Angiogenic Oligosaccharides of Hyaluronan Induce Multiple Signaling Pathways Affecting Vascular Endothelial Cell Mitogenic and Wound Healing Responses
2002; Elsevier BV; Volume: 277; Issue: 43 Linguagem: Inglês
10.1074/jbc.m109443200
ISSN1083-351X
AutoresMark Slevin, Shant Kumar, John Gaffney,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoHyaluronan (HA) is a large nonsulfated glycosaminoglycan and an important regulator of angiogenesis, in particular, the growth and migration of vascular endothelial cells. We have identified some of the key intermediates responsible for induction of mitogenesis and wound recovery. Treatment of bovine aortic endothelial cells with oligosaccharides of hyaluronan (o-HA) resulted in rapid tyrosine phosphorylation and plasma membrane translocation of phospholipase Cγ1 (PLCγ1). Cytoplasmic loading with inhibitory antibodies to PLCγ1, Gβ, and Gαi/o/t/z inhibited activation of extracellular-regulated kinase 1/2 (ERK1/2). Treatment with the Gαi/o inhibitor, pertussis toxin, reduced o-HA-induced PLCγ1 tyrosine phosphorylation, protein kinase C (PKC) α and β1/2 membrane translocation, ERK1/2 activation, mitogenesis, and wound recovery, suggesting a mechanism for o-HA-induced angiogenesis through G-proteins, PLCγ1, and PKC. In particular, we demonstrated a possible role for PKCα in mitogenesis and PKCβ1/2 in wound recovery. Using antisense oligonucleotides and the Ras farnesylation inhibitor FTI-277, we showed that o-HA-induced bovine aortic endothelial cell proliferation, wound recovery, and ERK1/2 activation were also partially dependent on Ras activation, and that o-HA-stimulated tyrosine phosphorylation of the adapter protein Shc, as well as its association with Sos1. Binding of Src to Shc was required for its activation and for Ras-dependent activation of ERK1/2, cell proliferation, and wound recovery. Neither Src nor Ras activation was inhibited by pertussis toxin, suggesting that their activation was independent of heterotrimeric G-proteins. However, the specific Src kinase inhibitor PP2 inhibited Gβ subunit co-precipitation with PLCγ1, suggesting a possible role for Src in activation of PLCγ1 and interaction between two distinct o-HA-induced signaling pathways. Hyaluronan (HA) is a large nonsulfated glycosaminoglycan and an important regulator of angiogenesis, in particular, the growth and migration of vascular endothelial cells. We have identified some of the key intermediates responsible for induction of mitogenesis and wound recovery. Treatment of bovine aortic endothelial cells with oligosaccharides of hyaluronan (o-HA) resulted in rapid tyrosine phosphorylation and plasma membrane translocation of phospholipase Cγ1 (PLCγ1). Cytoplasmic loading with inhibitory antibodies to PLCγ1, Gβ, and Gαi/o/t/z inhibited activation of extracellular-regulated kinase 1/2 (ERK1/2). Treatment with the Gαi/o inhibitor, pertussis toxin, reduced o-HA-induced PLCγ1 tyrosine phosphorylation, protein kinase C (PKC) α and β1/2 membrane translocation, ERK1/2 activation, mitogenesis, and wound recovery, suggesting a mechanism for o-HA-induced angiogenesis through G-proteins, PLCγ1, and PKC. In particular, we demonstrated a possible role for PKCα in mitogenesis and PKCβ1/2 in wound recovery. Using antisense oligonucleotides and the Ras farnesylation inhibitor FTI-277, we showed that o-HA-induced bovine aortic endothelial cell proliferation, wound recovery, and ERK1/2 activation were also partially dependent on Ras activation, and that o-HA-stimulated tyrosine phosphorylation of the adapter protein Shc, as well as its association with Sos1. Binding of Src to Shc was required for its activation and for Ras-dependent activation of ERK1/2, cell proliferation, and wound recovery. Neither Src nor Ras activation