Plasminogen activator inhibitor-1 promotes angiogenesis by stimulating endothelial cell migration toward fibronectin.

Increased expression of plasminogen activator inhibitor-1 (PAI-1) in cancer patients is associated with unfavorable outcome, and the reason for this paradox has been poorly understood. We have previously reported elevated levels of PAI-1 in primary tumors of advanced neuroblastomas (Y. Sugiura et al., Cancer Res., 59: 1327-1336, 1999). Here we demonstrate that PAI-1 is coexpressed with the angiogenesis marker alpha(v)beta3 integrin in blood vessels of primary neuroblastoma tumors, suggesting that PAI-1 plays a role in angiogenesis. Using human brain microvascular endothelial cells (HBMECs), we found that PAI-1 inhibits alpha(v)beta3 integrin-mediated cell adhesion to vitronectin but promotes alpha5beta1-mediated migration from vitronectin toward fibronectin. Inhibition of vitronectin adhesion by PAI-1 did not induce HBMEC apoptosis. PAI-1 also inhibited endothelial tube formation on Matrigel in the presence of vitronectin but had a stimulatory effect in the presence of fibronectin. This effect of PAI-1 on microvascular endothelial cells is primarily related to the ability of PAI-1 to bind to vitronectin via its NH2-terminal domain and to interfere with cell adhesion to vitronectin. We propose that PAI-1 acts as a positive switch for angiogenesis by promoting endothelial cell migration away from their vitronectin-containing perivascular space toward fibronectin-rich tumor tissue. These observations provide a novel explanation for the enhancing effect of PAI-1 in cancer progression.

Integrins alpha(v)beta3 and alpha(v)beta5 are expressed by endothelium of high-risk neuroblastoma and their inhibition is associated with increased endogenous ceramide.

Inhibition of the RGD-binding integrins, alpha(v)beta3 and alpha(v)beta5, prevents endothelial cell anchorage and induces endothelial apoptosis, which results in disruption of tumor angiogenesis and inhibition of tumor growth in animal models. In this study, we demonstrate by immunohistochemical analysis that integrin alpha(v)beta3 was expressed by 61% (mean) of microvessels in high-risk neuroblastomas (stage IV and MYCN-amplified stage III; n = 28) but only by 18% (mean) of microvessels in low-risk tumors (stages I and II and non-MYCN-amplified stage III; n = 12). Integrin alpha(v)beta5 was found on 60% (mean) of microvessels in 21 Stage IV tumors. These data suggest that neuroblastomas may be targeted for antiangiogenic treatment directed against endothelial integrins alpha(v)beta3 and alpha(v)beta5. In cell culture, inhibition of integrin-dependent endothelial cell anchorage to vitronectin by RGDfV, an RGD function-blocking cyclic peptide, induced apoptosis in bovine brain endothelial cells compared with the control peptide, RADfV (37.5% versus 8.7%, respectively), as detected by chromatin condensation and nuclear fragmentation. Treatment with RGDfV but not with RADfV, which prevented attachment of endothelial cells to vitronectin or fibronectin, was associated with up to a 50% increase in endogenous ceramide, a lipid second messenger that can mediate cell death. Furthermore, exogenous C2-ceramide was cytotoxic to bovine brain endothelial cells and induced activation of C-jun N-terminal kinase (JNK), a MAP kinase that can be activated in stress-induced apoptosis pathways. This suggests that ceramide may function in detachment-induced endothelial cell apoptosis, originating from inhibition of vitronectin binding to integrins such as alpha(v)beta3 and alpha(v)beta5. This is the first report to demonstrate expression of integrins alpha(v)beta3 and alpha(v)beta5 by microvascular endothelium of a childhood tumor and association of their expression with neuroblastoma aggressiveness. Furthermore, our data provide the first suggestion that inhibition of endothelial cell anchorage, resulting from specific blockade of RGD-binding integrins, increases endogenous ceramide, which may contribute to endothelial cell death.

Cbl functions downstream of Src kinases in Fc gamma RI signaling in primary human macrophages.

