Anti-CD45RO suppresses human immunodeficiency virus type 1 replication in microglia: role of Hck tyrosine kinase and implications for AIDS dementia.

Macrophages and microglia are productively infected by HIV-1 and play a pivotal role in the pathogenesis of AIDS dementia. Although macrophages and microglia express CD45, a transmembrane protein tyrosine phosphatase, whether modulation of its activity affects human immunodeficiency virus type 1 (HIV-1) replication is unknown. Here, we report that of the five human CD45 isoforms, microglia express CD45RB and CD45RO (RB > RO) and treatment of microglia with a CD45 agonist antibody alphaCD45RO (UCHL-1) inhibits HIV-1 replication. alphaCD45RO prevented HIV-1 negative factor (Nef)-induced autophosphorylation of hematopoietic cell kinase (Hck), a myeloid lineage-specific Src kinase. Recombinant CD45 protein also inhibited HIV-1-induced Hck phosphorylation in microglia. Antennapedia-mediated delivery of Hck Src homology domain 3 (SH3), a domain that binds to the Nef PxxP motif with high affinity, reduced HIV-1-induced Hck phosphorylation and HIV-1 production in microglia. HIV-1-induced LTR transactivation was observed in U38 cells stably overexpressing wild-type Hck but not kinase-inactive Hck. In microglia, alphaCD45RO reduced activation of transcription factors (NF-kappaB and CCAAT enhancer binding protein) necessary for LTR transactivation in macrophages. These results establish that in myeloid lineage cells, Nef interacts with the Hck SH3 domain, resulting in autophosphorylation of Hck and an increase in HIV-1 transcription. alphaCD45RO-mediated inhibition of HIV-1 replication in microglia identifies the CD45 protein tyrosine phosphatase as a potential therapeutic target for HIV-1 infection/AIDS dementia.

Comparison of the signaling mechanisms by which VEGF, H2O2, and phosphatase inhibitors activate endothelial cell ERK1/2 MAP-kinase.

VEGF-induced ERK1/2 activation is mediated by a signaling mechanism involving the sequential activation of PLCgamma-PKC-Raf1-MEK-ERK1/2. This signaling pathway is necessary, but not sufficient for ERK1/2 activation, as VEGF-induced generation of reactive oxygen species (ROS) is also required. The molecular interaction by which VEGF-induced ROS generation is coordinated with the PLCgamma plus PKC-dependent pathway is not certain, and the goal of this study was to clarify this issue. Prior investigations examining ROS-induced signaling have focused on the cellular protein tyrosine phosphatases (PTPs), and we asked whether a PTP participates in ERK1/2 activation in endothelial cells. We show that both the general PTP inhibitor vanadate, and a dominant negative inhibitor of SHP-1, mimics the effects of VEGF in activating ERK1/2. The phosphatase inhibitors induce ERK1/2 activation in endothelial cells lacking VEGF receptors, indicating that the inhibitors target a downstream effector. As is the case after VEGF treatment, the phosphatase inhibitors do lead to the activation of PLCgamma, and a pharmacological inhibitor of the Src kinases blocks this. These results lead to the conclusion that inhibition of a protein tyrosine phosphatase activates endothelial cell ERK1/2 by a signaling mechanism involving the sequential activation of Src-PLCgamma-PKC-Raf1-MEK-ERK1/2. VEGF treatment most likely activates this pathway by inhibiting SHP-1 through a ROS-dependent mechanism.

TEM8 expression stimulates endothelial cell adhesion and migration by regulating cell-matrix interactions on collagen.