was inhibited by pertussis toxin, suggesting that their activation was independent of heterotrimeric G-proteins. However, the specific Src kinase inhibitor PP2 inhibited Gβ subunit co-precipitation with PLCγ1, suggesting a possible role for Src in activation of PLCγ1 and interaction between two distinct o-HA-induced signaling pathways. Angiogenesis, the formation of new blood vessels, is essential for the growth and repair of tissues and is prevalent in a variety of pathological conditions. Excessive vascularization occurs in rheumatoid arthritis, diabetic retinopathy, psoriasis, and neoplasia (1Martin J.F. Hassall D.G. Warren J.B. The Endothelium: An Introduction to Current Research. Wiley-Liss Inc., New York1990: 95-105Google Scholar, 2Folkman J. Nat. Med. 1995; 1: 27-31Google Scholar). Conversely, in myocardial infarction and cerebrovascular diseases such as stroke, there is considerable destruction of the vasculature (3Krupinski J. Kaluza J. Kumar P. Kumar S. Wang J.M. Stroke. 1994; 25: 1794-1798Google Scholar). Further knowledge of the mechanisms that regulate angiogenesis is required for the development of strategies to control it. Hyaluronan (HA) 1The abbreviations used are: HA, hyaluronan; EC, endothelial cells, o-HA, oligosaccharides of hyaluronan; MAP, mitogen-activated protein kinase; RHAMM, receptor for hyaluronan mediated motility; ERK1/2, extracellular signal-regulated kinase; BAEC, bovine aortic endothelial cells; PLC, phospholipase C; FITC, fluorescein isothiocyanate; SPM, serum poor medium; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; pERK, phospho-ERK-1/ERK-2. is a nonsulfated, linear glycosaminoglycan, consisting of repeating units of (β,1–4)-d-glucuronic acid-(β,1–3)-N-acetyl-d-glucosamine. HA is found in its native state as a high molecular mass polymer (>106 kDa) in the extracellular matrix of almost all animal tissues and in significant quantities in the skin (dermis and epidermis), brain, and central nervous system (4Brink J. Heldin P. Exp. Cell Res. 1999; 252: 342-351Google Scholar). Apart from its role as an inert viscoelastic lubricant, essential for healthy joint function (5Scott J.E. J. Anat. 1995; 187: 259-269Google Scholar), HA has a crucial role in regulation of the angiogenic process. In particular, HA is a potent regulator of vascular endothelial cell (EC) function. Native high molecular weight HA is anti-angiogenic, inhibiting EC proliferation and migration (6West D.C. Kumar S. Ciba Found. Symp. 1989; 143: 187-207Google Scholar, 7West D.C. Kumar S. Lancet. 1988; 1: 715-716Google Scholar, 8West D.C. Kumar S. Int. J. Radiol. 1991; 61–62: 55-60Google Scholar) as well as capillary formation in a three-dimensional collagen gel model (9Watanabe M. Nakayasu K. Okisaki S. Nippon Ganka Gakkai Zasshi. 1993; 97: 1034-1039Google Scholar), whereas degradation products of specific size (3–10 disaccharide units; o-HA) stimulate EC proliferation (10West D.C. Kumar S. Exp. Cell Res. 1989; 183: 176-196Google Scholar, 11Deed R. Rooney P. Kumar P. Norton J.D. Freemont A.J. Kumar S. Int. J. Cancer. 1997; 71: 116-122Google Scholar), migration (12Sattar A. Rooney P. Kumar S. Pye D. West D.C. Scott I. Ledger P. J. Invest. Dermatol. 1994; 103: 576-579Abstract Full Text PDF Google Scholar), sprout formation (13Montesano R. Kumar S. Orci L. Pepper M.S. Lab. Invest. 1996; 75: 249-262Google Scholar), and result in angiogenesis in the chick chorioallantoic membrane (14West D.C. Hampson I.N. Arnold F. Kumar S. Science. 