Insulin‐like growth factor II binding and action in human fetal fibroblasts

Abstract

To investigate the role of insulin-like growth factor II (IGF-II) in human prenatal growth, IGF-II binding and biological action were studied in four lines of fetal and three lines of postnatal human fibroblasts. Specific binding of IGF-II was similar in both groups: 15.7% and 14.9% for fetal and postnatal fibroblasts, respectively. This was 5-10 times the amount of IGF-I binding found in these cells. IGF-I and IGF-II caused dose-dependent increases in [14C]aminoisobutyric acid (AIB) uptake. IGF-II was sevenfold less potent than IGF-I in stimulating this metabolic response in both fetal and postnatal fibroblasts. The maximal effect of IGF-II in stimulating [14C]AIB uptake approach that of IGF-I. Similar results were obtained when IGF-I and IGF-II stimulation of [3H]thymidine incorporation was compared in fetal and postnatal fibroblasts. Incubation in the presence of alpha IR-3, a monoclonal antibody to the type I IGF receptor, inhibited the ability of both IGF-I and IGF-II to stimulate [14C]AIB uptake and [3H]thymidine incorporation in fetal and postnatal cells. A monoclonal antibody to the insulin receptor did not affect IGF action. These data indicate that IGF-II is a potent metabolic and mitogenic stimulus for human fetal fibroblasts. However, despite the presence of abundant type II IGF receptors on both fetal and postnatal human fibroblasts, IGF-II stimulation of amino acid transport and DNA synthesis appears to be mediated through the type I rather than through its own type II IGF receptor.

A unique receptor-independent mechanism by which insulinlike growth factor I regulates the availability of insulinlike growth factor binding proteins in normal and transformed human fibroblasts.

“…Thus, these data suggested that the IGF-I-stimulated increase in IGFBPs might not be receptor-mediated. In concordance with this, aIR-3, a monoclonal antibody that inhibits IGF-I binding and receptor-mediated action in human fibroblasts (29,32), inhibited IGF-I receptor binding but failed to block the IGF-I-induced changes in IGFBPs in normal HF and SV40-HF media (Fig. 3).…”

Cell polarity of the insulin-like growth factor system in human intestinal epithelial cells. Unique apical sorting of insulin-like growth factor binding protein-6 in differentiated human colon cancer cells.

“…Confluent cell monolayers were washed three times with the same medium, but without FCS and containing 0.1% BSA, then incubated in this medium for 20 h at 37°C in the presence or not of 25 nM of IGF-I, IGF-ll, des(1 -3)IGF-I or des(1 -6)-IGF-LI added to the chamber either above (apical) or below (basolateral) the cell monolayer. "4C-AIB uptake (0.3 pCi/nil in 8.0 iM unlabeled AIB) was then measured for 12 min as described previously (21). After extensive washing with cold PBS containing 0.1% BSA, the cell-covered filters were cut, and counted in a liquid scintillation counter.…”

Insulin-like growth factor II-mediated proliferation of human neuroblastoma.

“…Together with our finding that IGF-II is expressed in cells of nonmalignant tissues associated with neuroblastoma tumor cells (Figs. 4 and 5), these data indicate that locally produced IGF-II may stimulate the proliferation of neuroblastoma.…”

“…SK-N-AS cells were continuously cultured in N2E medium without serum or growth factors (22 (24) was examined by Northern blot analysis using a 32P-labeled plasmid DNA probe containing a cDNA insert encoding rat IGF-II (pr-IGF-II-l, 780-bp insert in pUC12) (8,22) that was labeled to a specific activity of 2-4 X 108 cpm/.g DNA. Neuroblastoma and normal human adrenal gland tissues (6-uM sections) were processed and examined by in situ hybridization (25) using 5 x 105 cpm of a 31-bp synthetic DNA sequence specific for IGF-II (corresponding to nucleotides 535 to 565 [22,26] (4,5,22,27,28), a receptor which is detectable in RNA from all neuroblastoma cell lines (8 of 8) and tumors (8 of 8) we have examined, as well as in RNA from adrenal medulla, the tissue from which most neuroblastomas are thought to arise (22; and data not shown). We further pursued the role of IGF-II in the growth of neuroblastoma tumors for the following reasons: (a) neuroblastoma is an embryonal tumor and IGF-II is expressed at high levels in the human fetus whereas IGF-I is not (29); (b) IGF-II mRNA had been detected in 3 of 12 neuroblastomas previously examined (10,22,30,31); and (c) the growth ofone human neuroblastoma cell line, SK-N-AS, is mediated by endogenously produced IGF-II (22).…”

“…Although distinct cell surface receptors have been identified for IGF-I and IGF-II, both peptides are thought to stimulate growth by binding to the type I IGF receptor (2)(3)(4)(5). IGF-I mediates the growth promoting effects of growth hormone during postnatal growth (6,7).…”