PECAM-1 modulates thrombin-induced tissue factor expression on endothelial cells.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) (CD31) is known to inhibit platelet function and thrombus formation. The mechanisms involved in PECAM-1's roles as a modulator of hemostasis are still not completely understood. We examined the role of PECAM-1 as a regulator of tissue factor (TF) expression, a known important inducer of thrombosis. Wildtype and CD31KO mice underwent transient (30 min) renal ischemia followed by 24 h re-perfusion and their kidneys assessed for apoptosis, fibrin formation, and tissue factor expression. CD31KO mice exhibited increased tubular epithelial and endothelial apoptosis, increased fibrin deposition, and tissue factor expression. Human umbilical vein endothelial cells (HUVEC) transfected with antisense (AS) PECAM-1 oligonucleotides to downregulate PECAM-1 expression, exhibited greater induction of TF mRNA and protein expression as well as increased expression and nuclear localization of the transcription factor Egr-1 compared to scrambled AS PECAM-1 (Scr)-treated HUVEC following thrombin stimulation. TF induction was found to be mediated through thrombin receptor PAR-1 and the Galphai/o subunit of G-protein, confirmed by PAR-1 antagonist and pertussis toxin inhibition respectively. Thrombin-mediated TF induction was dependent on Rho Kinase activity, phosphorylation of p38(MAPK) and p85 & Akt dephosphorylation. The inverse correlation of PI3K-Akt phosphorylation with p38 (MAPK) phosphorylation was confirmed by pharmacological inhibition. These studies suggest that PECAM-1 is involved in regulating a signaling pathway, affecting PI3K and Akt activation, p38 (MAPK) phosphorylation, which in turn, affects Egr-1 expression and nuclear translocation, ultimately affecting TF expression. These findings provide new insights into the action of PECAM-1 as a modulator of thrombosis.

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

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.

Identification of the regions of PECAM-1 involved in beta- and gamma-catenin associations.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) binds tyrosine-phosphorylated beta-catenin and modulates beta-catenin localization and sequestration. The biological significance of this interaction, while still unclear, it has been postulated to be involved in modulating adherens junction dynamics in response to perturbants [J. Clin. Invest. 109 (2002) 383]. Here we demonstrate that tyrosine-phosphorylated beta-catenin, and to a lesser extent unphosphorylated beta-catenin, interact with a portion of the cytoplasmic domain of PECAM-1 encoded by exon 15. Using RT-PCR, we obtained products representing alternatively spliced PECAM-1 isoforms from mouse kidney total mRNA and generated PECAM-1-GST constructs expressing full length and naturally occurring alternatively spliced PECAM-1 variants. Co-precipitation assays revealed that the protein sequence encoded by exon 15 is necessary for beta-catenin binding. Transfections using deletion mutants confirmed the importance of the exon 15 sequence in this interaction. In contrast, gamma-catenin-PECAM-1 interactions are thought to be modulated by an as yet undefined PECAM-1 serine phosphorylation and appear to mediate dynamic PECAM-1 intermediate filament cytoskeletal interactions [J. Biol. Chem. 275 (2000) 21435]. Here we demonstrate that the PECAM-1-gamma-catenin interaction occurs via an exon 13-mediated process. GST-pull-down assays illustrated the importance of the exon 13 sequence in this interaction. Further, using site-directed mutagenesis of S(673) to C and D and S(669 and 670) to C, we confirmed the importance of S(673) and its phosphorylation state as a mediator of gamma-catenin-PECAM-1 binding. Our studies define the exons of the PECAM-1 cytoplasmic domain that is involved in mediating these PECAM-1-catenin family member interactions and will allow investigators to better define the biological functions resulting from these interactions.

Hepatocyte growth factor induces an endothelin-mediated decline in glomerular filtration rate.

Hepatocyte growth factor (HGF) is a multifunctional cytokine that plays a crucial role in renal development, injury, and repair. HGF also serves a protective role in chronic renal disease by preventing tissue fibrosis. Endothelin-1 (ET-1), produced primarily by endothelial cells, is a potent vasoconstrictor that also acts as a proinflammatory peptide, promoting vascular injury and renal damage. In addition to mediating a variety of epithelial cell responses, HGF also induces hemodynamic changes that are poorly understood. The aim of the present study was to study the acute and chronic effects of HGF on ET-1 production in the kidney. We hypothesized that hemodynamic changes upon HGF treatment are likely mediated by immediate ET-1 release, whereas protection from renal fibrosis in rats chronically treated with HGF is likely due to suppression of ET-1 production. Acute HGF infusion into rats caused a decline in blood pressure that was enhanced by pretreatment with bosentan (an endothelin A and B receptor antagonist). HGF infusion also resulted in a decline in glomerular filtration rate (GFR) that could be entirely prevented by bosentan, suggesting that HGF acutely increases production and/or release of ET-1, which then mediates the observed decline in GFR. In cultured glomerular endothelial cells, HGF induced ET-1 production in a dose-dependent manner. Moreover, although there was an initial increase in ET-1 production upon HGF treatment, longer administration suppressed ET-1 production. This finding was consistent with the observation in vivo of a decrease in ET-1 production in renal parenchyma of rats chronically treated with HGF. Our data suggest both a hemodynamic and biological role for HGF-mediated ET-1 regulation.