1985; 228: 1324-1327Google Scholar) and in myocardial infarction (15Kumar S. Ponting J. Rooney P. Kumar P. Pye D. Wang M. Angiogenesis: Molecular Biology and Clinical Applications. Plenum Press, New York1994: 219-231Google Scholar). Generation of this “angiogenic” o-HA from the naturally occurring HA polymer is mediated by the endoglycosidase hyaluronidase (16Lokeshwar V.B. Iida N. Bourguignon L.Y.W. J. Biol. Chem. 1996; 271: 23853-23864Google Scholar), in association with tissue damage, inflammatory disease, and in certain tumors (10West D.C. Kumar S. Exp. Cell Res. 1989; 183: 176-196Google Scholar, 16Lokeshwar V.B. Iida N. Bourguignon L.Y.W. J. Biol. Chem. 1996; 271: 23853-23864Google Scholar, 17Roden L. Campbell P. Fraser J.R.E. Laurent T.C. Pertoff H. Thompson J.N. Evered D. Whelan J. The Biology of Hyaluronan. Wiley and Sons, Chichester, UK1989: 60-86Google Scholar). In addition, the degree of invasiveness and metastasis of some tumors has been specifically linked to elevated HA expression (18Knudson W. Biswas C. Toole B.P. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6767-6771Google Scholar, 19Pauli B.U. Knudson W. Hum. Pathol. 1988; 19: 628-639Google Scholar, 20Delpech B. Chevallier B. Reinhardt N. Julien J.P. Dival C. Maingonnat C. Bastit P. Asselain B. Int. J. Cancer. 1990; 46: 388-390Google Scholar). Hyaluronidase produced by tumor cells could induce angiogenesis and be used by tumor cells as a “molecular saboteur” to depolymerize HA to facilitate invasion (21Liu D. Pearlman E. Guo E.D.K. Mori H. Haqqi S. Markowitz S. Willson J. Sy M.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7832-7837Google Scholar). Hyaluronan was intrinsically associated with metastasis in prostate and bladder cancer (22Lokeshwar V.B. Lokeshwar B.L. Pham H.T. Block N.L. Cancer Res. 1996; 56: 651-657Google Scholar, 23Pham H.T. Block N.L. Lokeshwar V.B. Cancer Res. 1997; 57: 778-783Google Scholar, 24Lokeshwar V.B. Young M.J. Goudarzi G. Iida N. Yudin A.I. Cherr G., N. Selzer M.G. Cancer Res. 1999; 59: 4464-4470Google Scholar) although high levels of o-HA were shown in children with a bone-metastasizing variant of renal tumor (25Ponting J. Rooney P. Kumar S. Current Perspectives in Molecular and Cellular Oncology. Jai Press, New York2002Google Scholar). The biological functions of HA/o-HA are thought to be initiated through cell surface receptors or HA-binding proteins, resulting in signal transduction activation and ultimately mitogenesis (26Hall C.L. Lange L.A. Prober D.A. Zhang S. Turley E.A. Oncogene. 1996; 13: 2213-2224Google Scholar, 27Rao C.M. Deb T.B. Datta K. Biochem. Mol. Biol. Int. 1996; 40: 327-337Google Scholar, 28Fieber C. Plug R. Sleeman J. Dall P. Ponta H. Hoffmann M. Gene (Amst.). 1999; 226: 41-50Google Scholar). Native HA binds to a 34-kDa member of the hyaladherins, and increases general protein tyrosine phosphorylation and that of PLCγ1 in a variety of cell lines, although the role of this protein in mediating cell behavioral effects is unknown (27Rao C.M. Deb T.B. Datta K. Biochem. Mol. Biol. Int. 1996; 40: 327-337Google Scholar). o-HA-induced Ras-dependent activation of mitogen-activated protein (MAP) kinase was shown in rat embryonic 3Y1 fibroblasts (29Serbulea M. Kakumu S. Thany A.A. Miyazaki K. Machida K. Senga T. Ohta S. Yoshioka K. Hotta N. Hamaguchi M. Int. J. Oncol. 1999; 14: 733-738Google Scholar). In T24 bladder carcinoma cells, o-HA induced activation of NF-κB via CD44 in a pathway involving Ras, protein kinase C (PKC) ζ, and a complex containing IκB kinase 1 and 2 (30Fitzgerald K.A. Bowie A.G. Skeffington B.S. O'Neill L.A.J. J. Immunol. 2000; 164: 2053-2063Google Scholar). In vascular EC, both CD44 (31Slevin M. Krupinski J. Kumar S. Gaffney J. Lab. Invest. 1998; 78: 987-1003Google Scholar,32Nandi A. Estess P. Siegelman M.H. J. Biol. Chem. 2000; 275: 14939-14948Google Scholar) and RHAMM (receptor for HA mediated motility) (33Lokeshwar V.B. Selzer M.G. J. Biol. Chem. 2000; 275: 27641-27649Google Scholar) have been identified as potential targets for transduction of o-HA-induced mitogenesis. Inhibition of the CD44/o-HA interaction using anti-CD44 antibodies (J173) reduced proliferation and migration of calf pulmonary artery EC and human microvessel EC (HMEC-1) (34Trochon V. Malibat C. Bertrand P. Legrand Y. SmadjaJoffe F. Soria C. Delpech B. Lu H. Int. J. Cancer. 