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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fig10: Endothelial-specific knockout of sAC blocks leukocyte transmigration in vivo. (a) Heart tissue and peripheral blood was collected from sAC-C2flox/flox and sAC-C2flox/flox/VE-Cre+ mice used in the following experiments (see Materials and methods). FACS was used to sort MHEC (CD31+/CD45−) and leukocyte (Ly6G+/CD45+) cell populations from the heart tissue and peripheral blood, respectively. MHEC and leukocyte DNA was isolated from each mouse. PCR was performed to assess the expression of either sAC WT allele (top band) or sAC C2-KO allele (bottom band). (b) The ears of sAC-C2flox/flox or sAC-c2flox/flox/VE-Cre+ mice (mixed background, see Materials and methods; Chen et al., 2013) were stimulated for 5 h with croton oil. Tissue was then harvested, stained, and analyzed. (c) Quantification of results above. (d) Quantification of site of arrest for sAC-C2flox/flox/VE-Cre+ mice. Percent of leukocytes extravasated within 50 µm of venule per field of view. (e) Our current model of how CD99 signals during TEM. Under resting conditions, CD99, sAC, PKA, and ezrin form a signaling complex at endothelial junctions. During TEM, homophilic engagement of endothelial CD99 with leukocyte CD99 (#1) signals through sAC (#2) to elevate cAMP (#3), to activate PKA, which works through a yet to be defined mechanism (#4) to induce LBRC membrane trafficking to sites of leukocyte–endothelial contact (#5). (f) Flow diagram of model detailed above. 100–200 cells were analyzed per ear. PMN per field of view, vessel length and vessel diameter were equivalent for all conditions tested (not depicted). Images were acquired with a 40× objective (n = 1.00). Insets show xz-orthogonal view (where yellow bar dissects the vessel) to demonstrate site of neutrophil arrest. Bars, 25 µm. Three mice per condition were used for each experiment. Images are representative of three (a and b) independent experiments. Data represent the average value of three (c and d) independent experiments. Error bars denote SEM (****, P < 0.0001; Student’s t test [c] and ANOVA [d]).
To test the effect of genetic deletion of sAC in ECs, we generated an endothelial-specific sAC knockout by crossing conditional sAC knockout mice (sAC-C2flox/flox; Chen et al., 2013) with VE-Cre mice (Alva et al., 2006). Because hemogenic endothelium bears VE-cadherin, there have been reports of variable numbers of leukocytes also being affected by this cre recombinase (Kim et al., 2005b). Therefore, we tested the specificity of the knockout in our hands. At the conclusion of the experiments, we isolated the peripheral blood and heart ECs (MHECs) from the mice used in the experiments below. Cells were then sorted by FACS into two populations: MHECs (CD31+/CD45−) and leukocytes (Ly6G+/CD45+). DNA was extracted from these populations and PCR was performed to assess the expression of the full-length (wild-type) sAC allele and the sAC-C2 knockout allele. The ECs in the VE-Cre+ mice bore the knockout allele, whereas the leukocytes from these mice had almost exclusively wild-type sAC (Fig. 10 a). In addition, we analyzed the expression of ICAM-1 on the postcapillary venules of carrier-treated and croton oil–treated sAC-C2flox/flox and sAC-C2flox/flox/VE-Cre+ mice. Deletion of sAC in ECs had no effect on the expression of ICAM-1 under either condition (unpublished data).