Cbl is a cytosolic protein that is rapidly tyrosine phosphorylated in response to Fc receptor activation and binds to the adaptor proteins Grb2, CrkL, and Nck. A few reports describe Cbl interactions in primary human hematopoietic cells. We show evidence that Cbl participates in signaling initiated by Fc gammaRI receptor cross-linking in human primary macrophages, and functions downstream of Src family kinases in this pathway. Fc gammaRI stimulation in human macrophages was associated with rapid and transient tyrosine phosphorylation of the Cbl adaptor protein. Immunoprecipitated Cbl was complexed with several tyrosine phosphorylated proteins, the most prominent of which was a 38-kDa band identified as the CrkL adaptor protein. CrkL associated with tyrosine-phosphorylated Cbl and itself became tyrosine phosphorylated after Fc gammaRI cross-linking. SLP-76, a recently cloned Grb2-associated protein, was strongly tyrosine phosphorylated after Fc gammaRI stimulation and was associated with both Cbl and Grb2. Grb2 and Cbl binding to SLP-76 were inducible after Fc gammaRI stimulation of the macrophages. Nck was inducibly bound to Cbl after Fc gammaRI stimulation, whereas Grb2 was constitutively associated with it. Shc was also inducibly tyrosine phosphorylated and bound to Grb2 after Fc gammaRI stimulation of the macrophages. PP1, a specific inhibitor of Src kinases, inhibited the Fc gammaRI-induced respiratory burst, as well as the tyrosine phosphorylation of Cbl and its inducible association with CrkL. These results suggest a fundamental role for the tyrosine phosphorylation of Cbl, CrkL, SLP-76, and Shc and the association of Cbl with CrkL, SLP-76, and Nck in Fc gammaRI signaling in human macrophages. Experiments performed with PP1, the specific Src kinase inhibitor, demonstrate the first evidence that Cbl and the Cbl-Crkl interaction are downstream targets for myeloid Src kinases required for the activation of myeloid NADPH oxidase activity.

Differential expression of Wnt genes in normal and flat variants of PC12 cells, a cell line responsive to ectopic Wnt1 expression.

Wnt genes encode secreted growth factor-like proteins that participate in growth regulation, differentiation and tumorigenesis. Ectopic expression of Wnt1 converts the PC12 neural crest-derived rat pheochromocytoma cell line from a round phenotype that express chromaffin markers to flat adherent cells (termed PC12/Wnt1) that do not express them. A pool of spontaneously flat variants of PC12 cells (PC12/flat) is phenotypically similar to the PC12/Wnt1 cells, but does not express Wnt1. Here we describe the expression of 13 Wnt genes in wild type PC12, PC12/flat and PC12/Wnt1 cells. Wild type PC12 expressed Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt6, Wnt10a and Wnt11. Compared with expression in wild type cells, both PC12/flat and PC12/Wnt1 cells lost most or all expression of Wnt3a and Wnt4 and gained expression of Wnt7b. Wnt5a and Wnt6 expression was higher in PC12/Wnt1 cells than in PC12 or PC12/flat. Wnt3 was expressed at low levels in both PC12 and PC12/flat, but was absent in PC12/Wnt1 cells. Wnt10a and Wnt11 were approximately equally expressed in the three groups, and Wnt2, Wnt5b, Wnt7a, Wnt10b and endogenous Wnt1 mRNAs were not detected. These results demonstrate that the expression of some Wnt genes changes in PC12 cells upon conversion to the flat phenotype, and suggest that Wnt1 may modulate expression of several other Wnt genes in these cells.

Prolonged clinical response to vincristine treatment in two patients with idiopathic hypereosinophilic syndrome.

Idiopathic hypereosinophilic syndrome (HES) is a rapidly progressive disease that can result in death within weeks to months of diagnosis in untreated patients. We report two pediatric cases of HES. One patient has remained clinically and hematologically stable for 9 years with vincristine and 6-thioguanine (6TG), and the other has received vincristine alone for 6 months with a good response. The use of long-term vincristine and 6TG or vincristine alone has not been previously reported in the management of HES in children. Vincristine administered every 3-4 weeks with or without 6TG can produce a reduction in the overall white cell count and improvement of symptoms of hypereosinophilia. Although the mortality of patients with HES is high, aggressive treatment can result in significant clinical benefit and improved prognosis in children with this syndrome.

Protein tyrosine phosphatase inhibitors in Fc gamma RI-induced myeloid oxidant signaling.