1996; 66: 664-668Google Scholar). In three types of primary human EC, o-HA bound to the RHAMM receptor and induced tyrosine phosphorylation of p125FAK, paxillin, and p42/44 extracellular signal-regulated kinase (ERK1/2) resulting in cell proliferation (33Lokeshwar V.B. Selzer M.G. J. Biol. Chem. 2000; 275: 27641-27649Google Scholar). We have previously demonstrated that o-HA but not native HA induced up-regulation of the immediate early response genes c-jun, junB, Krox 20,Krox 24, and c-fos in bovine aortic EC (BAEC) (11Deed R. Rooney P. Kumar P. Norton J.D. Freemont A.J. Kumar S. Int. J. Cancer. 1997; 71: 116-122Google Scholar, 35Rooney P. Kumar S. Ponting J. Wang M. Int. J. Cancer. 1995; 60: 632-636Google Scholar). Similarly, o-HA induced a rapid CD44- dependent activation of multiple isoforms of PKC (α, β, and ε), Raf-1 kinase, MEK-1, and ERK1/2 resulting in mitogenesis in these cells (31Slevin M. Krupinski J. Kumar S. Gaffney J. Lab. Invest. 1998; 78: 987-1003Google Scholar). These limited studies have so far failed to identify all of the key intermediates involved in transduction of the o-HA-induced mitogenic and wound recovery responses in vascular EC. This information could be important in the development of novel therapeutic strategies for treatment of angiogenic diseases. In this study, an extension of our findings from previous work (31Slevin M. Krupinski J. Kumar S. Gaffney J. Lab. Invest. 1998; 78: 987-1003Google Scholar), we have examined in detail rapid up-regulation of associated signaling proteins and have characterized the pathways responsible for o-HA-induced angiogenesis. Primary monoclonal antibodies to H-Ras, anti-phosphotyrosine (PY99), and phospho-ERK-1/ERK-2 (pERK1/2), polyclonal antibodies, and their specific blocking peptides against Gαi/o/t/z, Gαs/olf, Gαq/11, Gβ, PLCγ1, PLCγ2, PLCβ1–3, PLCδ1, PKCα, PKCβ1–2, PKCε, Sos, Shc, and α-actin as well as mouse B cell lymphoma cell lysate (WEHI-231) were obtained from Autogen Bioclear (Wiltshire, United Kingdom). PKC isoform-specific inhibitors (Go 6976 and PKCε translocation inhibitor peptide), H-Ras inhibitor FTI-277, Gq protein inhibitor GP antagonist-2A, Gαi/o inhibitor pertussis toxin, and PP2 Src family inhibitor were from Calbiochem. Manufacture of phosphorothioate antisense and matching sense oligonucleotides directed to bovine PKCα was by VHBio (Newcastle-upon-Tyne, UK). Antisense, matching scrambled, and FITC-labeled oligonucleotides directed against bovine PKCβ1-β2 and H-Raswere from Biognostik (Gottingen, Germany). Ras and ERK activity assay kits, as well as the Src substrate peptide (KVEKIGEGTYGVVYK), mouse monoclonal anti-Src family tyrosine kinase, and anti-phospho-Src antibodies were all from Upstate Biotechnology (Buckingham, UK). Thermanox plastic coverslips were from Nunc (Naperville, IL), ECL and ECL plus kits were from Amersham Biosciences (Bucks, UK) and the protein detection reagent was obtained from Bio-Rad. All other materials and chemicals were from Sigma. The method of preparation is described in full elsewhere (14West D.C. Hampson I.N. Arnold F. Kumar S. Science. 