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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fig3: Raising intracellular cAMP reverses anti-CD99 blockade of transmigration and restores TR of the LBRC. (a and b) Quantitative TEM assays were performed using HUVECs pretreated with anti–VE-cadherin (control) or anti-CD99 mAb (IgG1). After 50 min, 8-CPT (30 µM), Forskolin (30 µM), or DMSO (control) was added to the cells for 10 min. (c) Two-color TR assays were performed (as previously described). In brief, before warming monolayers to 37°C, 488-GαM IgG2a and 8-CPT, Forskolin, or DMSO were added. Cells were then incubated at 37°C for either 0 or 5 min and subsequently washed, fixed, and stained. Arrows denote LBRC enrichment around anti-CD99–arrested monocytes. (d and e) LBRC enrichment was quantified for both 550- or 488-GαM antibodies. (f) Quantitative TEM assays were performed using HUVECs pretreated with either anti–VE-cadherin (control) or anti-CD99 mAb. PBMCs were then added and allowed to transmigrate at 37°C for 50 min. 10 min before fixation, either 8-CPT (general cAMP analogue) or 007-AM (selective-Epac activator) was added to cells. (g) HUVECs were treated in parallel with either 8-CPT or 007-AM for 10 min and then lysed. Immunoblot analysis of pVASP-S157 normalized to total VASP was used to assess PKA activity induced by the drugs. (h) Quantification of results above. Bars, 10 µm. Images are representative of two (c) or three (g) independent experiments. Numerical values are the average of two (d and e) or three (a, b, f, and h) independent experiments. Error bars represent SD (h) or SEM (a, b, and d–f; **, P < 0.01; ***, P < 0.001; Student’s t test [a, b, and d–f] and ANOVA [h]).
Because there are cAMP-independent ways to activate PKA (Niu et al., 2001; Ferraris et al., 2002), we determined if elevating cAMP could restore transmigration in EC blocked with anti-CD99. We performed TEM assays in which HUVECs were pretreated with either nonblocking anti–VE-cadherin (control) or anti-CD99 mAb. Brief treatment with either 8-CPT (a cAMP analogue) or Forskolin (an activator of ACs) restored TEM in anti-CD99 blocked cells to control levels (Fig. 3, a and b). Next, we examined if 8-CPT and Forskolin could also rescued membrane trafficking from the LBRC. We found that within 5 min either 8-CPT or Forskolin restored TR to sites of TEM where leukocytes were arrested by anti-CD99 (Fig. 3, c–e). From these data, we inferred that the ability of 8-CPT and Forskolin to overcome anti-CD99 blockade of TEM was due to their ability to activate PKA to induce LBRC membrane trafficking to sites of leukocyte contact.