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• W2034281276 abstract "Previous studies have established that in response to wounding, the expression of amyloid precursor-like protein 2 (APLP2) in the basal cells of migrating corneal epithelium is greatly up-regulated. To further our understanding of the functional significance of APLP2 in wound healing, we have measured the migratory response of transfected Chinese hamster ovary (CHO) cells expressing APLP2 isoforms to a variety of extracellular matrix components including laminin, collagen types I, IV, and VII, fibronectin, and heparan sulfate proteoglycans (HSPGs). CHO cells overexpressing either of two APLP2 variants, differing in chondroitin sulfate (CS) attachment, exhibit a marked increase in chemotaxis toward type IV collagen and fibronectin but not to laminin, collagen types I and VII, and HSPGs. Cells overexpressing APLP2-751 (CS-modified) exhibited a greater migratory response to fibronectin and type IV collagen than their non-CS-attached counterparts (APLP2-763), suggesting that CS modification enhanced APLP2 effects on cell migration. Moreover, in the presence of chondroitin sulfate, transfectants overexpressing APLP2-751 failed to exhibit this enhanced migration toward fibronectin. The APLP2-ECM interactions were also explored by solid phase adhesion assays. While overexpression of APLP2 isoforms moderately enhanced CHO adhesion to laminin, collagen types I and VII, and HSPGs lines, especially those overexpressing APLP2-751, exhibited greatly increased adhesion to type IV collagen and fibronectin. These observations suggest that APLP2 contributes to re-epithelialization during wound healing by supporting epithelial cell adhesion to fibronectin and collagen IV, thus influencing their capacity to migrate over the wound bed. Furthermore, APLP2 interactions with fibronectin and collagen IV appear to be potentiated by the addition of a CS chain to the core proteins. Previous studies have established that in response to wounding, the expression of amyloid precursor-like protein 2 (APLP2) in the basal cells of migrating corneal epithelium is greatly up-regulated. To further our understanding of the functional significance of APLP2 in wound healing, we have measured the migratory response of transfected Chinese hamster ovary (CHO) cells expressing APLP2 isoforms to a variety of extracellular matrix components including laminin, collagen types I, IV, and VII, fibronectin, and heparan sulfate proteoglycans (HSPGs). CHO cells overexpressing either of two APLP2 variants, differing in chondroitin sulfate (CS) attachment, exhibit a marked increase in chemotaxis toward type IV collagen and fibronectin but not to laminin, collagen types I and VII, and HSPGs. Cells overexpressing APLP2-751 (CS-modified) exhibited a greater migratory response to fibronectin and type IV collagen than their non-CS-attached counterparts (APLP2-763), suggesting that CS modification enhanced APLP2 effects on cell migration. Moreover, in the presence of chondroitin sulfate, transfectants overexpressing APLP2-751 failed to exhibit this enhanced migration toward fibronectin. The APLP2-ECM interactions were also explored by solid phase adhesion assays. While overexpression of APLP2 isoforms moderately enhanced CHO adhesion to laminin, collagen types I and VII, and HSPGs lines, especially those overexpressing APLP2-751, exhibited greatly increased adhesion to type IV collagen and fibronectin. These observations suggest that APLP2 contributes to re-epithelialization during wound healing by supporting epithelial cell adhesion to fibronectin and collagen IV, thus influencing their capacity to migrate over the wound bed. Furthermore, APLP2 interactions