1985; 228: 1324-1327Google Scholar). Briefly, native rooster comb HA (500 mg) was dissolved in sodium acetate buffer (50 ml, 0.1 m, pH 5.4) containing 0.15 m NaCl and treated with 20,000 units of bovine testicular hyaluronidase at 37 °C. After 2, 4, 6, 8, and 24 h, aliquots (10 ml) were treated with 1 ml of trichloroacetic acid. Mixtures were centrifuged, and supernatants were dialyzed against distilled water for 24–48 h at 4 °C in Spectra-Por tubing (Pierce-Warriner, Chester, UK) with at least 4 changes. They were then re-centrifuged and lyophilized. The powder was dissolved in 20 ml of 0.1% acetic acid and applied to a G50 Sephadex column (2.6 × 180 cm). Fractions (10 ml) were collected, assayed for uronic acid, and combined to yield three pools (F1, F2, and F3). The size range of oligosaccharides in each pool was determined after incorporation of [3H]glucosamine-labeled HA, SDS-PAGE, and fluorography, as described previously (14West D.C. Hampson I.N. Arnold F. Kumar S. Science. 1985; 228: 1324-1327Google Scholar). Successive bands differed on SDS gels by one disaccharide unit, and precise definition of the size range was determined by comparison with labeled octa-, hexa-, and tetrasaccharides of known molecular size. F1, F2, and F3 fractions consisted of disaccharide units of >16, 10–16, and 3–10,i.e. of approximately >7200, 4500–7200, and 1350–4500 Da, respectively. Angiogenic activity was determined by adding freeze-dried samples of each fraction onto the chorioallantoic membrane of the chick embryo. Only fraction F3 (o-HA) produced a consistent angiogenic response (14West D.C. Hampson I.N. Arnold F. Kumar S. Science. 1985; 228: 1324-1327Google Scholar, 36Rooney P. Kumar S. Differentiation. 1993; 54: 1-9Google Scholar) and was used in this study. Angiogenic activity resided only in the hyaluronate fragments, because the activity of hyaluronan preparations digested with denatured hyaluronidase and a 24-h normal digest were found to lack biological activity, suggesting that there was no contamination with vascular permeability factor. Fraction F3 was further digested with Streptomyceshyaluronidase, and lost its biological activity, as determined by the chorioallantoic membrane of the chick embryo assay (14West D.C. Hampson I.N. Arnold F. Kumar S. Science. 1985; 228: 1324-1327Google Scholar). BAEC were obtained and characterized as endothelial by the presence of von Willebrand factor and the uptake of Dil-labeled acetylated low density lipoprotein, as described previously (12Sattar A. Rooney P. Kumar S. Pye D. West D.C. Scott I. Ledger P. J. Invest. Dermatol. 1994; 103: 576-579Abstract Full Text PDF Google Scholar). They were routinely cultured in Dulbecco's modified Eagle's medium containing 15% fetal calf serum, 1.5 mm glutamine, 100 IU/ml penicillin, and 50 ng/ml streptomycin (complete medium). Culture flasks were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. BAEC were seeded at a concentration of 2 × 104/ml in 2 ml of complete medium, in triplicate 6-well plates. After attachment, medium was replaced with serum poor medium (SPM), containing 2.5% fetal calf serum in which the cells grew at a significantly reduced rate (31Slevin M. Krupinski J. Kumar S. Gaffney J. Lab. Invest. 1998; 78: 987-1003Google Scholar). In some cases, specific enzyme inhibitors, pertussis toxin (10–500 ng/ml, 6 h), Go 6976 (1–100 nm, 24 h), ε-translocation inhibitor (εti, 1–20 μΜ, 24 h), FTI 277 (100 nm to 5 μm, 24 h), GP Ant-2A (100 nm-25 μm, 4 h), PP2 (10 nm to 10 μm), or sense/antisense oligonucleotides directed toward PKCα/β and H-Ras (10 μm, 72 h) were added before incubation with o-HA (1 μg/ml) for a further 72 h. Control wells contained appropriate concentrations of the vehicle (Me2SO or ethanol). Concentration ranges and preincubation times of inhibitors were based on previously published information and our own unpublished pilot studies. 