Different fibroblast growth factor 1 (FGF-1) transcripts in neural tissues, glioblastomas and kidney carcinoma cell lines

The significant factors in the development of the neurosurgery program at the University of Florida have been the funding for 10 endowed chairs and a Brain Institute, the achievement of departmental status in the College of Medicine, the collaborative research with a strong Department of Neuroscience, and the strong commitment by the faculty to subspecialty neurosurgery and to service in the national neurosurgical organizations.     

Over-expression of protein tyrosine phosphatase 1 (PTP1) alters IL-3-dependent growth and tyrosine phosphorylation

Interleukin-3 (IL-3) is a hematopoietic growth factor receptor which stimulates the proliferation of multilineage progenitor cells. It is known that IL-3 stimulates tyrosine phosphorylation while transducing a mitogenic signal. The signal transduction pathways activated by the IL-3 receptor, however, are not fully understood. In this study a protein tyrosine phosphatase has been over-expressed in the IL-3 dependent, murine myeloid progenitor cell line, 32D cl3 in order to test whether altering the levels of tyrosine phosphorylation would change IL-3 stimulated proliferation. These cells were transfected with a metal-inducible expression vector containing a rat cDNA encoding PTP1. A low basal level of rat PTP1 message and protein was detected in cells transfected with the PTP1 vector, and zinc treatment resulted in a three- to fourfold increase in the amount of PTP1 message, protein and catalytic activity. Over-expression of PTP1 resulted in a two- to threefold decrease in IL-3 stimulated proliferation. Cells over-expressing PTP1 also exhibited decreased levels of tyrosine phosphorylation; phosphorylation of the IL-3 receptor beta subunit and the Shc protein were both dramatically decreased. Thus, PTP1 over-expression negatively modulated IL-3 signal transduction. To identify potential substrates of PTP1, 32D cl3 cells were transfected with a catalytically inactive PTP1 mutant, PTP1(C/S). Three tyrosine-phosphorylated proteins of MW 140, 79 and 69 k coprecipitated with PTP1(C/S). We believe that the 140 kDa protein represents the beta subunit of the IL-3 receptor. In addition, a GST-fusion protein containing active PTP1 dephosphorylated the beta-subunit in an in vitro assay. By immunofluorescent microscopy over-expressed PTP1(C/S) co-localized largely with calnexin, an endoplasmic reticulum-associated protein. Immunofluorescent microscopy also indicated that PTP1(C/S) and the beta subunit co-localized at discrete sites at the plasma membrane and around a cytoplasmic organelle where most of the beta subunit was located. These observations suggest PTP1 over-expression may down-regulate the growth response to IL-3 through dephosphorylation of the IL-3 receptor, perhaps in an intracellular compartment, thereby inhibiting propagation of the IL-3 mitogenic signal.     

Epidermal growth factor receptor and the adaptor protein p52Shc are specific substrates of T-cell protein tyrosine phosphatase

T-cell protein tyrosine phosphatase (TCPTP) exists as two forms generated by alternative splicing: a 48-kDa endoplasmic reticulum (ER)-associated form (TC48) and a 45-kDa nuclear form (TC45). To identify TCPTP substrates, we have generated substrate-trapping mutants, in which the invariant catalytic acid of TCPTP (D182) is mutated to alanine. The TCPTP D182A substrate-trapping mutants were transiently overexpressed in COS cells, and their ability to form complexes with tyrosine-phosphorylated (pTyr) proteins was assessed. No pTyr proteins formed complexes with wild-type TCPTP. In contrast, TC48-D182A formed a complex in the ER with pTyr epidermal growth factor receptor (EGFR). In response to EGF, TC45-D182A exited the nucleus and accumulated in the cytoplasm, where it bound pTyr proteins of approximately 50, 57, 64, and 180 kDa. Complex formation was disrupted by vanadate, highlighting the importance of the PTP active site in the interaction and supporting the characterization of these proteins as substrates. Of these TC45 substrates, the approximately 57- and 180-kDa proteins were identified as p52Shc and EGFR, respectively. We examined the effects of TC45 on EGFR signaling and observed that it did not modulate EGF-induced activation of p42Erk2. However, TC45 inhibited the EGF-induced association of p52Shc with Grb2, which was attributed to the ability of the PTP to recognize specifically p52Shc phosphorylated on Y239. These results indicate that TC45 recognizes not only selected substrates in a cellular context but also specific sites within substrates and thus may regulate discrete signaling events.     

