Additionally, HUVECs were pretreated with diphenhydramine (H1-R antagonist, 10 µM), ranitidine (H2-R antagonist, 10 µM), or JNJ-10191584 (H4-R antagonist, 10 µM) or DMSO (carrier). After 50 min, histamine (10 µM) or dimaprit (H2-R agonist, 10 µM) was added to samples for 10 min. (c) Immunoblot analysis of phospho-VASP S157 and phospho-CREB S133 activity after anti-CD99 or anti-PECAM mAb (control) cross-linking (XL) in resting HUVECs. (d and e) Quantification of results in c, pVASP and pCREB signals were normalized to total VASP and total CREB, respectively. Values were then normalized to CD99 XL. (f) Antibody-coated polystyrene bead recruitment of CD99 and activation of phospho-PKA. Beads precoupled with mIgG1, anti-CD99 mAb, or anti-PECAM mAb were added to HUVEC monolayers expressing hCD99-GFP. Monolayers were subsequently stained with anti–VE-cadherin and anti-pPKA T197 PKA antibodies. Arrows indicate beads bound to HUVECs. (g and h) Quantification of data; percent of beads in the field of view with either hCD99-GFP or pPKA T197 enrichment. Bars, 10 µm. Images are representative of three (f) or four (c) independent experiments. Numerical values are the mean of three (a, b, e, g, and h) or four (d) independent experiments. Error bars represent SD (d and e) or SEM (a, b, g, and h; ***, P < 0.001; ****, P < 0.0001; Student’s t test [a, b, d, and e] and ANOVA [g and h]).
The cytoplasmic tail of CD99 is bereft of traditional signaling domains. To find potential signaling pathways responsible for restoring TR, we investigated whether compounds known to stimulate EC membrane movement, such as histamine (Feng et al., 1996), were sufficient to induce TR and rescue TEM in the presence of anti-CD99. We performed TEM assays in which HUVECs were blocked with anti-PECAM or anti-CD99 and then briefly treated with histamine. Histamine selectively overcame the CD99 blockade and significantly restored TEM (Fig. 2 a). Because histamine acts on three receptors in EC (H3-receptor is predominately neuronal), we used pharmacologic antagonists of each receptor to show that blocking the EC H2-receptor (H2-R) specifically prevented the ability of histamine to overcome the CD99 blockade (Fig. 2 b). The H2-R agonist dimaprit reproduced the effect of histamine by overcoming anti-CD99 treatment. We conclude that the ability of histamine to override the anti-CD99 blockade of TEM was a result of the activation of a common downstream signaling pathway shared by H2-R and CD99.