2M. Slevin, S. Kumar, and J. Gaffney, unpublished data. Trypan blue exclusion studies confirmed that the inhibitors were not cytotoxic to cells at the concentrations tested (data not included) and wells treated with inhibitors without o-HA were included as controls. Fresh medium and inhibitors were added every 72 h where necessary. After 72 h, cells were washed in PBS, detached in 0.05% trypsin/PBS, and counted on a Coulter counter (Coulter Electronics, Hialeah, FL) set to a threshold of 30 μm. Statistical significance was determined by one-way analysis of variance. Semiconfluent cells cultured on Thermanox plastic coverslips in 24-well plates were grown to confluence in SPM (24–48 h). Medium was replaced with Dulbecco's modified Eagle's medium containing 0.1% fetal calf serum and cells were incubated for a further 48 h with or without inhibitors/antisense reagents, in triplicate, as described above. Coverslips were washed in PBS, the cellular layer wounded using a mechanical wounder (37Lauder H. Sellers L.A. Fan T.P.D. Feniuk W. Humphrey P.P.A. Br. J. Pharmacol. 1997; 122: 663-670Google Scholar), rinsed again in PBS to remove loose and dislodged cells, and placed into a fresh 24-well plate containing inhibitors or the appropriate vehicles. Some coverslips were immediately fixed in 100% ethanol (time zero controls). o-HA (0.5 μg/ml) was added to some of the wells and the plate was incubated at 37 °C for 18 h. Pilot studies demonstrated that BAEC wounded under these conditions underwent negligible proliferation up to 24 h (even in the presence of o-HA), however, cell movement resulting in wound closure was almost complete in cells treated with o-HA at this concentration after 18 h (native HA had no effect, data not shown). The coverslips were rinsed (×3) in PBS, fixed in 100% ethanol (5 min), and allowed to air dry. The mechanical wounder produced 11 parallel lesions 400-μm wide across the cell monolayer. Movement of cells into the denuded area was quantified using a Seescan computerized image analysis system (Manchester, UK). Each field of view covered ∼2% of the total coverslip area. For each coverslip, 10 fields of view (each ∼2 mm by 1.45 mm) were examined at random. The lesion area in each field of view was measured and using the data from time 0 (T 0), the wound area was then converted to give mean % recovery from 3 identically treated coverslips (%r) using the equation: %r = [1 − (wound area at T t/wound area atT 0)] × 100. Where T t is the wounded area 18 h post-injury. Statistical significance was determined by one-way analysis of variance. Semiconfluent BAEC were cultured in 6-well plates in SPM for 48 h and the medium was replaced with Ca2+- and Mg2+-free bicarbonate buffer (pH 7.3) containing glycerol (1.2 m) at 37 °C (38Shea T.B. Perrone-Bizzozero N.I. Beermann M.L. Benowitz L.I. J. Neurosci. 1991; 11: 1685-1690Google Scholar, 39Yassin R.R. Abrams J.T. Peptides. 1998; 19: 47-55Google Scholar). Cells were placed immediately on ice for 10 min and the plasma cell membrane made transiently permeable by addition of chilledl-α-lysophosphatidylcholine (40 μg/ml) for a further 7 min. Pre-warmed SPM (1 ml, 37 °C), containing 5 μg of rabbit anti-mouse control IgG, FITC-labeled IgG, or antibodies to PLCγ1–2, PLCβ1–3, Gαi/o/t/z, Gβ, Gαs/olf, or Gαq/11 was added and the cells were incubated at 37 °C for a further 1 h. Cells regained their impermeability during this phase as determined by the recovery of trypan blue exclusion (data not shown). Uptake and cytoplasmic expression of antibodies after 1 h was confirmed by fluorescent microscopy of FITC-labeled cells, using a Leitz microscope (Leica, Bensheim, Germany) and the appropriate filter, and by FACS analysis (FACScan, BD Biosciences) following washing (PBS), trypsinization, and fixation of cells in 4% formaldehyde, PBS. After a 1-h recovery, o-HA (1 μg/ml, 5 min) was added to some of the wells that were then washed in PBS and cell lysates were collected in 0.5 ml of ice-cold radioimmunoprecipitation (RIPA) buffer containing 10 mm Tris-HCl (pH 