Activation of latent transforming growth factor‐β1 is induced by mannose 6‐phosphate/insulin‐like growth factor‐II receptor

This study was conducted to further explore the mechanism of transforming growth factor β1 (TGF‐β1) activation, which plays a critical role in many physiological and pathological conditions. We have previously shown that the large (270 kDa), but not small (40 kDa), mannose 6‐phosphate receptors facilitate the cellular response to latent TGF‐β1 released from genetically modified cells. In this study, we explored the role of cell membrane associated transglutaminase and plasmin in mannose 6‐phosphate receptor induced latent TGF‐β activation using MS and MS‐9 cells bearing either no receptors or the 270 kDa mannose 6‐phosphate/insulin‐like growth factor II receptors, respectively. As a source of latent TGF‐β1, PA317 cells were transfected with either pLin‐TGF‐β1 vector or pLin retroviral vector with no TGF‐β1 insert using calcium phosphate precipitation. The latency and bioactivity of TGF‐β1 in conditioned medium derived from transfected PA317 cells were evaluated by enzyme‐linked immunosorbent assay and mink lung epithelial cell growth inhibition assay, respectively. The level of latent TGF‐β1 was 13‐fold higher (20.1 ± 0.4 vs. 1.5 ± 0.03 ng/ml) in conditioned medium from pLin‐TGF‐β1 transfected cells than that of control. The latency and bioactivity of TGF‐β1 released from pLin‐TGF‐β1 transfected cells were confirmed by evaluation of 3 H‐thymidine incorporation in Mv1Lu epithelial cells treated with non‐ and heat‐activated 10% conditioned medium. The results showed a significantly lower 3 H‐thymidine incorporation in Mv1Lu epithelial cells treated with heat‐activated PA317 conditioned medium (4% of control) relative to those treated with either control or nonheated conditioned medium. This inhibition was abrogated by addition of 40 μg/ml of TGF‐β1 neutralizing antibody. The level of 3 H‐thymidine incorporation was then evaluated in MS‐9 cells receiving Dulbecco's modified Eagle medium containing either 0% 10%, 30% or 50% volumes of nonactivated PA317 conditioned medium for 24 hours. The results showed a markedly lower proliferation in response to 30% and 50% conditioned medium used in MS‐9 cells. Under similar experimental conditions, addition of only mannose 6‐phosphate, but not fructose 6‐phosphate or mannose 1‐phosphate, at 1 mM concentration restored the MS‐9 cell proliferative response to latent TGF‐β1. The inhibitory effects of latent TGF‐β1 on MS‐9 cell proliferation were restored by addition of either TGF‐β1 neutralizing antibody or cystamine, a transglutaminase inhibitor. In contrast, addition of aprotinin, a plasmin inhibitor, had a marginal influence on inhibitory effects of latent TGF‐β1 on MS‐9 cell proliferation. Interestingly, a mixture of latent TGF‐β1 + MS‐9 cell membranes, but not MS cell membranes, also inhibited the mink lung epithelial cell proliferation (34% of control). These findings indicate that mannose 6‐phosphate/insulin‐like growth factor II receptors are involved in latent TGF‐β activation and that is at least partly dependent on cell membrane associated transglutaminase, but not on plasmin.