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

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 p...

Complex epithelial–mesenchymal interactions modulate transforming growth factor‐β expression in keloid‐derived cells

“…Each TGF‐β isoform is synthesized as a latent complex consisting of a 25‐kDa dimeric mature protein and the dimeric N‐terminal precursor peptide known as the latency associated peptide. The latent form of TGF‐β is activated through interactions with the M6P/IGF‐IIR 8,15 . In our coculture system, we have demonstrated that coculture of KKs and KFs also results in increased expression of M6P/IGF‐IIR in fibroblasts correlating with the increased levels of active TGF‐β in the conditioned medium.…”

“…Mannose‐6‐phosphate/IGF insulin‐like growth factor II receptor (M6P/IGF‐IIR) plays an important role in activation of latent TGF‐β 8,15 . We demonstrate that KFs expressed four times M6P/IGF‐IIR than NFs without the presence of keratinocytes.…”

“…Mannose-6-phosphate/IGF insulin-like growth factor II receptor (M6P/IGF-IIR) plays an important role in activation of latent TGF-b. 8,15 We demonstrate that KFs expressed four times M6P/IGF-IIR than NFs without the presence of keratinocytes. The presence of either KKs or NKs stimulated fibroblast expression of the M6P/IGF-IIR and KF expression of M6P/IGF-IIR remained approximately fourfold higher than NF under all coculture conditions ( Figure 4C).…”

Inhibition of transient receptor potential canonical channels impairs cytokinesis in human malignant gliomas

“…While this search for TRPC channels involved a number of widely used cell lines and patient biopsies we chose one cell line in particular, D54MG, for our biophysical studies. D54MG glioma cells are derived from a high‐grade (WHO grade IV) glioma and have been widely used and extensively described in the literature (Ghahary et al . 2002) and in many of our previous studies (Ullrich et al .…”

CCL18‐stimulated upregulation of collagen production in lung fibroblasts requires Sp1 signaling and basal Smad3 activity

“…To confirm the ELISA data on active TGF-b, mink lung epithelial cell (Mv1Lu, ATCC, Manassas, VA) proliferation assays were employed. The effects of fibroblast cell culture supernatants or titrated dilutions of rhTGF-b on 3 H-thymidine incorporation were tested with and without neutralizing anti-pan-TGF-b antibody (clone 1D11 from R&D Systems), following the standard protocol (Ghahary et al, 2002). Before testing, the fibroblast cell culture supernatants were cleared of cellular debris by centrifugation, to avoid possible cell membrane-dependent activation of TGF-b (Ghahary et al, 2002).…”