The TEM8 gene is selectively expressed in tumor versus normal blood vessels, though its function in endothelial cell biology is not known. Towards the goal of clarifying this function, we tested whether TEM8 overexpression, or blocking TEM8's function with a dominant negative protein, would modulate endothelial cell activities. We found that TEM8-expressing endothelial cells migrated at a rate 3-fold greater than control cells in a monolayer denudation assay. Also, the addition of recombinant TEM8 extracellular domain (TEM8-ED) specifically inhibited both chemokinetic and chemotactic migration on collagen in the denudation and Boyden chamber assays, respectively. The TEM8-ED binds preferentially to collagen, and TEM8 expression enhanced endothelial adhesion to collagen 3-fold; the latter response was antagonized by the TEM8-ED. Consistent with the TEM8-ED acting as a dominant negative inhibitor of endogenously expressed protein were data showing that the TEM8-ED had no effect on the activation of beta1 integrin. TEM8 protein is present in human umbilical vein in situ and is expressed in low passage HUVEC in vitro. TEM8 protein expression in HUVEC was increased 5-fold by the initiation of tube formation, correlating expression of TEM8 with the angiogenic response. Taken together, these results indicate that TEM8 plays a positive role in endothelial cell activities related to angiogenesis.

ATR/TEM8 is highly expressed in epithelial cells lining Bacillus anthracis' three sites of entry: implications for the pathogenesis of anthrax infection.

Anthrax is a disease caused by infection with spores from the bacteria Bacillus anthracis. These spores enter the body, where they germinate into bacteria and secrete a tripartite toxin that causes local edema and, in systemic infections, death. Recent studies identified the cellular receptor for anthrax toxin (ATR), a type I membrane protein. ATR is one of the splice variants of the tumor endothelial marker 8 (TEM8) gene. ATR and TEM8 are identical throughout their extracellular and transmembrane sequence, and both proteins function as receptors for the toxin. ATR/TEM8 function and expression have been associated with development of the vascular system and with tumor angiogenesis. TEM8 is selectively upregulated in endothelial cells during blood vessel formation and tumorigenesis. However, selective expression of TEM8 in endothelial cells contradicts the presumably ubiquitous expression of the receptor. To resolve this controversial issue, we evaluated the distribution of ATR/TEM8 in a variety of tissues. For this purpose, we generated and characterized a novel anti-ATR/TEM8 polyclonal antibody. Here, we show that this novel antibody recognizes all three ATR/TEM8 isoforms, which are widely and differentially expressed in various tissue types. We found that ATR/TEM8 expression is not only associated with tumor endothelial cells, as previously described. Indeed, ATR/TEM8 is highly and selectively expressed in the epithelial cells lining those organs that constitute the anthrax toxin's sites of entry, i.e., the lung, the skin, and the intestine. In fact, we show that ATR/TEM8 is highly expressed in the respiratory epithelium of the bronchi of the lung and is particularly abundant in the ciliated epithelial cells coating the bronchi. Furthermore, immunostaining of skin biopsies revealed that ATR/TEM8 is highly expressed in the keratinocytes of the epidermis. Finally, we show that the epithelial cells lining the small intestine strongly express ATR/TEM8 isoforms. This is the first demonstration that the ATR/TEM8 protein is highly expressed in epithelial cells, which represent the primary location for bacterial invasion. These results suggest that the ATR/TEM8 expression pattern that we describe here is highly relevant for understanding the pathogenesis of anthrax infection.

Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation.

We had previously shown that Rap1 mediates certain of the signaling pathways involved in VEGF-induced endothelial cell migration, although the downstream Rap1 effectors are not known. Towards the goal of identifying those effectors, we utilized a commercially available antibody array filter to identify proteins that either directly interact with Rap1 or interact indirectly through a multi-protein complex. The protocol identified 10 possible Rap1-interacting proteins, including the Bmx non-receptor tyrosine kinase. The conclusion that VEGF treatment leads to a Rap1/Bmx complex was confirmed by an experiment in which cell lysates from VEGF and control cells were immunoprecipitated with Bmx antibodies and Western blotting was done using anti-Rap1 antibodies. VEGF treatment led to the recruitment of Bmx to the CAS scaffolding protein, and inhibition of the Bmx kinase blocked VEGF-induced cell migration. Formation of a Rap1/Bmx complex was not observed in cells transfected with an expression vector for a dominant-negative Rap1, indicating that Bmx is a downstream Rap1 effector in VEGF-induced endothelial cell activation.

Nck and Crk mediate distinct VEGF-induced signaling pathways that serve overlapping functions in focal adhesion turnover and integrin activation.

