Cell proliferating effect of latent transforming growth factor‐β1 is cell membrane dependent

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Abstract
The mechanism of in vivo activation of transforming growth factor-beta1 (TGF-beta1), which is critical to its role in many physiological and pathological conditions, is not fully understood. To explore the mechanism by which dermal fibroblasts respond to latent TGF-beta1 directly, the efficacy of either latent TGF-beta1 (LTGF-beta1) alone or LTGF-beta1 plus cell membranes isolated from fibroblasts, mink lung, and one skin-related (Sk23) and two skin-unrelated (U251 and D54MG) transformed cell lines was examined using the mink lung epithelial cell (Mv1Lu) inhibition assay. As a source of LTGF-beta1, PA317 cells were transfected with previously constructed pLin-TGF-beta1 or pLin vectors with no TGF-beta1 insert. LTGF-beta1 expressing PA317 cells were then enriched by growth in the presence of 0.5 mg G-418 for 6-10 days. Eight out of 53 colonies of cells expressing high levels of LTGF-beta1 were selected and their conditioned media were removed after 3 days and used to evaluate the latency and bioactivity of TGF-beta1 using ELISA and Mv1Lu growth inhibition assay, respectively. The level of TGF-beta1 was 19-fold greater (21.4 +/- 0.4 vs. 1.1 +/- 0.2 ng/ml) in conditioned medium derived from pLin-TGF-beta1 transfected cells than that of control. These conditioned media were then used for the subsequent cell proliferating experiments. The results showed that latent TGF-beta1, which proved to be inactive in an Mv1Lu inhibition assay, significantly stimulates fibroblast cell proliferation compared to that of control in a dose-dependent fashion. In another set of experiments, cells were treated with either active (acidified/neutralized) or latent TGF-beta1 and the results showed a significant increase in cell proliferation in response to low concentrations of active TGF-beta1. However, high concentrations of active TGF-beta1 markedly suppressed fibroblast proliferation. These dual effects were in contrast to a steady increase in fibroblast proliferation found in response to latent TGF-beta1. To explore why LTGF-beta1 has a differential proliferating effect on epithelial and fibroblast cell proliferation, cell membranes from these cells were isolated and incubated with PA317-conditioned medium containing LTGF-beta1 and then added to mink lung cells. Only isolated fibroblast cell membranes incubated with LTGF-beta1 inhibited Mv1Lu cells. To examine whether the LTGF-beta1 cell proliferating activity is unique to dermal fibroblasts or is a general phenomenon, in similar experimental conditions cell membranes from several cell lines, U251, D54MG, and SK23, were isolated, incubated with LTGF-beta1, and then added to an Mv1Lu inhibition assay. The proliferation of Mv1Lu epithelial cells was significantly (1547 +/- 269 vs. 3568 +/- 23) inhibited with SK23, but not U251 cell membranes plus LTGF-beta1 relative to that of control. The inhibitory effect of SK23 plus LTGF-beta1 was cell membrane dose-dependent. In conclusion, the result of this study shows that LTGF-beta1 may directly modulate cell proliferation of those cells that possess a cell membrane associated LTGF-beta1 activation mechanism.

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