Cellular response to latent TGF-beta1 is facilitated by insulin-like growth factor-II/mannose-6-phosphate receptors...

This study was conducted to explore the mechanism of activation of TGF-beta1 which is critical to its role in many physiological and pathological conditions. We have previously demonstrated that latent TGF-beta1 modulates ECM through interaction with IGF-II/M6P receptors on dermal fibroblasts. In this report, we provide evidence that large (270 kDa) but not small (46 kDa) M6P receptors facilitate the cellular response to LTGF-beta1 released from genetically modified cells. As a source of LTGF-beta1, PA317 cells were transfected with either pLin-TGF-beta1 vector or pLin vector with no TGF-beta1 insert using calcium phosphate precipitation. Conditioned medium from transfected cells was removed after 3 days and used to evaluate the latency and bioactivity of TGF-beta1 using ELISA and mink lung epithelial cell growth inhibition assay, respectively. The level of TGF-beta1 was 20-fold greater (2142 +/- 369 vs 102 +/- 23 pg/ml) in conditioned medium derived from pLin-TGF-beta1-transfected cells than in that of controls. Various volumes of this conditioned medium were then used to treat MS-9, SR-2, and MS cells bearing the large, small, and no IGF-II/M6P receptors, respectively, for 24 h. [(3)H]Thymidine incorporation, used as an index for cell proliferation, showed a markedly lower level of proliferation in MS-9 cells in response to a given concentration of LTGF-beta1 than was seen in SR-2 and MS cells. Interestingly, under similar experimental conditions, either addition of M6P at 1 mM concentration or anti-TGF-beta1 antibody abrogated the MS-9 cell proliferative response to LTGF-beta1. In contrast, the inhibitory response of these three cell strains to heat-activated conditioned medium was the same. As another measure of LTGF-beta1-induced cellular response, the expression of mRNA for pro alpha1(I) of type I collagen was also evaluated. A marked increase in expression of this transcript in MS-9 cells in response to LTGF-beta1 was observed. To further examine the possible correlation between the large IGF-II/M6P receptors and cellular responses to LTGF-beta1, expression of IGF-II/M6P receptors at the protein and mRNA levels were evaluated by ligand binding and RT-PCR, respectively. Using (125)I-IGF-II as a ligand, the number of specific IGF-II/M6P receptors was found to be threefold greater on MS-9 than on SR-2 and MS cells. This finding was consistent with the level of IGF-II/M6P receptor mRNA detected by RT-PCR in MS-9 cells. In conclusion, the result of this study shows a direct link between large but not small IGF-II/M6P receptors on MS-9 cells and their response to LTGF-beta1.