We have asked whether the Nck and Crk adaptor proteins play important roles in the vascular endothelial growth factor (VEGF)-induced signaling pathways that lead to an enhancement in cell migration. The introduction into human umbilical vein endothelial cells of a dominant-negative inhibitor for either Nck or Crk blocked the recruitment of both endogenous proteins to the KDR VEGF receptor subtype indicating that both proteins are recruited to the same docking site. The Nck and Crk dominant-negatives led to the formation of abnormally large focal adhesion, blocked VEGF-induced integrin activation, and blocked VEGF-induced actin dynamics. The dominant-negatives had no effects on these properties in cells expressing constitutively active Rac1 or RhoA. Since a DN to either Nck or Crk blocks the cellular responses mediated by both proteins, we performed experiments directed at clarifying signaling pathways specifically mediated by each protein. Inhibition of the interaction between Nck with its downstream effector PAK led to abnormally large focal adhesions, but had no effect on integrin activation or cell adhesiveness. Evidence is presented that Crk complexes with C3G in control cells, and VEGF treatment leads to the recruitment of the complex to the cell surface. Inhibition of the C3G downstream effector Rap1 leads to enlarged focal adhesions and blocks VEGF-induced integrin activation. We conclude that Nck and Crk mediate distinct VEGF-induced signaling pathways that serve overlapping functions in cell migration.

VEGF treatment induces signaling pathways that regulate both actin polymerization and depolymerization.

The angiogenic growth factor vascular endothelial growth factor (VEGF) enhances endothelial cell migration through the activation of multiple signaling transduction pathways. Actin reorganization is an important component in VEGF-induced migration, yet the signaling pathways mediating this process remain unclear. Actin reorganization involves both actin polymerization and depolymerization, and in this study we demonstrate that VEGF-treatment regulates both of these activities. With respect to actin polymerization, our results indicate that the actin nucleation promoting factors (NPF) neural Wiskott-Aldrich syndrome protein (N-WASP) binds the SH2- plus SH3-domain containing adaptor protein Nck in both control and VEGF-treated cells. We had previously showed that VEGF treatment leads to the recruitment of Nck to activated receptor, and our current results indicate a VEGF-dependent redistribution of N-WASP to the cell surface. A Nck dominant-negative blocked Nck recruitment to receptor, blocked N-WASP cellular redistribution and attenuated actin stress fiber formation. With respect to actin depolymerization, VEGF-treatment led to the rapid phosphorylation of the actin depolymerization factor cofilin, and its upstream regulator, LIM-kinase (LIMK). Unlike what is observed in certain other cell types, the p21-activated kinase (PAK), a Nck binding protein, does not mediate VEGF-induced LIMK phosphorylation, as a PAK dominant-negative had no effect on this activity. The PAK dominant-negative also did not affect VEGF-induced actin reorganization. Pharmacological inhibitors of phosphoinositide-3 kinase (PI3-K) and the rho-activated kinase (ROCK) attenuated VEGF-induced LIMK phosphorylation, indicating a role for (PI3-K) and ROCK in the signaling pathways leading to regulation of LIMK activity.

Characterization of a new alternatively spliced neuropilin-1 isoform.

Neuropilin-1 (NP1) and neuropilin-2 (NP2) are receptors for semaphorins, which act as axonal chemorepellents, and for members of the vascular endothelial growth factor (VEGF) family of angiogenic growth factors. The NP1 and NP2 genes consist of 17 exons, and protein isoforms are expressed because of alternative transcript splicing. Here we report the identification of a new NP1 transcript (designated NRP1(Delta exon16)) that contains an arginine codon in place of exon 16-derived sequences at a locus between the c-domain and membrane spanning domain. NRP1(Delta exon16) is expressed in endothelial cells, astrocytes, and various tumor cell lines, and accounts for 30% of the total NRP1 transcript. After cellular expression of NRP1(Delta exon16), we found (unlike the two previously identified alternatively spliced NRP1 isoforms) no evidence that the extracellular domain of NRP1(Delta exon16) is secreted from cells as a soluble protein. (125)I-VEGF bound with high affinity to NRP1 and NRP1(Delta exon16) expressing cells, and VEGF treatment led to the formation of complexes between VEGFR-2 and either NRP1 or NRP1(Delta exon16). It is concluded that NRP1 and NRP1(Delta exon16) mediate VEGF-induced signaling in a similar manner.