with fibronectin and collagen IV appear to be potentiated by the addition of a CS chain to the core proteins. amyloid protein precursor amyloid precursor-like protein 2 Chinese hamster ovary chondroitin sulfate chondroitin sulfate proteoglycan Dulbecco's modified Eagle's medium extracellular matrix heparan sulfate proteoglycan fibronectin phosphate-buffered saline bovine serum albumin Amyloid precursor protein (APP)1 is the precursor of 39–43 amino acid polypeptides-Aβ, the major component of cerebrovascular and neuritic plaque amyloid deposits found in the brains of Alzheimer's patients. APP is a member of a protein family including amyloid precursor-like proteins (APLP)-1 and -2 (1Kang J. Lemaire H. Masters C. Grzeschik K. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3951) Google Scholar, 2Wasco W. Gurubhagavatula S. Paradis M.D. Romano D.M. Sisodia S.S. Hyman B.T. Neve R.L. Tanzi R.E. Nature Genet. 1993; 5: 95-100Crossref PubMed Scopus (322) Google Scholar, 3Wasco W. Bupp K. Magendantz M. Gusella J.F. Tanzi R.E. Solomon F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10857-10861Crossref PubMed Scopus (323) Google Scholar, 4Sprecher C.A. Grant F.J. Grimm G. OHara P.J. Norris F. Norris K. Foster D.C. Biochemistry. 1993; 32: 4481-4486Crossref PubMed Scopus (162) Google Scholar, 5Slunt H.H. Thinakaran G. Von Koch C. Lo A.C. Tanzi R.E. Sisodia S.S. J. Biol. Chem. 1994; 269: 2637-2644Abstract Full Text PDF PubMed Google Scholar). Members of the APP/APLP family are type I integral membrane proteins that contain a single membrane-spanning domain with a large extracellular N-terminal domain and a short C-terminal cytoplasmic domain (1Kang J. Lemaire H. Masters C. Grzeschik K. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3951) Google Scholar, 2Wasco W. Gurubhagavatula S. Paradis M.D. Romano D.M. Sisodia S.S. Hyman B.T. Neve R.L. Tanzi R.E. Nature Genet. 1993; 5: 95-100Crossref PubMed Scopus (322) Google Scholar). Both APP and APLP2 are ubiquitously expressed in mammalian tissues and cells and their in vivo roles largely remain to be determined (6Selkoe D. Ann. Rev. Cell Biol. 1994; 10: 373-403Crossref PubMed Scopus (745) Google Scholar, 7Sisodia S. Price D. FASEB J. 1995; 9: 366-370Crossref PubMed Scopus (224) Google Scholar). APP and APLP2 are encoded by alternatively spliced mRNAs (2Wasco W. Gurubhagavatula S. Paradis M.D. Romano D.M. Sisodia S.S. Hyman B.T. Neve R.L. Tanzi R.E. Nature Genet. 1993; 5: 95-100Crossref PubMed Scopus (322) Google Scholar, 4Sprecher C.A. Grant F.J. Grimm G. OHara P.J. Norris F. Norris K. Foster D.C. Biochemistry. 1993; 32: 4481-4486Crossref PubMed Scopus (162) Google Scholar, 5Slunt H.H. Thinakaran G. Von Koch C. Lo A.C. Tanzi R.E. Sisodia S.S. J. Biol. Chem. 1994; 269: 2637-2644Abstract Full Text PDF PubMed Google Scholar). One of the spliced exons has structural/functional homology to the Kunitz-type serine protease inhibitors (8Van-Nostrand W. Schmaier A. Neiditch B. Siege R. Raschke W. Sisodia S. Wagner S. Biochim. Biophys. Acta. 1994; 1209: 165-170Crossref PubMed Scopus (34) Google Scholar). The other spliced exon encodes a 15- (APP) or 12- (APLP2) amino acid insert that disrupts a consensus sequence required for the addition of a chondroitin sulfate (CS) chain (9Thinakaran G. Slunt H.H. Sisodia S.S. J. Biol. Chem. 1995; 270: 16522-16525Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 10Thinakaran G. Sisodia S.S. J. Biol. Chem. 1994; 269: 22099-22104Abstract Full Text PDF PubMed Google Scholar). Hence, the isoforms of APP and APLP2 lacking these small polypeptide inserts are subject to CS modification. We previously showed that the majority of APLP2 molecules in rat corneal epithelium and in olfactory sensory axons are modified by the addition of a CS glycosaminoglycan (CSPG) chain (11Guo J. Thinakaran G. Guo Y. Sisodia S. Yu F. Invest. Ophthalmol. & Visual Sci. 1998; 39: 292-300PubMed Google Scholar, 12Thinakaran G. Kitt C. Roskams A. Slunt H. Masliah E. Koch C.V. Ginsberg S. Ronnett G. Reed R. Price D. Sisodia S. J. Neurosci. 