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Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration.

Watson RL, Buck J, Levin LR, Winger RC, Wang J, Arase H, Muller WA - J. Exp. Med. (2015)

Bottom Line: 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.

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Affiliation: Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60208.

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Inhibition of sAC prevents CD99 activation of PKA. (a) HUVECs were pretreated with 50 µM KH7 (sAC inhibitor), 25 µM ddAdo (tmAC inhibitor), or DMSO (control). HUVECs were then cross-linked with mIgG1 control or anti-CD99 and lysed. Immunoblot analysis was performed for pVASP-S157 and total VASP. (b) Quantification of immunoblots. Values denote pVASP-S157 signal normalized to total VASP signal. Values were then normalized to the CD99/DMSO condition. (c) Amine-modified polystyrene latex beads were precoupled with mIgG1, anti-CD99, or anti-PECAM. HUVECs expressing hCD99-GFP were pretreated with DMSO, PKI, KH7, or ddAdo. Beads were added to HUVECs for 20 min at 37°C. Samples were then fixed and stained for VE-cadherin (not depicted) and pPKA-T197. Arrows indicate where polystyrene beads bound to HUVECs. (d and e) Data were quantified for percent of beads in the field of view with either hCD99-GFP or pPKA-T197 enrichment. (f) HUVECs were pretreated with DMSO, KH7, or ddAdo. PGE2 (100 ng/ml) was added to HUVECs for 10 min. Cells were then lysed and immunoblot analysis was performed for pCREB-S133 and total CREB. (g) Quantification of immunoblots. Values denote pCREB-S133 signal normalized to total CREB signal. Values for all conditions were then normalized to DMSO/PGE2 condition. Bars, 10 µm. Images are representative of two (c), three (f), or four (a) independent experiments. Numerical values are the average of two (d and e), three (g) four (b) independent experiments. Error bars represent SD (b) or SEM (d, e, and g; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test [b] and ANOVA [d, e, and g]).
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fig4: Inhibition of sAC prevents CD99 activation of PKA. (a) HUVECs were pretreated with 50 µM KH7 (sAC inhibitor), 25 µM ddAdo (tmAC inhibitor), or DMSO (control). HUVECs were then cross-linked with mIgG1 control or anti-CD99 and lysed. Immunoblot analysis was performed for pVASP-S157 and total VASP. (b) Quantification of immunoblots. Values denote pVASP-S157 signal normalized to total VASP signal. Values were then normalized to the CD99/DMSO condition. (c) Amine-modified polystyrene latex beads were precoupled with mIgG1, anti-CD99, or anti-PECAM. HUVECs expressing hCD99-GFP were pretreated with DMSO, PKI, KH7, or ddAdo. Beads were added to HUVECs for 20 min at 37°C. Samples were then fixed and stained for VE-cadherin (not depicted) and pPKA-T197. Arrows indicate where polystyrene beads bound to HUVECs. (d and e) Data were quantified for percent of beads in the field of view with either hCD99-GFP or pPKA-T197 enrichment. (f) HUVECs were pretreated with DMSO, KH7, or ddAdo. PGE2 (100 ng/ml) was added to HUVECs for 10 min. Cells were then lysed and immunoblot analysis was performed for pCREB-S133 and total CREB. (g) Quantification of immunoblots. Values denote pCREB-S133 signal normalized to total CREB signal. Values for all conditions were then normalized to DMSO/PGE2 condition. Bars, 10 µm. Images are representative of two (c), three (f), or four (a) independent experiments. Numerical values are the average of two (d and e), three (g) four (b) independent experiments. Error bars represent SD (b) or SEM (d, e, and g; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test [b] and ANOVA [d, e, and g]).

Mentions: We next investigated how CD99 engagement led to increased cAMP and activated PKA. In mammalian cells, two classes of AC catalyze the formation of cAMP: G protein–regulated transmembrane ACs (tmACs) and sACs (sACs). KH7 is an sAC-specific inhibitor, whereas 2’5′-didexoyadenosine (ddAdo), when used at or below 50 µM, is a tmAC-selective inhibitor (Bitterman et al., 2013). HUVECs were pretreated with KH7, ddAdo, or dimethylsulfoxide (DMSO; control) and cross-linked with anti-CD99 or mIgG1 control. Immunoblot analysis for phospho-VASP was performed to assess PKA activity. The activation of PKA by cross-linking CD99 was significantly attenuated in EC treated with KH7, whereas inhibition of tmACs with ddAdo had no effect (Fig. 4, a and b). To confirm this observation, we used antibody-coated beads and IF staining to spatially assess PKA activity. As seen previously, anti-CD99–coated beads were locally enriched with phospho-PKA staining. Furthermore, activation of PKA was significantly blocked in HUVECs pretreated with KH7 but not with ddAdo. As an additional control, we demonstrated that CD99-coated bead activation of PKA was blocked by the PKA inhibitor myristolyated protein kinase inhibitor peptide (PKI; Fig. 4, c–e). From these data, we conclude that CD99 cross-linking activated PKA via stimulation of sAC.


Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration.

Watson RL, Buck J, Levin LR, Winger RC, Wang J, Arase H, Muller WA - J. Exp. Med. (2015)

Inhibition of sAC prevents CD99 activation of PKA. (a) HUVECs were pretreated with 50 µM KH7 (sAC inhibitor), 25 µM ddAdo (tmAC inhibitor), or DMSO (control). HUVECs were then cross-linked with mIgG1 control or anti-CD99 and lysed. Immunoblot analysis was performed for pVASP-S157 and total VASP. (b) Quantification of immunoblots. Values denote pVASP-S157 signal normalized to total VASP signal. Values were then normalized to the CD99/DMSO condition. (c) Amine-modified polystyrene latex beads were precoupled with mIgG1, anti-CD99, or anti-PECAM. HUVECs expressing hCD99-GFP were pretreated with DMSO, PKI, KH7, or ddAdo. Beads were added to HUVECs for 20 min at 37°C. Samples were then fixed and stained for VE-cadherin (not depicted) and pPKA-T197. Arrows indicate where polystyrene beads bound to HUVECs. (d and e) Data were quantified for percent of beads in the field of view with either hCD99-GFP or pPKA-T197 enrichment. (f) HUVECs were pretreated with DMSO, KH7, or ddAdo. PGE2 (100 ng/ml) was added to HUVECs for 10 min. Cells were then lysed and immunoblot analysis was performed for pCREB-S133 and total CREB. (g) Quantification of immunoblots. Values denote pCREB-S133 signal normalized to total CREB signal. Values for all conditions were then normalized to DMSO/PGE2 condition. Bars, 10 µm. Images are representative of two (c), three (f), or four (a) independent experiments. Numerical values are the average of two (d and e), three (g) four (b) independent experiments. Error bars represent SD (b) or SEM (d, e, and g; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test [b] and ANOVA [d, e, and g]).
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fig4: Inhibition of sAC prevents CD99 activation of PKA. (a) HUVECs were pretreated with 50 µM KH7 (sAC inhibitor), 25 µM ddAdo (tmAC inhibitor), or DMSO (control). HUVECs were then cross-linked with mIgG1 control or anti-CD99 and lysed. Immunoblot analysis was performed for pVASP-S157 and total VASP. (b) Quantification of immunoblots. Values denote pVASP-S157 signal normalized to total VASP signal. Values were then normalized to the CD99/DMSO condition. (c) Amine-modified polystyrene latex beads were precoupled with mIgG1, anti-CD99, or anti-PECAM. HUVECs expressing hCD99-GFP were pretreated with DMSO, PKI, KH7, or ddAdo. Beads were added to HUVECs for 20 min at 37°C. Samples were then fixed and stained for VE-cadherin (not depicted) and pPKA-T197. Arrows indicate where polystyrene beads bound to HUVECs. (d and e) Data were quantified for percent of beads in the field of view with either hCD99-GFP or pPKA-T197 enrichment. (f) HUVECs were pretreated with DMSO, KH7, or ddAdo. PGE2 (100 ng/ml) was added to HUVECs for 10 min. Cells were then lysed and immunoblot analysis was performed for pCREB-S133 and total CREB. (g) Quantification of immunoblots. Values denote pCREB-S133 signal normalized to total CREB signal. Values for all conditions were then normalized to DMSO/PGE2 condition. Bars, 10 µm. Images are representative of two (c), three (f), or four (a) independent experiments. Numerical values are the average of two (d and e), three (g) four (b) independent experiments. Error bars represent SD (b) or SEM (d, e, and g; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test [b] and ANOVA [d, e, and g]).
Mentions: We next investigated how CD99 engagement led to increased cAMP and activated PKA. In mammalian cells, two classes of AC catalyze the formation of cAMP: G protein–regulated transmembrane ACs (tmACs) and sACs (sACs). KH7 is an sAC-specific inhibitor, whereas 2’5′-didexoyadenosine (ddAdo), when used at or below 50 µM, is a tmAC-selective inhibitor (Bitterman et al., 2013). HUVECs were pretreated with KH7, ddAdo, or dimethylsulfoxide (DMSO; control) and cross-linked with anti-CD99 or mIgG1 control. Immunoblot analysis for phospho-VASP was performed to assess PKA activity. The activation of PKA by cross-linking CD99 was significantly attenuated in EC treated with KH7, whereas inhibition of tmACs with ddAdo had no effect (Fig. 4, a and b). To confirm this observation, we used antibody-coated beads and IF staining to spatially assess PKA activity. As seen previously, anti-CD99–coated beads were locally enriched with phospho-PKA staining. Furthermore, activation of PKA was significantly blocked in HUVECs pretreated with KH7 but not with ddAdo. As an additional control, we demonstrated that CD99-coated bead activation of PKA was blocked by the PKA inhibitor myristolyated protein kinase inhibitor peptide (PKI; Fig. 4, c–e). From these data, we conclude that CD99 cross-linking activated PKA via stimulation of sAC.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60208.

Show MeSH
Related in: MedlinePlus