Epidermal growth factor stimulates the tyrosine phosphorylation of SHPS-1 and association of SHPS-1 with SHP-2, a SH2 domain-containing protein tyrosine phosphatase

SHPS-1 is a 120 kDa glycosylated receptor-like protein that contains immunoglobulin-like domains in its extracellular region and four potential tyrosine phosphorylation for SH2 domain binding sites in its cytoplasmic region. Epidermal growth factor (EGF) stimulated the rapid tyrosine phosphorylation of SHPS-1 and subsequent association of SHPS-1 with SHP-2, a protein tyrosine phosphatase containing SH2 domains, in Chinese hamster ovary cells overexpressing human EGF receptors. In the cells overexpressing SHPS-1, the tyrosine phosphorylation of SHPS-1 was more evident than that observed in parent cells. However, overexpression of SHPS-1 alone did not affect the activation of MAP kinase in response to EGF. These results suggest that SHPS-1 may be involved in the recruitment of SHP-2 from the cytosol to the plasma membrane in response to EGF.     

Identification of the cytoplasmic regions of fibroblast growth factor (FGF) receptor 1 which play important roles in induction of neurite outgrowth in PC12 cells by FGF-1

Fibroblast growth factor 1 (FGF-1) induces neurite outgrowth in PC12 cells. Recently, we have shown that the FGF receptor 1 (FGFR-1) is much more potent than FGFR-3 in induction of neurite outgrowth. To identify the cytoplasmic regions of FGFR-1 that are responsible for the induction of neurite outgrowth in PC12 cells, we took advantage of this difference and prepared receptor chimeras containing different regions of the FGFR-1 introduced into the FGFR-3 protein. The chimeric receptors were introduced into FGF-nonresponsive variant PC12 cells (fnr-PC12 cells), and their ability to mediate FGF-stimulated neurite outgrowth of the cells was assessed. The juxtamembrane (JM) and carboxy-terminal (COOH) regions of FGFR-1 were identified as conferring robust and moderate abilities, respectively, for induction of neurite outgrowth to FGFR-3. Analysis of FGF-stimulated activation of signal transduction revealed that the JM region of FGFR-1 conferred strong and sustained tyrosine phosphorylation of several cellular proteins and activation of MAP kinase. The SNT/FRS2 protein was demonstrated to be one of the cellular substrates preferentially phosphorylated by chimeras containing the JM domain of FGFR-1. SNT/FRS2 links FGF signaling to the MAP kinase pathway. Thus, the ability of FGFR-1 JM domain chimeras to induce strong sustained phosphorylation of this protein would explain the ability of these chimeras to activate MAP kinase and hence neurite outgrowth. The role of the COOH region of FGFR-1 in induction of neurite outgrowth involved the tyrosine residue at amino acid position 764, a site required for phospholipase C gamma binding and activation, whereas the JM region functioned primarily through a non-phosphotyrosine-dependent mechanism. In contrast, assessment of the chimeras in the pre-B lymphoid cell line BaF3 for FGF-1-induced mitogenesis revealed that the JM region did not play a role in this cell type. These data indicate that FGFR signaling can be regulated at the level of intracellular interactions and that signaling pathways for neurite outgrowth and mitogenesis use different regions of the FGFR.     

