PECAM-1 affects GSK-3beta-mediated beta-catenin phosphorylation and degradation

Abstract

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) regulates a variety of endothelial and immune cell biological responses. PECAM-1-null mice exhibit prolonged and increased permeability after inflammatory insults. We observed that in PECAM-1-null endothelial cells (ECs), beta-catenin remained tyrosine phosphorylated, coinciding with a sustained increase in permeability. Src homology 2 domain containing phosphatase 2 (SHP-2) association with beta-catenin was diminished in PECAM-1-null ECs, suggesting that lack of PECAM-1 inhibits the ability of this adherens junction component to become dephosphorylated, promoting a sustained increase in permeability. beta-Catenin/Glycogen synthase kinase 3 (GSK-3beta) association and beta-catenin serine phosphorylation levels were increased and beta-catenin expression levels were reduced in PECAM-1-null ECs. Glycogen synthase kinase 3 (GSK-3beta) serine phosphorylation (inactivation) was blunted in PECAM-1-null ECs after histamine treatment or shear stress. Our data suggest that PECAM-1 serves as a critical dynamic regulator of endothelial barrier permeability. On stimulation by a vasoactive substance or shear stress, PECAM-1 became tyrosine phosphorylated, enabling recruitment of SHP-2 and tyrosine-phosphorylated beta-catenin to its cytoplasmic domain, facilitating dephosphorylation of beta-catenin, and allowing reconstitution of adherens junctions. In addition, PECAM-1 modulated the levels of beta-catenin by regulating the activity of GSK-3beta, which in turn affected the serine phosphorylation of beta-catenin and its proteosomal degradation, affecting the ability of the cell to reform adherens junctions in a timely fashion.

Figure 1

Endothelial cells devoid of PECAM-1 (KO) and endothelial cells reconstituted with PECAM-1 (RC) express similar levels of the H1 histamine receptor. Top panel: Representative Western blots of lysates derived from lung microvascular endothelial cells isolated from CD31 KO mice (KO) and similar CD31 KO endothelial cells transfected with and stably expressing full-length wild-type PECAM-1 (RC) express similar levels of H1 histamine receptor. Bottom panel: β-Actin was used to normalize the protein loads.

Figure 2

Histamine induces prolonged increase in permeability in endothelial cells lacking PECAM-1. A: A graph illustrating the quantitation of the changes in the permeability of reconstituted (RC; open boxes) and PECAM-1-deficient (KO; dark shaded boxes) microvascular endothelial cell confluent monolayers after exposure to histamine. Permeability of the monolayers to Evans blue dye was measured by collecting medium from the lower wells of the transwell chambers and measuring the absorbance at 650 nm. n = 5; *P < 0.05. B: A graph illustrating the quantitation of the changes in tyrosine-phosphorylated β-catenin expression in reconstituted (RC; open boxes) and PECAM-1-deficient (KO; dark shaded boxes) microvascular endothelial cell confluent monolayers after exposure to histamine. Samples were immunoprecipitated with anti-β-catenin antibodies, followed by anti-phosphotyrosine (PY) antibody immunoblotting, stripping, and anti-β-catenin immunoblotting. Phosphotyrosine band intensities were normalized to β-catenin band intensities. Vertical bars represent SD. n = 4; *P < 0.05. C: Immunofluorescence micrographs illustrating changes in the cell periphery β-catenin staining in reconstituted (RC; A, C, and E) and PECAM-1-deficient (KO; B, D, and F) microvascular endothelial cell cultures before (A and B) and after exposure to histamine at 5-minute (C and D) and 15-minute (E and F) exposure times. Confluent cultures were labeled with anti-β-catenin. At the zero time point, both WT and KO cultures exhibited uniform, continuous peripheral staining outlining individual cells. Five minutes after histamine treatment, both WT and KO endothelial monolayers exhibited discontinuous peripheral β-catenin staining, consistent with disruption of monolayer integrity. At 15 minutes, the WT cultures exhibited re-formation of continuous peripheral β-catenin staining, whereas the KO cultures demonstrated a persistence of discontinuous, peripheral β-catenin staining, consistent with sustained disruption of monolayer integrity. Scale bar = 25 μm; arrows indicate discontinuities in the peripheral β-catenin staining.

