Does the Overexpression of Pro‐Insulin‐Like Growth Factor‐II in Transfected Human Embryonic Kidney Fibroblasts Increase the Secretion of Lysosomal Enzymes?

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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 μg and 1.0 μg immunoreactive IGF‐II/ml and 22X106 cells and 24X106 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, β‐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 β‐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 neoglyoprotein 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 β‐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.

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