DOES THE OVEREXPRESSION OF PRO-INSULIN-LIKE GROWTH FACTOR-II IN TRANSFECTED HUMAN EMBRYONIC KIDNEY FIBROBLASTS INCREASE THE SECRETION OF LYSOSOMAL-ENZYMES

Insulin-like growth factor-II (IGF-LI) and lysosomal enzymes bearing t he mannose 6-phosphate (Man6P) recognition marker, bind to two distinc t 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-leng th human IGF-II cDNA or a control cDNA. Solution hybridization/RNase p rotection experiments using a human IGF-II riboprobe showed that two t ransfectants expressed large quantities of IGF-II mRNA, whereas the no n-transfected cells did not. The analysis of conditioned media reveale d that these cells secrete approximately 0.15 mu g and 1.0 mu g immuno reactive IGF-II/ml and 22X10(6) cells and 24X10(6) cells within 24 hou rs. Immunoreactive IGF-II was shown by Western blotting to represent 1 7-kDa pro-IGF-II. The amount of the lysosomal enzyme, beta-hexosaminid ase, 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-hexosaminidas e was not affected by pro-IGF-II overexpression. In addition, the basa l amount of another newly synthesized lysosomal enzyme, the cathepsin D precursor, was also twofold higher in pro-IGF-II overexpressing cell s than in control cells. In contrast, the surface binding and cellular uptake rate of a Man6P-containing neoglyoprotein did not differ betwe en the cell lines. The results indicate that the overexpression of pro -IGF-II doubles the secretion and/or reduces the re-uptake of beta-hex osaminidase and cathepsin D to approximately 20% of the total synthesi zed 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 l ysosomal enzymes, mainly by partially enhancing the secretion of newly synthesized enzymes and, in addition, possibly by affecting the re-up take mechanism.