7.5), 50 mm NaCl, 0.5% sodium deoxycholate, 0.5% (w/v) Nonidet P-40, 0.1% SDS, 1 mmNa3VO4, and 5 μg/ml aprotinin (40Vainikka S. Joukou V. Wennistrom S. Bergman M. Pelicci P. Alitalo K. J. Biol. Chem. 1994; 269: 18320-18326Google Scholar). After sonication, lysates were centrifuged (10,000 × g, 15 min at 4 °C) and the supernatants were collected and stored at −70 °C. Phosphorothioate oligonucleotides corresponding to bovine PKCβ1−β2 and H-Ras were synthesized by Biognostik (GmbH, Gottingen, Germany) using R.A.D.A.R sequence design. Sequences of antisense nucleotides for PKCβ1−β2 were 5′-TCAGCTGGAATCTAAATG and matched scrambled sense/FITC-labeled nucleotides were 5′-ACTACTACACTAGACTAC (41Coussens L. Parker P.J. Rhee L. Yang-Feng T.L. Chen E. Waterfield M.D. Francke U. Ullrich A. Science. 1986; 233: 859-866Google Scholar), whereas H-Ras antisense nucleotides were 5′-GCTTATACTCCGTCATTG and matched sense nucleotides were 5′-GTTACTGCCTCATATTCG (42McCaffery R.E. Coggins L.W. Doherty I. Kennedy I. O'Prey M. McColl L. Campo M.S. Oncogene. 1989; 4: 1441-1448Google Scholar). Antisense and control sense oligonucleotides directed toward bovine PKCα were synthesized by VHBio (Newcastle upon Tyne, UK). Antisense sequences were 5′-GTCCCTCGCCGCCTCCTG-3′ and sense, 5′-GTCCTCCGCCGCTCCCTG-3′ as described elsewhere (43Xia P. Aiello L.P. Ishii H. Jiang Z.Y. Park D, J. Robinson G.S. Takagi H. Newsome W.P. Jirousek M.R. King G.L. J. Clin. Invest. 1996; 98: 2018-2026Google Scholar). Semiconfluent BAEC were cultured at 37 °C in 24-well plates in SPM with or without oligonucleotides (10 μm/72 h) together with LipofectAMINE 2000 (Invitrogen, 10 μg/ml). Cell lysates were collected at 4 °C in RIPA buffer (300 μl) and processed as described previously (31Slevin M. Krupinski J. Kumar S. Gaffney J. Lab. Invest. 1998; 78: 987-1003Google Scholar). Delivery of oligonucleotides into the cell cytoplasm was monitored by addition of FITC-labeled PKCα oligonucleotides (10 μm, 4–72 h) to semiconfluent BAEC cultured on glass coverslips in SPM. Pilot studies were carried out to optimize the effects of oligonucleotides, and showed a notable reduction in specific protein expression determined by Western blotting, after 72 h exposure to 10 μm antisense oligonucleotide. Semiconfluent BAEC cultured in 6- or 24-well plates in SPM (48 h) were incubated with specific enzyme inhibitors or oligonucleotides before addition of o-HA (1 μg/ml, 2–10 min) as described previously. Total cell lysates were collected in RIPA buffer and processed as described earlier, whereas plasma cell membrane and cytoplasmic fractions were separated using a digitonin-based buffer system (31Slevin M. Krupinski J. Kumar S. Gaffney J. Lab. Invest. 1998; 78: 987-1003Google Scholar). Briefly, after washing in ice-cold PBS, cells in 6-well plates were agitated at 4 °C for 5 min in 300 μl/well of ice-cold buffer containing 140 mm NaCl, 25 mm KCl, 5 mm MgCl2, 2 mm EDTA and EGTA, 10 μg/ml leupeptin and pepstatin, 1 mm phenylmethylsulfonyl fluoride, 20 mmTris-HCl (pH 7.5), and 0.5 μg/ml digitonin (44Farese R.V. Cooper D.R. Siddle K. Hutton J.C. Lipid-related Second Messengers. IRL Press Ltd., Oxford, UK1991: 205-206Google Scholar). The buffer, now containing the cytoplasmic contents was removed and stored at 4 °C. The remaining “membrane” fraction was again rinsed in ice-cold PBS and solubilized in 300 μl of the same buffer containing 1% (w/v) Triton X-100. Both fractions were centrifuged (10,000 ×g, 15 min at 4 °C) to remove insoluble debris, and stored at −70 °C. Complete separation of cytoplasmic and membrane fractions was demonstrated using the lactate dehydrogenase assay. Protein concentration of cell lysates was determined using a modification of the Bradford assay (Bio-Rad) and equal quantities of