Biomedical subjects

Simone C Spring

Publications and source records attributed to Simone C Spring.

5 recordsLinked to original sources

PubMed2005

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.

Adipose Tissue↗

PubMed2005

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.

Cells, Cultured↗

PubMed2003

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.

Alternative Splicing↗

PubMed2002

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.

Adaptor Proteins, Signal Transducing↗

PubMed2002

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.

Amino Acid Sequence↗