Figure 3

Histamine induces tyrosine phosphorylation of PECAM-1, and PECAM-1 promotes interactions between β-catenin and SHP-2. A: Confluent RC monolayers were treated with histamine for 10 minutes, and lysates were immunoprecipitated with anti-phosphotyrosine antibody followed by Western blotting for PECAM-1. An increase in tyrosine-phosphorylated PECAM-1 is noted 10 minutes after histamine treatment. B: Lysates of confluent RC and KO monolayers were normalized for protein, run on SDS-polyacrylamide gel electrophoresis, and immunoblotted for SHP-2, revealing essentially equal levels of expression of this phosphatase. C: Confluent RC and KO monolayers were treated with histamine for 15 minutes, and lysates were immunoprecipitated with anti-SHP-2 antibody followed by Western blotting for β-catenin. Very little β-catenin is observed to be associated with SHP-2 prior histamine treatment in the RC cell cultures. However, after histamine treatment, a significant increase in β-catenin/SHP-2 association was observed after histamine treatment. In contrast, very little β-catenin is observed to be associated with SHP-2 before and after histamine treatment in the KO cell cultures. Expression of SHP-2 was similar in both RC and KO cultures. D: Averages of four independent experiments assessing β-catenin/SHP-2 association in RC and KO cultures before and after histamine treatment illustrating significant (P < 0.05) increases in β-catenin/SHP-2 association in RC cultures after histamine treatment, whereas no significant changes (P > 0.05) in the modest β-catenin/SHP-2 association were noted in the KO cultures. Vertical lines represent SD.

Figure 4

Histamine treatment elicits increased β-catenin/PECAM-1 association. Representative Western blots of RC cultures treated with histamine for 0, 5, 15, and 60 minutes. The RC endothelial cells exhibited increased β-catenin/PECAM-1 association when assayed by immunoprecipitation with anti-PECAM-1 followed by Western blotting with anti-β-catenin (top panel) and PECAM-1 (bottom panel).

Figure 5

PECAM-1 blunts GSK-3β activity, abrogates β-catenin-GSK-3β association, and abrogates β-catenin serine phosphorylation, diminishing β-catenin degradation. A: Representative Western blot of RC and KO endothelial cell cultures labeled with anti-pSer9-GSK-3β (pGSK-3β) (top panel) and anti-GSK-3β illustrating an increased pSer9-GSK-3β fraction of pS-GSK-3β in the RC cultures. B: Representative Western blot of RC and KO endothelial cell cultures labeled with anti-pS-β-catenin (anti-Ser33, Ser37, and Thr41-β-catenin), illustrating increased pS-β-catenin in the KO cultures. C: Representative Western blot of RC and KO endothelial cell cultures labeled with anti-β-catenin (top panel) and anti-GSK-3β after an immunoprecipitation with anti-GSK-3β, illustrating increased GSK-3β-associated β-catenin in the KO cultures. D: Quantitation of Western blots of brain WT (open boxes) and KO (gray filled boxes) endothelial cell cultures, before and after histamine treatment (0, 5, and 10 minutes), labeled with anti-pS-GSK-3β and anti-GSK-3β and normalized to total GSK-3β. Note the increase in the WT pS-GSK-3β fraction at the 5-minute time point. n = 3. E: Quantitation of Western blots of lung RC (open boxes) and KO (gray filled boxes) endothelial cell cultures, before and after histamine treatment (0, 5, and 10 minutes), labeled with anti-pS-GSK-3β and anti-GSK-3β and normalized to total GSK-3β. Note the increase in the WT pS-GSK-3β fraction at the 10 minutes time point. n = 3. F: Representative Western blots of lung CD31 KO lysates with and without a 6-hour pretreatment with lactacystin (10 μmol/L) followed by a 10-minute treatment with vehicle or before a 10-minute histamine treatment (100 μmol/L). The blot was labeled with anti-phospho-serine β-catenin (anti-Ser33, Ser37, and Thr41-β-catenin), illustrating a robust increase in pS-β-catenin in the cells pretreated with lactacystin followed by histamine treatment at the 10-minute time point (top panel). When stripped and re-blotted with anti-β-catenin, the blot also revealed a robust increase in β-catenin in the cells pretreated with lactacystin followed by histamine treatment at the 10-minute time point (bottom panel). The samples were normalized to protein load. G: Representative Western blots of HUVEC lysates before and after histamine treatment (0, 5, and 15 minutes) labeled with anti-GSK-3β (top panel), anti-pSer9-GSK-3β GDK-3β) (second panel), illustrating a transient increase in pSer9-GSK-3β after histamine treatment at the 5-minute time point. The blots were normalized to Erk-2 (third panel), and the pS-GSK-3β/GSK-3β ratios were calculated (bottom panel).

