Distinct sites within the vascular cell adhesion molecule-1 (VCAM-1) cytoplasmic domain regulate VCAM-1 activation of calcium fluxes versus Rac1 during leukocyte transendothelial migration

Abstract

Vascular adhesion molecules regulate the migration of leukocytes from the blood into tissue during inflammation. Binding of leukocytes to vascular cell adhesion molecule-1 (VCAM-1) activates signals in endothelial cells, including Rac1 and calcium fluxes. These VCAM-1 signals are required for leukocyte transendothelial migration on VCAM-1. However, it has not been reported whether the cytoplasmic domain of VCAM-1 is necessary for these signals. Interestingly, the 19-amino acid sequence of the VCAM-1 cytoplasmic domain is 100% conserved among many mammalian species, suggesting an important functional role for the domain. To examine the function of the VCAM-1 cytoplasmic domain, we deleted the VCAM-1 cytoplasmic domain or mutated the cytoplasmic domain at amino acid N724, S728, Y729, S730, or S737. The cytoplasmic domain and S728, Y729, S730, or S737 were necessary for leukocyte transendothelial migration. S728 and Y729, but not S730 or S737, were necessary for VCAM-1 activation of calcium fluxes. In contrast, S730 and S737, but not S728 or Y729, were necessary for VCAM-1 activation of Rac1. These functional data are consistent with our computational model of the structure of the VCAM-1 cytoplasmic domain as an α-helix with S728 and Y729, and S730 and S737, on opposite sides of the α-helix. Together, these data indicate that S728 and Y729, and S730 and S737, are distinct functional sites that coordinate VCAM-1 activation of calcium fluxes and Rac1 during leukocyte transendothelial migration.

Figure 1. Schematic of the highly conserved 19 amino acid cytoplasmic domain of VCAM-1 and the VCAM-1/ICAM-1 (V/I) chimeric proteins

A) The VCAM-1 19 amino acid cytoplasmic domain sequences for the species listed were obtained from Entrez-Pubmed and Ensemble. Conserved amino acids are underlined and in italics. B) The V/I chimeric molecules were created by replacing the first two immunoglobulin-like domains of VCAM-1 with the first two immunoglobulin-like domains of ICAM-1. This chimeric receptor is designated as V/I wild type (V/I WT). C) The V/I chimeric receptor was mutated to delete the cytoplasmic domain or insert specific amino acid substitutions. The V/I receptor with the cytoplasmic domain deletion (V/I ΔCD) was created by inserting a “stop” codon after the alanine (A723) (white arrow). Alternatively, the VCAM-1 cytoplasmic domain serines, tyrosine, or an asparagine were selected for single point mutations as indicated by the black arrows. The serines and asparagine were mutated to alanines and the tyrosine was mutated to a phenylalanine. mHEV cells were transfected with plasmids containing these mutants and selected for stable expression. At least two separately derived clones were generated for each V/I construct. The V/I in the mHEV clones were completely sequenced to ensure nucleotide sequence accuracy (data not shown).

Figure 2. Expression of VCAM-1 and V/I receptors by the endothelial cells

A) The mHEV clones with stable expression of V/I WT, V/I ΔCD, V/I N724A, V/I S728A, V/I Y729A, V/I S730A, or V/I S737A receptors were immuno-labeled with anti-VCAM-1 primary antibodies (solid lines) or isotype control antibodies (dotted lines) and FITC-conjugated anti-rat IgG secondary antibodies to determine surface VCAM-1 expression by flow cytometry. Shown is a representative graph of one of two separately derived clonal cell lines expressing each V/I chimeric receptor; the flow cytometry profile for the other clone of each chimera was similar (data not shown). B) The clones expressing V/I WT and mutant V/I were immuno-labeled with a FITC-conjugated anti-ICAM-1 antibody to determine V/I expression by flow cytometry. Shown is one representative graph of at least two separately derived clonal cell lines. Dotted lines are the isotype antibody controls. C) The relative molecular weight of VCAM-1 and the V/I proteins were examined by western blot. VCAM-1 or V/I receptors were immuno-precipitated from the endothelial cell clones as indicated and separated by SDS/PAGE. The blots were probed with anti-VCAM-1. Then, the blots were stripped and reprobed with anti-ICAM-1 directed against domain 1 of ICAM-1 to detect the V/I proteins. The V/I WT or the V/I proteins with single amino acid mutations or deletion of the short cytoplasmic domain were relatively the same overall molecular weight as VCAM-1 on SDS/PAGE when compared to the molecular weight standards.

Figure 3. The VCAM-1 cytoplasmic domain amino acids S728, Y729, S730, and S737 but not N724 are necessary for leukocyte transendothelial migration on VCAM-1

Leukocyte transendothelial migration across endothelial cells expressing A) V/I WT, B) V/I ΔCD, C) V/I N724A, D) V/I S728A, E) V/I Y729A, F) V/I S730A, or G) V/I S737A was examined under laminar flow in a parallel plate flow chamber. The endothelial cells were pretreated for 15 minutes with the indicated blocking antibodies against VCAM-1, ICAM-1, or both. Secondary antibodies were not added to the cells so the receptors were not activated by antibody crosslinking. Spleen leukocytes were added to each slide and allowed to settle for 5 minutes to initiate leukocyte-endothelial cell contact in the chamber as detailed in the methods. Then, for transendothelial migration, laminar flow was applied for 15 min at 2 dynes/cm². Migrated cells were determined as phase dark by phase contrast microscopy. Comparisons can only be made within an experiment because total migration can vary somewhat among experiments with the same cell line as previously reported ⁽¹²⁾. Shown is one representative experiment of 2–3 experiments. The other separately derived clone for each V/I chimera had similar results (data not shown). N = 3 slides per treatment. *, p <0.05 compared to anti-VCAM-1-treated group and anti-VCAM-1+anti-ICAM-1-treated group. **, p<0.05 compared to all groups. #, p<0.05 compared to anti-VCAM-1-treated group and anti-ICAM-1-treated group.

