title: Does the overexpression of pro-insulin-like growth factor-II in transfected human embryonic kidney fibroblasts increase the secretion of lysosomal enzymes?
abstract: Insulin-like growth factor-II (IGF-II) and lysosomal enzymes bearing the mannose 6-phosphate (Man6P) recognition marker, bind to two distinct binding sites of the IGF-II/M6P receptor. The two classes of ligands reciprocally modulate the binding of the other class of ligand to the receptor [Kiess, W., Thomas, C. L., Greenstein, L., Lee, L., Sklar, M. M., Rechler, M. M., Sahagian, G. G. & Nissley, S. P. (1989) J. Biol. Chem. 264, 4710-4714]. We asked whether or not overexpression of pro-IGF-II by cells in culture leads to missorting of lysosomal enzymes. Human embryonal kidney fibroblasts were transfected with the full-length human IGF-II cDNA or a control cDNA. Solution hybridization/RNase protection experiments using a human IGF-II riboprobe showed that two transfectants expressed large quantities of IGF-II mRNA, whereas the non-transfected cells did not. The analysis of conditioned media revealed that these cells secrete approximately 0.15 micrograms and 1.0 micrograms immunoreactive IGF-II/ml and 22 x 10(6) cells and 24 x 10(6) cells within 24 hours. Immunoreactive IGF-II was shown by Western blotting to represent 17-kDa pro-IGF-II. The amount of the lysosomal enzyme, beta-hexosaminidase, was approximately twofold increased in the conditioned media from pro-IGF-II overexpressing cells compared with control media, as shown by Western-blot analysis and immunoprecipitation of media extracts of metabolically labeled cells. The synthesis rate of beta-hexosaminidase was not affected by pro-IGF-II overexpression. In addition, the basal amount of another newly synthesized lysosomal enzyme, the cathepsin D precursor, was also twofold higher in pro-IGF-II overexpressing cells than in control cells. In contrast, the surface binding and cellular uptake rate of a Man6P-containing neoglycoprotein did not differ between the cell lines. The results indicate that the overexpression of pro-IGF-II doubles the secretion and/or reduces the re-uptake of beta-hexosaminidase and cathepsin D to approximately 20% of the total synthesized enzymes in human embryonal kidney fibroblasts compared to control cells. We hypothesize that, in cells synthesizing high amounts of pro-IGF-II, the growth factor may modulate the targeting of a portion of lysosomal enzymes, mainly by partially enhancing the secretion of newly synthesized enzymes and, in addition, possibly by affecting the re-uptake mechanism.
authors.affiliation: Children's Hospital, Dept. General Paediatrics & Neonatology, University of Giessen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Hoeflich
authors.forename: E
authors.initials: E
authors.lastname: Wolf
authors.forename: T
authors.initials: T
authors.lastname: Braulke
authors.forename: G
authors.initials: G
authors.lastname: Koepf
authors.forename: U
authors.initials: U
authors.lastname: Kessler
authors.forename: G
authors.initials: G
authors.lastname: Brem
authors.forename: W
authors.initials: W
authors.lastname: Rascher
authors.forename: W
authors.initials: W
authors.lastname: Blum
authors.forename: W
authors.initials: W
authors.lastname: Kiess
mesh_terms: D002402:Cathepsin D / Q000378:metabolism; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D018014:Gene Transfer Techniques; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000096:biosynthesis* / Q000235:genetics; D007668:Kidney / Q000196:embryology / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism*
chemical_list: D017077:Culture Media, Conditioned; D007335:Insulin-Like Growth Factor II; D001619:beta-N-Acetylhexosaminidases; D002402:Cathepsin D