1995; 15: 6314-6326Crossref PubMed Google Scholar). Following wounding, the levels of APLP2 mRNA and protein are increased markedly in the basal epithelial cells that are actively migrating (11Guo J. Thinakaran G. Guo Y. Sisodia S. Yu F. Invest. Ophthalmol. & Visual Sci. 1998; 39: 292-300PubMed Google Scholar), implicating a role(s) for APLP2 in mediating epithelial migration during re-epithelialization. Cell migration plays a central role in many biological processes, including embryonic development, wound healing, immunoresponses, and tumor metastasis. Cell migration requires a dynamic interaction between the cell, its substrate, and the cytoskeleton-associated motile apparatus (13Palecek S.P. Loftus J.C. Ginsberg M.H. Lauffenburger D.A. Horwitz A.F. Nature. 1997; 385: 537-540Crossref PubMed Scopus (1189) Google Scholar, 14Huttenlocher A. Sandborg R.R. Horwitz A.F. Curr. Opin. Cell Biol. 1995; 7: 697-706Crossref PubMed Scopus (449) Google Scholar). Cell surface adhesion receptors serve to connect the substratum with the cytoskeleton, and thus they are central to the migratory process (14Huttenlocher A. Sandborg R.R. Horwitz A.F. Curr. Opin. Cell Biol. 1995; 7: 697-706Crossref PubMed Scopus (449) Google Scholar). The best characterized cell surface receptors for matrix components are the integrin family of proteins (15Hynes R. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar). Integrins play a key role in cell migration, both as receptors connecting the ECM to intracellular cytoskeletal proteins and as receptors transducing information from ECM to affect cell behavior (15Hynes R. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar,16Yamada K. Gailit J. Clark R. Clark R. The Molecular and Cellular Biology of Wound Repair. Plenum Press, New York1996: 51-93Google Scholar). Another group of cell surface molecules capable of binding ECM components are cell surface proteoglycans including the syndecans, CD44 and NG2. Syndecans, via their covalently attached heparan sulfate chains, bind fibronectin (FN), interstitial collagens, thrombospondin, and tenascin (17Thesleff I. Vainio S. Salmivirta M. Jalkanen M. Cell Differ. Dev. 1990; 32: 383-389Crossref PubMed Scopus (17) Google Scholar, 18Saunders S. Bernfield M. J. Cell Biol. 1988; 106: 423-430Crossref PubMed Scopus (247) Google Scholar, 19Corless C.L. Mendoza A. Collins T. Lawler J. Dev. Dyn. 1992; 193: 346-358Crossref PubMed Scopus (64) Google Scholar). Syndecans are thought to play important roles in cell-matrix and cell-cell adhesion, migration, and proliferation (20Carey D.J. Biochem. J. 1997; 327: 1-16Crossref PubMed Scopus (605) Google Scholar). CD44 binds several ECM components including hyaluronan (21Clark R.A. Alon R. Springer T.A. J. Cell Biol. 1996; 134: 1075-1087Crossref PubMed Scopus (126) Google Scholar), FN (22Verfaillie C.M. Benis A. Iida J. McGlave P.B. McCarthy J.B. Blood. 1994; 84: 1802-1811Crossref PubMed Google Scholar), and collagen type IV (23Knutson J.R. Iida J. Fields G.B. McCarthy J.B. Mol. Biol. Cell. 1996; 7: 383-396Crossref PubMed Scopus (128) Google Scholar), and it is linked to the cytoskeleton by ezrin (24Tsukita S. Oishi K. Sato N. Sagara J. Kawai A. Tsukita S. J. Cell Biol. 1994; 126: 391-401Crossref PubMed Scopus (683) Google Scholar) and ankyrin (25Bourguignon L. Lokeshwar V. He J. Chen X. Bourguignon G. Mol. Cell. Biol. 1992; 12: 4464-4467Crossref PubMed Scopus (73) Google Scholar). Another cell surface CSPG is NG2 (26Iida J. Meijne A.M. Spiro R.C. Roos E. Furcht L.T. McCarthy J.B. Cancer Res. 1995; 55: 2177-2185PubMed Google Scholar, 27Lin X. Dahlin-Huppe K. Stallcup W. J. Cell. Biochem. 