Involvement of phosphatase activities in the run-down of GABA(A) receptor function in rat cerebellar granule cells in culture

PURPOSE: To compare the magnetic resonance (MR) imaging appearance of the anal sphincter in patients with fecal incontinence and scleroderma with that in patients with fecal incontinence alone, scleroderma alone, or neither. MATERIALS AND METHODS: The study population comprised 14 patients with fecal incontinence and scleroderma, four with scleroderma alone, 13 with incontinence alone, and six with neither. T1- and T2-weighted spin-echo, magnetization transfer contrast-weighted, and dynamic gadolinium-enhanced images were obtained and analyzed for the integrity, thickness, and length of sphincter components. Magnetization transfer contrast ratios and T2 were calculated to assess fibrosis of the internal sphincter. The percentage enhancement above baseline was calculated at 30-second intervals for the internal and the external sphincter. RESULTS: Eleven patients with incontinence and scleroderma showed descent of rectal air and feces into the anterior anal canal, with forward deviation of the significantly (P < .05) atrophied internal sphincter, which showed a slower gadolinium-enhancement pattern compared with that in other groups. Patients with incontinence alone showed no evidence of internal sphincter deviation or altered vascularity but had a significant reduction (P < .05) in deep external sphincter bulk. CONCLUSION: In patients with fecal incontinence and scleroderma, endoanal MR imaging helps delineate the anterior sphincter deformity and shows the slower gadolinium-enhancement pattern on dynamic studies of the internal sphincter.     

Continuous administration of basic fibroblast growth factor (FGF-2) accelerates bone induction on rat calvaria--an application of a new drug delivery system

Some studies have shown that locally applied basic fibroblast growth factor (FGF-2) enhances bone regeneration at a fracture site, while others have not been in agreement. We developed a new continuous FGF-2 delivery system designed to accelerate cytokine-induced new bone formation. A subperiosteal pocket was surgically formed in 36 eight-week-old male Wistar rats. The rats were administered 0, 1, 10, or 100 ng of FGF-2 contained in a collagen minipellet, mixed with allogeneic demineralized bone matrix in a dome-shaped Millipore filter and then placed into the pocket. New bone formation in the dome was evaluated at 2, 4, and 8 wks after placement. Soft x-ray radiographs disclosed an apparently larger radiopaque region in the 1-ng group at 4 wks compared with those in the other groups. Morphometrical analysis revealed that the new bone area in the 1-g group was significantly larger than that in the 0-g group (p<0.01). In the 100-ng FGF-2 group, new bone formation seemed suppressed. We concluded that continuous slow administration of a small amount of FGF-2 accelerates bone-derived osteogenic cytokine-induced new bone formation.     

alphavbeta3 integrin mediates the cell-adhesive capacity and biological activity of basic fibroblast growth factor (FGF-2) in cultured endothelial cells

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38-61 and 82-101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-alphavbeta3 antibodies prevent cell adhesion to FGF-2. Also, purified human alphavbeta3 binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-alphavbeta3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with alphavbeta3 integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.     

Role of tyrosine kinase and phosphotyrosine phosphatase in growth of the intestinal crypt cell (IEC-6) line

Antibodies to hepatitis C virus (HCV) were measured in 1,580 Ethiopian subjects representing urban and rural populations. Sera found positive by a repeated second generation enzyme immunoassay (EIA) were subjected to three additional confirmatory tests. The overall confirmed seroprevalence was 2.0%. Less than 1% were confirmed to be seropositive in rural communities, with 1.4% positive among blood donors, 1.6% positive among patients with dermatologic disorders, 3.6% among leprosy patients, and 6.0% among patients attending a University Hospital clinic for neurologic disorders. The patients in the groups with leprosy and neurologic disorders have most likely been in ill health for many years and have sought relief by traditional healers or treatment at poorly equipped clinics. This group of patients demonstrated a high prevalence of antibodies to HCV. In Ethiopia, especially in small clinics, there is a shortage of syringes and needles and they have to be reused many times often with inadequate sterilization. Therefore, these syringes and needles may be contaminated, thus being a risk factor for HCV and HIV infection.     