Hepatocyte growth factor ameliorates renal interstitial inflammation in rat remnant kidney by modulating tubular expression of macrophage chemoattractant protein-1 and RANTES.

Hepatocyte growth factor (HGF) has been shown to reduce renal injury in a variety of animal models of chronic renal disease. Suggested mechanisms to explain this action include prevention of tubular cell apoptosis, blocking epithelial-to-mesenchymal transition, and promotion of extracellular matrix degradation. Inflammation is another common finding in kidneys that progress to end-stage renal failure; however, the effect of HGF on inflammation has hardly been investigated. For examining this issue, beginning 2 wk after subtotal nephrectomy, rats received a continuous infusion of recombinant HGF, neutralization of endogenous HGF by daily injection of an anti-HGF antibody, or preimmune IgG for an additional 2 wk. HGF infusion halted the progression of proteinuria and decreased renal collagen accumulation. Renal inflammation in both glomeruli and tubulointerstitium was significantly attenuated, associated with reductions in the tubular expression of the chemokines macrophage chemoattractant protein-1 (MCP-1) and RANTES (regulated upon expression normal T cell expressed and secreted). In contrast, HGF neutralization worsened renal fibrosis, aggravated renal inflammation, and enhanced tubular expression of MCP-1 and RANTES. In vitro, HGF suppressed basal and TNF-alpha-induced expression of these chemokines at both the mRNA and protein levels in a time- and dose-dependent manner in proximal tubular epithelial cells. HGF also blunted TNF-alpha-induced nuclear translocation and activation of NF-kappaB, a pivotal transcription factor that regulates chemokine expression. Immunohistochemistry showed that activated NF-kappaB was evident in tubules in remnant kidneys and increased remarkably with anti-HGF treatment. HGF infusion markedly suppressed expression of activated NF-kappaB in remnant kidneys. These findings suggest that the beneficial effect of HGF in chronic renal disease is attributable, at least in part, to a direct anti-inflammatory action, likely via NF-kappaB, on tubular epithelial cells.

PECAM-1 promotes beta-catenin accumulation and stimulates endothelial cell proliferation.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) binds tyrosine phosphorylated beta-catenin and modulates beta-catenin localization [J. Immunol. 158 (7) (1997) 3408; J. Cell Sci. 112 (Pt 18) (1999) 3005]. To elucidate functional consequences of this interaction, we studied endothelial cells from PECAM-1 knockout animals and compared them to PECAM-1 expressing endothelial cells [Mol. Biol. Cell 11 (9) (2000) 3109]. We noted an increase in the expression of beta-catenin protein in PECAM-1 expressing endothelial cells. Further, by immunofluorescence, beta-catenin localized to the cell membrane as well as to the nucleus in PECAM-1 positive endothelial cells, whereas cells not expressing PECAM-1 stained for beta-catenin only at the membrane. Additionally, we demonstrate that PECAM-1 lacking the majority of the cytoplasmic domain promotes significantly less accumulation of transcriptionally active beta-catenin than full-length PECAM-1. Finally, we note an increased proliferative rate in the PECAM-1 reconstituted cells compared to the endothelial cells lacking PECAM-1. Taken together, our data suggest that PECAM-1, an adhesion molecule, affects cell proliferation via accumulation of transcriptionally active beta-catenin.

Lack of platelet endothelial cell adhesion molecule-1 attenuates foreign body inflammation because of decreased angiogenesis.

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kd member of the immunoglobulin superfamily of proteins, expressed on endothelial cells, leukocytes, and platelets. Antibody-blocking studies have implicated it in modulating leukocyte transmigration and angiogenesis. However, the generation of the PECAM-1 knockout mouse has shown that its function can be compensated for by similarly acting proteins because most acute inflammatory models proceed in a comparable manner in wild-type and knockout animals. We decided to examine the function of PECAM-1 in the chronic process of foreign body inflammation. We show that PECAM-1-deficient mice exhibit attenuated neutrophil infiltration in and around a subcutaneous polyvinyl acetyl implant. Bone marrow engraftment studies indicate that the lack of CD31 expression on the endothelium determines the diminished leukocyte accumulation in the knockout implants. Specifically, we find that decreased angiogenesis (as manifested by lower vessel density, decreased hemoglobin content, and less laminin deposition) correlates with lower neutrophil accumulation in the knockout animals. This study indicates that the absence of endothelial PECAM-1 results in decreased angiogenesis and therefore in diminished delivery of leukocytes to the foreign body implants.