1996; 63: 463-477Crossref PubMed Scopus (57) Google Scholar). NG2-expressing B28 glioma cells exhibited a greater migratory response toward type VI collagen than do non-NG2 expressing cells (28Burg M. Nishiyama A. Stallcup W. Exp. Cell Res. 1997; 235: 254-264Crossref PubMed Scopus (83) Google Scholar). These cell surface proteins, by serving as adhesion molecules, are thought to promote cell migration during normal development, in vitro tumor invasion, and wound repair. The APP family of proteins is known to interact with selected ECM proteins such as heparin sulfate proteoglycan (29Narindrasorasak S. Lowery D. Gonzalez D.P. Poorman R.A. Greenberg B. Kisilevsky R. Brain Res. Mol. Brain Res. 1991; 10: 173-178Crossref PubMed Scopus (93) Google Scholar), laminin (30Narindrasorasak S. Lowery D.E. Altman R.A. Gonzalez D.P.A. Greenberg B.D. Kisilevsky R. Neurosci. Lett. 1992; 144: 46-48Crossref PubMed Scopus (9) Google Scholar), FN (31Narindrasorasak S. Altman R.A. Gonzalez D.P. Greenberg B.D. Kisilevsky R. Lab. Invest. 1995; 72: 272-282PubMed Google Scholar), and collagen (32Beher D. Hesse L. Masters C.L. Multhaup G. J. Biol. Chem. 1996; 271: 1613-1620Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). APP has also been shown to promote adhesion of a number of cell types in culture (33Chen M. Yankner B. Neurosci. Lett. 1991; 125: 223-226Crossref PubMed Scopus (88) Google Scholar, 34Breen K.C. Bruce M. Anderton B.H. J. Neurosci. Res. 1991; 28: 90-100Crossref PubMed Scopus (222) Google Scholar, 35Ghiso J. Rostagno A. Gardella J.E. Liem L. Gorevic P.D. Frangione B. Biochem. J. 1992; 288: 1053-1059Crossref PubMed Scopus (129) Google Scholar). Thus, the APP family of proteins might be components of a multidimensional mechanism for the regulation of spatial and temporal cell-matrix interactions during tissue morphogenesis and wound healing. To investigate the role of APLP2 in cell adhesion and cell migration, we analyzed Chinese hamster ovary (CHO) cell lines overexpressing APLP2 isoforms and report in this article that APLP2 overexpression caused a significant increase in migratory response of CHO cells to FN and type IV collagen. The ability of APLP2 transfectants to migrate toward FN and type IV collagen is closely related to the increases in cell adhesion to these ECM proteins. These findings suggest that APLP2-ECM interaction may play a functional role in cell behavior. They also provide support for APLP2 to function in epithelial wound healing. Because the APP family of proteins is highly conserved and similarly processed (2Wasco W. Gurubhagavatula S. Paradis M.D. Romano D.M. Sisodia S.S. Hyman B.T. Neve R.L. Tanzi R.E. Nature Genet. 1993; 5: 95-100Crossref PubMed Scopus (322) Google Scholar), information about the biology of APLP2 should add to our knowledge regarding the functional role of these proteins. CHO cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) (complete medium). The stable CHO lines expressing APLP2-751 and APLP2-763 were generated by co-transfecting APLP2 expression vector pSVAPLP2-751 (10Thinakaran G. Sisodia S.S. J. Biol. Chem. 1994; 269: 22099-22104Abstract Full Text PDF PubMed Google Scholar) and pSVAPLP2-763 (9Thinakaran G. Slunt H.H. Sisodia S.S. J. Biol. Chem. 1995; 270: 16522-16525Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) with a neovector, respectively, and were maintained in complete DMEM containing 200 μg/ml Geneticin. Three APLP2-751-transfected cell lines, B2, C1, and D1 (10Thinakaran G. Sisodia S.S. J. Biol. Chem. 1994; 