Figure 4. Transfection with V/I mutants does not alter leukocyte adhesion

Leukocyte binding to the endothelial cells expressing V/I WT, V/I ΔCD, V/I N724A, V/I S728A, V/I Y729A, V/I S730A, or V/I S737A was determined at 5 minutes using an adhesion assay. Shown is the mean ±SEM from 3–4 experiments. The other separately derived clone for each V/I chimera had similar results (data not shown). In each experiment, an average was obtained from triplicate wells. *, p <0.05 compared to anti-VCAM-1-treated group and anti- VCAM-1+anti-ICAM-1-treated group.

Figure 5. The VCAM-1 cytoplasmic domain amino acids S730, and S737 but not S728, Y729, or N724 are necessary for Rac1 signaling

To crosslink and activate the V/I chimeric receptors, anti-ICAM-1, which is specific for domain 1 of ICAM-1, was incubated with a secondary antibody for five minutes and then added to endothelial cells expressing A) V/I WT, B) V/I ΔCD, C) V/I N724A, D) V/I S728A, E) V/I Y729A, F) V/I S730A, or G) V/I S737A for 30, 60, and 120 seconds. Then, Rac1 activity was determined. As a positive control, an anti-VCAM-1 plus a secondary antibody was added to the cells to crosslink and activate the endogenous VCAM-1 for 30 or 60 seconds. N = 2–4 experiments for each V/I chimeric receptor. The blots are representative blots. The data in the graphs are the average of the two separately derived clones for each V/I chimeric receptor. *, p <0.05 compared to isotype antibody control.

Figure 6. The VCAM-1 cytoplasmic domain amino acids S730, and S737 but not S728, Y729, or N724 are necessary for calcium fluxes

Endothelial cells expressing V/I WT, V/I ΔCD, V/I N724A, V/I S728A, V/I Y729A, V/I S730A, or V/I S737A were loaded with fluo4 and examined for V/I or VCAM-1-induced calcium fluxes. To crosslink and activate the V/I chimeras, anti-ICAM-1, which is specific for domain 1 of ICAM-1, was incubated with a secondary antibody for five minutes and then added to flou4-loaded endothelial cells. Then, relative fluo-4 fluorescence was examined. As a positive control, an anti-VCAM-1 plus a secondary antibody was added to the cells to crosslink and activate the endogenous VCAM-1 for 30 or 60 seconds. Shown are representative calcium fluxes. Also, data from triplicate experiments are presented as the magnitude of the calcium response (peak height; mean± S.E.M.). The other separately derived clone for each V/I chimera had similar results (data not shown). *, p <0.05 compared to isotype control.

Figure 7. Model of the VCAM-1 cytoplasmic domain structure and function

A) The VCAM-1 cytoplasmic domain 19 amino acid sequence (RKANMKGSYSLVEAQKSKV) was submitted to the I-TASSER database for structural prediction. Shown is the model with the highest predictive accuracy score. The three images show three different side-views rotated on the vertical axis of the VCAM-1 cytoplasmic domain with the membrane proximal region at the top and the carboxyl terminus at the bottom of the image. The serines, tyrosine and asparagine are indicated. The I-TASSER program color coded the amino acids as follows: blue for basic amino acids R and K; grey for A and G; light (lt) blue for polar/uncharged amino acids N and Q; yellow for M; purple for Y; red for acidic amino acid E; amber for S; and green for nonpolar amino acids L and V. The S728 and Y729 are on a horizontal plane and on the opposite side of the helix are located S730 and S737 in a vertical plane. B) Schematic for VCAM-1 signaling. Upon antibody crosslinking of VCAM-1, VCAM-1 S728/Y729 function in the activation of calcium fluxes and VCAM-1 S730/S737 function in the activation of Rac-1. The calcium and Rac1 then activate endothelial cell NOX2. Nox2 catalyzes the production of superoxide that then dismutates to H₂O₂. VCAM-1 induces the production of only 1 μM H₂O₂. H₂O₂ activates endothelial cell-associated matrix metalloproteinases (MMPs) that degrade extracellular matrix and endothelial cell surface receptors in cell junctions. H₂O₂ also diffuses through membranes to oxidize and transiently activates endothelial cell protein kinase C-α (PKCα). PKCα phosphorylates and activates protein tyrosine phosphatase 1B (PTP1B) on the endoplasmic reticulum (ER). PTP1B is not oxidized. PTP1B activates signals that induce phosphorylation and activation of ERK1/2. These signals through reactive oxygen species (ROS), MMPs, PKCα, PTP1B, and ERK1/2 are required for VCAM-1-dependent leukocyte transendothelial migration.