Characterization of the extracellular domain in vascular endothelial growth factor receptor-1 (Flt-1 tyrosine kinase)

Flt-1 tyrosine kinase, vascular endothelial growth factor (VEGF) receptor-1, binds VEGF and a new VEGF-related ligand, placenta growth factor, but KDR/Flk-1 (VEGF receptor-2) binds only VEGF. To characterize the functional regions in the Flt-1 extracellular domain such as the ligand binding region and the dimer formation of the receptor, we constructed a series of mutants of the Flt-1 extracellular domain as soluble forms in a baculovirus system. We found that a region carrying the N-terminal 1st to 3rd immunoglobulin (Ig)-like domains of Flt-1 binds both ligands with high affinity. However, for dimer formation of soluble Flt-1, a region further downstream in the Flt-1 extracellular domain was required. Mutant Flt-1 receptors expressed in COS cells confirmed the requirement of the 4th to 7th Ig region for the activation of Flt-1 tyrosine kinase. Soluble Flt-1 carrying the N-terminal 1st to 3rd Ig region suppressed VEGF-dependent endothelial proliferation in vitro to the same level as the larger forms of soluble Flt-1, suggesting that the binding of one soluble Flt-1 molecule to one subunit of the VEGF homodimer may be sufficient to block the VEGF activity.     

heartless encodes a fibroblast growth factor receptor (DFR1/DFGF-R2) involved in the directional migration of early mesodermal cells in the Drosophila embryo

After invagination of the mesodermal primordium in the gastrulating Drosophila embryo, the internalized cells migrate in a dorsolateral direction along the overlying ectoderm. This movement generates a stereotyped arrangement of mesodermal cells that is essential for their correct patterning by later position-specific inductive signals. We now report that proper mesodermal cell migration is dependent on the function of a fibroblast growth factor (FGF) receptor encoded by heartless (htl). In htl mutant embryos, the mesoderm forms normally but fails to undergo its usual dorsolateral migration. As a result, cardiac, visceral, and dorsal somatic muscle fates are not induced by Decapentaplegic (Dpp), a transforming growth factor beta family member that is derived from the dorsal ectoderm. Visceral mesoderm can nevertheless be induced by Dpp in the absence of htl function. Ras1 is an important downstream effector of Htl signaling because an activated form of Ras1 partially rescues the htl mutant phenotype. The evolutionary conservation of htl function is suggested by the strikingly similar mesodermal migration and patterning phenotypes associated with FGF receptor mutations in species as diverse as nematode and mouse. These studies establish that Htl signaling provides a vital connection between initial formation of the embryonic mesoderm in Drosophila and subsequent cell-fate specification within this germ layer.     

Src homology 2 protein tyrosine phosphatase (SHPTP2)/Src homology 2 phosphatase 2 (SHP2) tyrosine phosphatase is a positive regulator of the interleukin 5 receptor signal transduction pathways leading to the prolongation of eosinophil survival

Interleukin-5 (IL-5) regulates the growth and function of eosinophils. It induces rapid tyrosine phosphorylation of Lyn and Jak2 tyrosine kinases. The role of tyrosine phosphatases in IL-5 signal transduction has not been investigated. In this study, we provide first evidence that SH2 protein tyrosine phosphatase 2 (SHPTP2) phosphotyrosine phosphatase plays a key role in prevention of eosinophil death by IL-5. We found that IL-5 produced a rapid activation and tyrosine phosphorylation of SHPTP2 within 1 min. The tyrosine phosphorylated SHPTP2 was complexed with the adapter protein Grb2 in IL-5-stimulated eosinophils. Furthermore, SHPTP2 appeared to physically associate with beta common (betac) chain of the IL-5 receptor (IL-5betacR). The association of SHPTP2 with IL-5betacR was reconstituted using a synthetic phosphotyrosine-containing peptide, betac 605-624, encompassing tyrosine (Y)612. The binding to the phosphotyrosine-containing peptide increased the phosphatase activity of SHPTP2, whereas the same peptide with the phosphorylated Y612--> F mutation did not activate SHPTP2. Only SHPTP2 antisense oligonucleotides, but not sense SHPTP2, could inhibit tyrosine phosphorylation of microtubule-associated protein kinase, and reverse the eosinophil survival advantage provided by IL-5. Therefore, we conclude that the physical association of SHPTP2 with the phosphorylated betac receptor and Grb2 and its early activation are required for the coupling of the receptor to the Ras signaling pathway and for prevention of eosinophil death by IL-5.     