269: 22099-22104Abstract Full Text PDF PubMed Google Scholar) and three APLP2-763-transfected cell lines D + 127, 8, and 16 were used. For control, untransfected parental and mock-transfected CHO cells were used. Cells were allowed to grow to confluence in 25-cm2 flasks. The conditioned medium from each cell line was collected and cells were then washed twice with PBS. To determine relative levels of APLP2 in transfectants, CHO cells were lysed with a buffer containing 50 mm Tris, pH 8.0, 150 mmNaCl, 5 mm EDTA, 0.5% Triton X-100, and protease inhibitors (50 μg/ml pepstatin, 50 μg/ml leupeptin, 10 μg/ml aprotinin, and 0.25 mm phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 10,000 × g to remove nuclei and insoluble debris. Protein concentration was determined using Pierce Micro BCA Protein assay Reagent kit. Ten-microgram aliquots of detergent lysates were digested with 0.05 unit of protease-free chondroitinase ABC (Seikagaku America, Inc., Rockville, MD) in 100 mm Tris-HCl, pH 8.0, and 30 mm sodium acetate for 1 h at 37 °C. Samples were prepared in Laemmli sample buffer and boiled for 5 min. The proteins were fractionated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose (Bio-Rad) and the blots were probed with 1:2500 dilution of APLP2-specific antiserum, D2II (5Slunt H.H. Thinakaran G. Von Koch C. Lo A.C. Tanzi R.E. Sisodia S.S. J. Biol. Chem. 1994; 269: 2637-2644Abstract Full Text PDF PubMed Google Scholar). Western blotting was carried out using horseradish peroxidase-conjugated IgG (goat anti-rabbit, Bio-Rad; 1:1000) as a secondary antibody and the Amersham Enhanced Chemiluminescence System (Amersham, Arlington Heights, IL) for detection. To detect APLP2 at the cell surface, monolayer CHO cells in 100 mm-tissue culture dishes were washed three times with PBS and incubated with sulfo-NHS-SS-biotin (Pierce Chemical) at 200 μg/ml, 5 min at room temperature. After biotinylation, cells were washed once with ice-cold PBS/glycine and twice with ice-cold PBS and then scraped from the culture dishes and homogenized in 1 ml of PBS with proteinase inhibitors. The homogenates were centrifuged at 1000 ×g for 5 min to yield low speed pellets. The supernatant was centrifuged at 80,000 × g for 1 h at 4 °C in a Beckman TL-100 ultracentrifuge to yield a cytosolic fraction and high speed pellet containing membrane-associate proteins. The low and high speed pellets were resuspended in a solution containing 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 0.05% SDS, 0.1% Triton X-100. The biotinylated proteins were precipitated with avidin-agarose (Pierce Chemical) overnight at 4 °C. After five washes in 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, biotinylated proteins were released from avidin-agarose by boiling for 5 min in Laemmli sample buffer and fractionated by SDS-polyacrylamide gel electrophoresis. Cell surface APLP2 was detected by Western blotting as described above. To determine the ratio of modified/non-modified APLP2, the image of autoradiograph was captured by the BDS Image System and analyzed by NIH Image 1.55-Gel Plotting Micros. Each desired band was marked; the image was acquired and plotted. The area beneath each plotted curve (APLP2 band intensity) was automatically calculated. Human plasma FN, laminin, and type I collagen were purchased from Collaborative Biomedical (Becton Dickinson, Franklin Lakes, NJ); types IV and VII collagen and heparan sulfate proteoglycan (HSPG) were purchased from Sigma. Migration assays were carried out in 12- or 48-well Neuroprobe chemotaxis chambers (Cabin John, MD). CHO cells were harvested by trypsinization, washed once with PBS + 10% FCS, and then washed twice with DMEM. Cells were resuspended in DMEM + 0.1% BSA