Cell proliferating effect of latent transforming growth factor‐β1 is cell membrane dependent
Authors: Ghahary Aziz , Tredget Edward E. , Ghahary Ali , Bahar Mohammad A. , Telasky Christopher 
Published: March 2003

The mechanism of in vivo activation of transforming growth factor‐β1 (TGF‐β1), 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‐β1 directly, the efficacy of either latent TGF‐β1 (LTGF‐β1) alone or LTGF‐β1 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‐β1, PA317 cells were transfected with previously constructed pLin‐TGF‐β1 or pLin vectors with no TGF‐β1 insert. LTGF‐β1 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‐β1 were selected and their conditioned media were removed after 3 days and used to evaluate the latency and bioactivity of TGF‐β1 using ELISA and Mv1Lu growth inhibition assay, respectively. The level of TGF‐β1 was 19‐fold greater (21.4 ± 0.4 vs. 1.1 ± 0.2 ng/ml) in conditioned medium derived from pLin‐TGF‐β1 transfected cells than that of control. These conditioned media were then used for the subsequent cell proliferating experiments. The results showed that latent TGF‐β1, 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‐β1 and the results showed a significant increase in cell proliferation in response to low concentrations of active TGF‐β1. However, high concentrations of active TGF‐β1 markedly suppressed fibroblast proliferation. These dual effects were in contrast to a steady increase in fibroblast proliferation found in response to latent TGF‐β1. To explore why LTGF‐β1 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‐β1 and then added to mink lung cells. Only isolated fibroblast cell membranes incubated with LTGF‐β1 inhibited Mv1Lu cells. To examine whether the LTGF‐β1 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‐β1, 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‐β1 relative to that of control. The inhibitory effect of SK23 plus LTGF‐β1 was cell membrane dose‐dependent. In conclusion, the result of this study shows that LTGF‐β1 may directly modulate cell proliferation of those cells that possess a cell membrane associated LTGF‐β1 activation mechanism. (WOUND REP REG 2002;10:328–335)