Fc-receptor stimulation in myeloid cells results in increased oxygen consumption, termed the respiratory burst, which is coupled to a rapid and transient increase in tyrosine phosphorylation of cellular proteins. In a previous paper in this journal we showed that the protein tyrosine phosphatase (PTPase) inhibitors sodium orthovanadate and phenylarsine oxide (PAO) block the Fc gamma RI-induced respiratory burst in interferon-gamma-differentiated U937 cells (U937IF) while augmenting the Fc gamma RI-induced tyrosine phosphorylation of cellular proteins. Herein we examine the effects of PTPase inhibitors on specific molecules involved in Fc gamma RI signaling. We show that orthovanadate and PAO augmented the Fc gamma RI-induced tyrosine phosphorylation of the adaptor protein CBL. CBL interactions with other phosphoproteins, among them SHC and CRKL, were also augmented in response to pretreatment with the PTPase inhibitors. SHC was tyrosine phosphorylated in response to Fc gamma RI stimulation of U937IF cells and bound to the SH2 domain of GRB2 in a stimulation-dependent manner. In fusion protein pull-down experiments the interaction of SHC with the SH2 domain of GRB2 was increased in PTPase inhibitor pretreated U937IF cells in response to Fc gamma RI stimulation. Our data support the hypothesis that a tyrosine dephosphorylation event is required for effective transmission of the Fc gamma RI signal to result in activation of the myeloid respiratory burst response.

Characterization of Cbl-Nck and Nck-Pak1 interactions in myeloid FcgammaRII signaling.

Fc receptors modulate inflammatory processes, including phagocytosis, serotonin and histamine release, superoxide production, and secretion of cytokines. Aggregation of FcgammaRIIa, the low-affinity receptor for monomeric IgG, activates nonreceptor protein tyrosine kinases such as Lyn, Hck, and Syk, potentially driving the phosphorylation of the downstream adaptor proteins, including Cbl and/or Nck. Previous work from our laboratory using interferon-gamma-differentiated U937 (U937IF) myeloid cells investigated mechanisms which regulate Fcgamma receptor-induced assembly of adaptor complexes. Herein we report that FcgammaRII receptor signaling in U937IF and HEL cells involves Cbl and Nck, suggesting that Cbl-Nck interactions may link FcgammaRII to downstream activation of Pak kinase. FcgammaRII crosslinking induced the phosphorylation of Cbl and Nck on tyrosine. The alphaCbl immunoprecipitations revealed constitutive binding of Nck and Grb2 to Cbl and FcgammaRII-inducible binding of CrkL to Cbl. The interactions of Cbl with Nck and CrkL were phosphorylation dependent since dephosphorylation of cellular proteins with potato acid phosphatase abrogated binding. GST-Nck fusion protein pulldown experiments show that Cbl and Pak1 bind to the second SH3 domain of Nck. A specific Src inhibitor, PP1, was shown to completely abrogate the FcgammaR-induced superoxide response, correlating with a decrease in Cbl and Nck tyrosine phosphorylation. Our results provide the first evidence that Src is required for FcgammaR activation of the respiratory burst in myeloid cells and suggest that Cbl-Nck, Cbl-Pak1, and Nck-Pak1 interactions may regulate this response.

High affinity IgG receptor activation of Src family kinases is required for modulation of the Shc-Grb2-Sos complex and the downstream activation of the nicotinamide adenine dinucleotide phosphate (reduced) oxidase.

We used the U937 cell line to examine the modulation of adaptor protein interactions (Shc, Grb2, and Cbl) after high affinity IgG receptor (FcgammaRI) cross-linking, leading to the formation of the Grb2-Sos complex, the activation of Ras, and the regulation of the respiratory burst. Cross-linking of FcgammaRI induced the conversion of GDP-Ras to GTP-Ras reaching a maximum 5 min after stimulation. Concomitant with Ras activation, Sos underwent an electrophoretic mobility shift and the Sos-Grb2 association was increased (6-fold). The Grb2-Sos complex was present only in the membrane fraction and was augmented after FcgammaRI stimulation. Tyrosine-phosphorylated Shc, mainly the p52 isoform, was observed to transiently onload to the membrane Grb2-Sos complex on FcgammaRI stimulation. Cross-linking of FcgammaRI induces the tyrosine phosphorylation of Cbl, which forms a complex with Grb2 and Shc via the Cbl C terminus. Kinetic experiments confirm that Cbl-Grb2 is relatively stable, whereas Grb2-Sos, Grb2-Shc, and Cbl-Shc interactions are highly inducible. The Src family tyrosine kinase inhibitor, PP1, was shown to completely inhibit Shc tyrosine phosphorylation, the Shc-Grb2 interaction, and the FcgammaR-induced respiratory burst. Our results provide the first evidence that the upstream activation of Src kinases is required for the modulation of the Shc-Grb2 interaction and the myeloid NADPH oxidase response.