and then added to the upper chamber (1.8 × 104 cells for 12-well chambers and at 8 × 103 cells for 48-well chambers). For blocking assay, chondroitin sulfate were preincubated with the cells before they were added to the upper compartment of the Boyden chamber. The lower compartment was filled with either DMEM containing 0.1% BSA as control, or with various extracellular matrix proteins at 20 μg/ml (Fig. 2). For subsequent fibronectin and collagen IV studies, 10 μg/ml protein or as otherwise indicated (see figure legends) were used. The two compartments of the Boyden chamber were separated by a polycarbonate filter (8 mm pore size, Poretics, Livermore, CA). Cells were allowed to migrate for 2–10 h at 37 °C in a humidified atmosphere containing 5% CO2. The membranes were briefly fixed with methanol and stained with Diff Quick Stain (Dade Diagnostics, Aguada, Puerto Rico). Cells on the upper side of the filter were removed mechanically. The filters were mounted on glass slides and the number of cells that had migrated to the lower surface were counted or photographed and then counted on the micrograph. A random microscope field (× 200 magnification) was counted per well. Each assay was carried out in 12 wells and repeated at least once. Conditioned medium from CHO B2 cells was obtained from a confluent CHO culture (100-mm dish) that was maintained in 6 ml of serum-free medium (Opti-DMEM, Life Sciences) for 24 h. Under this condition, the maximum amount of secreted APLP2-751 molecules was accumulated in the media. One milliliter of collected medium was dialyzed twice against 200 ml of DMEM + 0.1% BSA. The dialyzed medium was used to resuspend CHO cells and added to the lower chamber to determine the effects of secreted APLP2 molecules on cell migration. Purified matrix proteins (10 μg/ml, Fig. 7) tested included collagen I, collagen IV, collagen VII, FN, laminin, and HSPG. To test concentration dependence, 0.5–10 μg/ml fibronectin or collagen IV was used with 1% BSA as control. Matrix proteins were diluted in PBS and 100-μl aliquots were added to the wells of Nunc-Immuno plates. Proteins were allowed to absorb overnight at 4 °C. The wells were then washed three times with PBS and nonspecific binding sites were blocked for 1 h with 1% BSA in PBS at 37 °C. Cells were detached by treatment with 0.25% trypsin-EDTA, washed once with 10% serum in PBS, twice with serum-free DMEM, and then suspended in serum-free DMEM containing 1% BSA, and added to the plates (104 cells/well). Plates were kept at 37 °C in a humidified incubator containing 5% CO2 for 0, 30, and 60 min and then washed twice with PBS to remove unbound cells. Attached cells were fixed with 75% ethanol, and stained with 0.5% crystal violet in 20% methanol, 80% water for 20 min each. Excess dye was removed by rinsing the plates with PBS. Bound stain was extracted with 0.1 m sodium citrate, pH 4.2, for 30 min and the optical density (OD) was read at 570 nm in a microtiter enzyme-linked immunosorbent assay reader (Bio-Tek Instrument, Winooski, VT). The background OD value from a control well with DMEM + 1% BSA was subtracted from the OD values for each well. Data are presented as mean ± S.E. Student's t test was used to determine if there was a significant difference between the two groups (p < 0.05). When multiple means were compared, analysis of variance (ANOVA) was used to compare means between arms of each experiment. If found to be significant (p < 0.01), Scheffe's multiple comparison procedure was performed to compute adjusted p values for pairwise comparisons (e.g. CHO versus APLP2-751, CHO versus APLP2-763, and APLP2-751 versus APLP2-763). Naive CHO cells express detectable levels of APLP2 molecules. We previously