Figure 6

Shear stress increases GSK-3β inactivation and abrogates β-catenin serine phosphorylation in PECAM-1-expressing endothelial cells but not in PECAM-1-null cells. A: Representative Western blots of lung-derived CD31 KO (top two panels) and CD31RC (bottom two panels) cultures labeled with anti-serine9 phosphorylated GSK-3β (pGSK-3β) and anti-GSK-3β (Tot GSK) before and after 0, 5, 10, 30, and 60 minutes of shear stress. The bottom panel is a quantitation of the percent change in serine-phosphorylated GSK-3β in the CD31KO (squares) and CD31RC (circles) cultures before and after various time periods of shear stress. Vertical lines represent SEM. n = 4. B: Representative Western blots of lung-derived CD31 KO (top two panels) and CD31RC (bottom two panels) cultures labeled with anti-serine-phosphorylated β-catenin (anti-Ser33, Ser37, and Thr41-β-catenin) (pβcat) and anti-β-catenin (Tot βcat) before and after 0, 5, 10, 30, and 60 minutes of shear stress. The bottom panel is a quantitation of the percent change in serine/threonine phosphorylated β-catenin in the CD31KO (squares) and CD31RC (circles) cultures before and after various time periods of shear stress. Vertical lines represent SEM. n = 4.

Figure 7

Levels of pP85 (pPI3K), pAkt, and pS-GSK-3β correlate directly with PECAM-1 expression levels. A: Representative Western blots of HUVECs treated with either a scrambled PECAM-1 oligonucleotide (scrambled CD31) and/or antisense PECAM-1 oligonucleotide (antisense CD31), revealing a significant knockdown of PECAM-1 expression in the antisense CD31-treated cultures, which correlated with significant knockdowns of tyrosine-phosphorylated (active) PI3K and Akt. n = 3. B: Quantitation of three independent experiments consisting of HUVECs treated with either a scrambled PECAM-1 oligonucleotide (scrambled CD31) and/or antisense PECAM-1 oligonucleotide (antisense CD31), illustrating significant knockdown of phospho-Akt and phospho-PI3K. C: Representative Western blots of three independent experiments consisting of lung-derived RC and CD31 KO and brain-derived WT and CD31 KO endothelial cell cultures, illustrating decreased phospho-PI3K and phospho-Akt levels in the CD31 KO cultures compared with the WT and RC cultures.

Figure 8

Working model illustrating the effects of presence or absence of PECAM-1 on GSK-3β-mediated β-catenin phosphorylation and degradation. A: In PECAM-1-expressing endothelial cells, tyrosine phosphorylated β-catenin is efficiently complexed with SHP-2 in a tripartite complex with immunoreceptor tyrosine-based activation motif tyrosine phosphorylated PECAM-1, dephosphorylated, and made available to participate in reformation of adherens junctional complexes and translocation to the nucleus where it modulates gene expression on complexing with Lef/Tcf. PECAM-1 expression is also associated with increased activity (phosphorylation) of PI3K and Akt, which increase the serine phosphorylation of GSK-3β, inactivating it, thus reducing the fraction of β-catenin that is serine/threonine phosphorylated and thus targeted for proteosomal degradation. B: In the absence/reduction of PECAM-1 expression, tyrosine-phosphorylated β-catenin is inefficiently complexed with and dephosphorylated by SHP-2, reducing its ability to participate in the reformation of adherens junctions (dashed lines). In addition, the absence/reduction of PECAM-1 expression is also associated with a reduction in PI3K and Akt activity (phosphorylation), leading to a persistent high activity of GSK-3β (dashed line), resulting in an increased serine/threonine phosphorylation of β-catenin and its targeting for proteosomal degradation (heavy solid lines) and reduced nuclear translocation (light solid lines).