A possible role for multidrug resistance-associated protein in the secretion of basic fibroblast growth factor by osteogenic sarcoma cell line (MG-63)

Basic fibroblast growth factor (bFGF) lacks signal sequence and therefore the mechanism of its secretion is unknown. Multidrug resistance-associated protein (MRP) has been shown to transport a variety of molecules. Therefore, in this study we examined the role of MRP in the secretion of bFGF by osteogenic sarcoma MG-63 cells which spontaneously secrete bFGF. We show that MG-63 cells express MRP both at the protein and at the mRNA level. Furthermore, probenecid (a putative inhibitor of transport activity of MRP), in a concentration-dependent manner, inhibited secretion of bFGF from MG-63 cells with concomitant increase in intracellular contents of bFGF. These results suggest that MRP may have a possible role in the secretion of bFGF.     

Inhibition of growth of primary human tumour cell cultures by a 4-anilinoquinazoline inhibitor of the epidermal growth factor receptor family of tyrosine kinases

The epidermal growth factor receptor (EGFR) is thought to mediate the action of the mitogens EGF and tumour growth factor-alpha (TGF-alpha) in a variety of cancers, including those of the lung, breast and ovary. A number of new selective inhibitors of EGFR tyrosine kinase have now been developed as potential new antitumour agents. We used a potent inhibitor of this tyrosine kinase, 6-amino-4-[(3-bromophenyl)amino]-7-(methylamino)quinazoline (SN 25531; PD 156273), to determine the responses of primary cultures derived from patients with cancer of the lung, ovary, breast, cervix and endometrium. Cells were cultured in 96-well plates and proliferation assessed by incorporation of 3H-thymidine. Measured growth inhibitory concentrations IC50 values) varied from 1 nM to 14 microM with a 1000-fold differential between sensitive and resistant cultures. Results were compared with rates of proliferation, estimated using a paclitaxel-based method. We also measured the IC50 values for the tyrosine kinase inhibitor using a number of established human cell lines, and compared them with EGFR content using fluorescent antibody staining and flow cytometry. The presence of EGFR was found to be necessary, but not sufficient, for in vitro response. Only a small number of cell lines (3 of 7 for lung, 1 of 7 for ovarian, 2 of 3 squamous cell and 0 of 12 for melanoma) were sensitive to the tyrosine kinase inhibitor. In contrast, 40 of the 50 primary cultures (including 14 of 15 lung cancer samples and 14 of 19 ovarian cancer samples) were sensitive.     

Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) tyrosine phosphorylation state changes during vasculogenesis in the murine conceptus

Vasculogenesis, the differentiation of mesodermal cells to angioblasts and the subsequent formation of blood islands and blood vessels by angioblasts in the conceptus, is a dynamic process modulated, in part, by cell-extracellular matrix and cell-cell interactions in the presence of a variety of growth factors and morphogens. In this report we demonstrate differential tyrosine phosphorylation of platelet-endothelial cell adhesion molecule-1 (PECAM-1) during the formation of blood islands and vessels from clusters of extraembryonic and embryonic angioblasts in the murine conceptus. In addition, we identify the phosphorylation of a particular tyrosine residue in the PECAM-1 cytoplasmic domain, Tyr686, which has the potential of mediating binding to Src homology 2 domain-containing proteins, affecting PECAM-1 cellular localization and endothelial cell migration.