reported that permanent CHO cell lines transfected with cDNAs encoding mouse APLP2-751 and APLP2-763 have markedly increased levels of APLP2 (see Refs. 9Thinakaran G. Slunt H.H. Sisodia S.S. J. Biol. Chem. 1995; 270: 16522-16525Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar and 10Thinakaran G. Sisodia S.S. J. Biol. Chem. 1994; 269: 22099-22104Abstract Full Text PDF PubMed Google Scholar, and also, Fig.3). APLP2-751 in transfected cells were modified by addition of CS chains of various lengths (10Thinakaran G. Sisodia S.S. J. Biol. Chem. 1994; 269: 22099-22104Abstract Full Text PDF PubMed Google Scholar, 11Guo J. Thinakaran G. Guo Y. Sisodia S. Yu F. Invest. Ophthalmol. & Visual Sci. 1998; 39: 292-300PubMed Google Scholar). Furthermore, the cell lines overexpressing APLP2 also release ectodomain fragments derived from corresponding APLP2 precursor isoforms into the conditioned media (10Thinakaran G. Sisodia S.S. J. Biol. Chem. 1994; 269: 22099-22104Abstract Full Text PDF PubMed Google Scholar). Cell surface localization of APLP2 in CHO transfectants was determined by experiments in which cell surface proteins were biotinylated briefly (5 min at room temperature) and precipitated with avidin-agarose from high speed pellet or cytosol fractions (Fig.1). While no APLP2 immunoreactivity was detected in cytosol, APLP2 was detected for three cell lines at the surface of live CHO cells (Fig. 1, membrane, lanes 1, 2, and 3). The levels of APLP2 in the fraction of cell surface proteins from APLP2 transfectants (Fig. 1, cell line D + 1216, lanes 2 and line B2, lane 3) were markedly higher than that from untransfected cells (lane 1). Band intensity analysis with original x-ray film revealed in lane 3 (membrane) ∼60% of APLP2 staining was in ∼120–200 kDa (CS modified) forms. Similar results were obtained when biotinylation was performed at 4 °C for 30 min (data not shown).Figure 1Immunoblot detection of cell surface APLP2. To detect cell surface APLP2, CHO cells without transfection (lane 1), transfected with APLP2-763 (line D + 1216, lane 2) or APLP2-751 (line B2, lane 3) from one confluent 100-mm tissue culture dish were surface biotinylated. Biotinylated proteins were precipitated from subcellular fractions (cytosol, 100,000 × g supernatant; membrane, 100,000 × g pellet) with avidin-Sepharose and separated by SDS-polyacrylamide gel electrophoresis. APLP2 was detected by immunoblotting with rabbit polyclonal APLP2 antiserum D2II (1:1000 dilution) using enhanced chemiluminescence. The positions of molecular markers (myosin, 202 kDa; β-galactosidase, 109 kDa) are indicated at the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our in vivo wound healing study suggested that APLP2 isoforms might be involved in mediating epithelial sheet migration (11Guo J. Thinakaran G. Guo Y. Sisodia S. Yu F. Invest. Ophthalmol. & Visual Sci. 1998; 39: 292-300PubMed Google Scholar). To determine whether APLP2 affects the ability CHO cells to migrate in response to ECM proteins, we used Boyden chamber assays to compare the relative ability of parental or mock-transfected cells, cells overexpressing APLP2-763 (non-CS-modified) and cells overexpressing APLP2-751 (CS-modified) to migrate toward a variety of ECM components (Fig.2). For these assays, ECM proteins were placed in the lower chamber of the Boyden apparatus to serve as a soluble chemoattractant. As shown in Fig. 2, ECM proteins collagen type I and VII, laminin, and HSPG had low stimulative effects on CHO cell migration. Overexpression of APLP2 did not alter the migratory response of CHO cells toward these proteins. On the other hand, while the control cells exhibited low migratory responses to type IV collagen, APLP2-transfected cell lines