Sck is expressed in endothelial cells and participates in vascular endothelial growth factor-induced signaling.

Sck, a member of the Shc family of cell signaling proteins, has only been studied in neuronal cells, though previous studies have demonstrated its expression in tissues other than brain. Using RT-PCR and RNase protection assays, we detected Sck mRNA expression in endothelial cells, and Sck protein was detected by Western blotting using polyclonal and monoclonal antibodies targeting the Sck CH1 domain. Immunohistochemistry protocols demonstrate that Sck is expressed in KDR and PECAM positive cells found in the mouse retina, mouse heart and human umbilical chord. Treatment of human umbilical vein endothelial (HUVE) cells with vascular endothelial growth factor (VEGF) leads to the recruitment of Sck to the KDR VEGF receptor and an enhanced Sck tyrosine phosphorylation. Sck is recruited to KDR tyrosine 1175, as co-immunoprecipitation of KDR and Sck is not observed in VEGF-treated porcine aortic endothelial cells expressing a receptor mutated at this autophosphorylation site. The Sck and Shc SH2 domains, and not the PTB domain, mediates its interactions with KDR, as recombinant Sck SH2 domain binds to a tyrosine phosphorylated KDR 1175-derived synthetic peptide, but not to a peptide synthesized without tyrosine phosphate. Recombinant PLCgamma SH2 domain also interacts with the phosphotyrosine 1175 containing peptide. VEGF-induced MAPK activation is dependent upon PLCgamma activity, and chimeric proteins consisting of the Shc or Sck SH2 domains fused with a cellular internalization sequence attenuated this activation. Taken together, these results demonstrate that Sck is expressed in vascular endothelial cells, and participates in VEGF-induced signal transduction.

Fibroblast growth factor receptor substrate 2 participates in vascular endothelial growth factor-induced signaling.

Vascular endothelial growth factor (VEGF) activates endothelial cells, in part, by interacting with the kinase insert domain-containing receptor (KDR) receptor tyrosine kinase. Although progress has been made in the identification of cell-signaling proteins that participate in the VEGF-induced response, questions remain concerning the molecular interactions that allow coupling of receptor activation with an increased cellular response. Evidence is provided in this manuscript that indicates a role for the fibroblast growth factor receptor substrate 2 (FRS2) in VEGF-induced signal transduction. VEGF treatment of human umbilical vein endothelial cells (HUVECs) and KDR-transfected porcine aortic endothelial cells leads to the rapid tyrosine phosphorylation of FRS2. FRS2 is associated constitutively with KDR, and VEGF treatment has no effect on this interaction. VEGF treatment of KDR-expressing cells leads to the recruitment of Nck, p21-activated kinase, Crk, Grb2, and protein kinase C l to FRS2. The ability of FRS2 to recruit cell-signaling proteins to the cell is significant because it provides a mechanism for enhancing the repertoire of VEGF-induced signaling pathways.

Identification of a syndecan 4 pseudogene.

The syndecan family of heparan sulfate proteoglycans participates in cellular activation through interactions with growth factors, extracellular matrix, and other molecules. The family consists of four proteins that share sequence homology within their cytosolic domains. Here we report that a 5.8 kb region of human chromosome 22q12.2 contains multiple segments that share greater than 80% sequence homology to the syndecan 4 transcript, including homology to 443 nucleotides of the syndecan 4 coding region. Three pieces of evidence indicate that the chromosome 22 sequences are a syndecan 4 pseudogene. First, single nucleotide gaps need to be inserted into the chromosome 22 sequence in order to maintain maximal alignment to the syndecan 4 coding sequence, and this introduces stop codons into a deduced amino acid sequence. Second, the total length of chromosome 22 containing the homologous sequences is compressed when compared to the genomic organization of the complementary syndecan 4 sequences. Third, the 5.8 kb chromosome 22 sequence contains multiple Alu and other repetitive sequences, and this is a property of pseudogenes. Both RT-PCR and RNase protection assays indicated that the syndecan 4 pseudogene is transcribed in human umbilical vein endothelial cells.