protein (15 μg) were mixed with 2× Laemmli sample buffer, vortex mixed, and boiled in a water bath for 15 min. Samples were separated along with prestained molecular weight markers (32,000–200,000) by 12% SDS-PAGE. Proteins were electroblotted (Hoefer, Bucks, UK) onto nitrocellulose filters (1 h) and the filters were blocked for 1 h at room temperature in TBS-Tween (pH 7.4) containing 5% (w/v) de-fatted milk (PKC antibodies) or containing 1% (w/v) bovine serum albumin (all other antibodies). Filters were stained with the following primary antibodies diluted in the appropriate blocking buffer, overnight at 4 °C on a rotating mixer: rabbit polyclonal anti-PLCγ1–2, PLCβ1–3, PLCδ1 (1:500); Gαi/o/t/z, Gβ, Gαs/olf, Gαq/11 (1:750); PKCα, PKCβ1–2, PKCε (1:100); H-Ras (1:400), Shc and Sos (1:1000); mouse monoclonal antibodies to pERK1/2, Src, and phospho-Src (1:1000); phosphotyrosine PY99 (1:1500), and phospho-myelin basic protein (1:1500). After washing (5× 10 min in TBS-Tween at room temperature), filters were stained with either goat anti-rabbit or rabbit anti-mouse horseradish peroxidase-conjugated secondary antibodies diluted in TBS-Tween containing 5% (w/v) de-fatted milk (1:1000, 1 h, room temperature) with continuous mixing. After a further 5 washes in TBS-Tween, proteins were visualized using ECL chemiluminescent detection. Equal concentrations of protein (15 μg) from cell lysates were resolved in duplicate by 12% SDS-PAGE as described above. Polyclonal antibodies (1 μg/ml) were incubated with or without matching blocking peptides specific for a particular epitope (Santa Cruz) (10 μg/ml) overnight at 4 °C on a rotating mixer. Antibodies were then diluted in the appropriate blocking buffer, and identical blots were stained with antibody, with or without peptide treatment using the method described previously. Specificity of Src antibodies was assessed by comparing staining in total BAEC extracts separated by Western blotting (as described above) with positive control cell lysates (WEHI-231). We have previously characterized all remaining antibodies (31Slevin M. Krupinski J. Kumar S. Gaffney J. Lab. Invest. 1998; 78: 987-1003Google Scholar). Equal concentrations of protein from total cell lysates (100 μg in 0.5 ml of RIPA buffer) were incubated with 2 μg of primary antibody overnight at 4 °C on a rotating mixer. Antibodies were then attached to protein A/G-agarose beads (20 μl, 30 min, 4 °C with continuous mixing). Alternatively, cell lysates were mixed directly with antibodies already conjugated to protein-agarose beads (Shc, PLCγ1, 20 μl, overnight, 4 °C). The beads were pelleted by centrifugation (13,000 × g/10 min, 4 °C), the supernatant was removed, and the pellet washed 3× in ice-cold RIPA buffer (0.5 ml). Excess buffer was removed from the beads and protein-antibody complexes were solubilized in 50 μl of 2× Laemmli sample buffer and subjected, in duplicate, to 12% SDS-PAGE followed by blotting as described previously. One of the blots was stained with the original immunoprecipitating primary antibody to confirm equality of protein loading. The assay kit was supplied by Upstate Biotechnology, and the protocol was as per the manufacturers instructions. Briefly, semiconfluent BAEC, cultured in 6-well plates in SPM (48 h), were treated with the appropriate enzyme inhibitors or neutralizing antibodies as described earlier, prior to addition of o-HA (1 μg/ml, 5 min). ERK1/2 was immunoprecipitated from the cell lysates, attached to an agarose complex, and then incubated with a substrate mixture containing myelin basic protein. After SDS-PAGE, blots were stained with anti-myelin basic protein antibody and developed using ECL plus. Based on the method described previously (3
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