exhibited significantly increased migration toward this basement membrane-specific collagen (p < 0.0001). We observed approximately 1.7 times more cells crossing the membrane for APLP2-763 cells and ∼3.6 times more for cells expressing APLP2-751, compared with control CHO cells. Among the ECM proteins tested, FN had the highest stimulatory effects on CHO migration. CHO cells overexpressing APLP2 displayed markedly enhanced migration toward FN (p < 0.0001), being 1.8 times greater for APL-763 and 2.5 times greater for APLP2-751 cells when compared with parental or mock-transfected cells (Fig. 2). Furthermore, while overexpression of both isoforms of APLP2 promoted CHO cell migration toward collagen IV and FN, cells overexpressing APLP2-751 exhibited a significantly greater (∼2.1 times toward collagen IV and ∼1.4 times toward FN, p < 0.0001) migratory response to these ECM proteins than those overexpressing APLP2–763. Several lines overexpressing each isoform were generated. Although these cell lines expressed different levels of APLP2 as determined by chondroitinase digestion and Western blotting (Fig.3 A), all cell lines expressing the same isoform (lines D + 127, 8 and 16 expressing APLP2-763; lines B2, C1, and D1 expressing APLP2-751) exhibited similar levels of enhanced cell migration toward FN (Fig. 3 B), indicating that the migratory response of these cell lines correlates with the isoform expressed but not with the levels of APLP2 overexpression. Fig. 4 shows migration of CHO cells toward different concentrations of FN. All three cell lines exhibited increasing migration with increasing FN concentration. The pattern of APLP2 isoforms promoting CHO migration, APLP2-751 > APLP2-763 > control cells, was observed at all concentrations tested. Fig. 5 shows that the increased migration of APLP2-transfected CHO cell lines, when compared with control cells, toward type IV collagen can be observed within the first 2 h in the modified Boyden chamber assays, but was more obvious after longer incubations (6–10 h). This effect was much more pronounced in cells overexpressing CS-modified APLP2 molecules.Figure 5Time course of migration toward type IV collagen of CHO cells expressing APLP2 isoforms. Cell migration assays were performed for control (Δ) and two cell lines expressing APLP2 isoforms (⋄, APLP2–751 line B2; ○, APLP2-763 line D + 1216) using 10 μg/ml type IV collagen. After various times as indicated, cells that migrated to the underside of the membrane were quantified. The results are averages of at least 12 random fields; error bars show standard errors.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To test whether CS modification contributes to the effects of APLP2 molecules on promoting CHO cell migration, we preincubated cell suspensions with chondroitin sulfate (20 μg/ml) before adding cells to the upper chambers in the migration assays (Fig.6). Clearly, chondroitin sulfate interfered with FN-mediated migration of CHO cells expressing CS-APLP2 (2.84 times reduction, p < 0.005), but had no effect on parental or cells expressing APLP2-763. These results suggest that modification of APLP2 by addition of CS chains potentiates the effects of APLP2 core protein on CHO cell migration" @default.
• W2034281276 created "2016-06-24" @default.
• W2034281276 creator A5006901857 @default.
• W2034281276 creator A5010416927 @default.
• W2034281276 creator A5032254385 @default.
• W2034281276 creator A5080421638 @default.
• W2034281276 date "1999-09-01" @default.
• W2034281276 modified "2023-10-17" @default.
• W2034281276 title "Amyloid Precursor-like Protein 2 Promotes Cell Migration toward Fibronectin and Collagen IV" @default.
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