The TGF-betas are important in cell growth, matrix formation, immune system modulation, and apoptosis. Defects in TGF-beta function are associated with pathological conditions including tumor growth and metastasis. Because TGF-betas are released in an inactive complex, their activity is controlled at the level of cytokine presentation as the dissociation of TGF-beta from its complex is a critical step in regulating the concentration and action of active TGF-beta. The processes controlling latent TGF-beta activation are poorly understood. To address how is TGF-beta released from its complex, we developed a genetic screen for activator identification. The screen relies upon cells that constitutively produce latent TGF-beta. The cells are engineered to express green fluorescent protein (GFP) in the presence of TGF-beta. The reporter cells an infected with retroviruses containing a cDNA library prepared from cells that activate latent TGF-beta. The infected cells are sorted for GFP expression. The protein responsible for enhanced GFP expression is identified by retrieval of the cDNA or isolation of the overexpressed protein. The TGF-beta activator activity of the candidate protein is validated in independent assays. We performed proof-of-principle experiments with a known activator, beta6 integrin. Using a cDNA library from glioblastoma cells that activate latent TGF-beta, we identified cell clones that activate endogenous latent TGF-beta. We propose to extend this work in two specific aims.
In Aim 1, we will analyze the clones that we have obtained to identify and characterize the novel activating molecule in the U1240 glioblastoma cell line.
In Aim 2, we will repeat the isolation procedure using cDNA libraries from other two tumor cell lines that activate latent TGF-beta by uncharacterized mechanisms. These experiments will enhance our understanding of TGF-beta biology and potentially result in the development of inhibitors of TGF-beta activation. Although there is a high risk in these studies, the potential reward is great.
