Abstract
TGFβ is part of a growth factor superfamily which modulates cell growth, differentiation, adhesion, migration, ECM synthesis and apoptosis (Massague, 1998; Siegel and Massague, 2003). Free TGFβ binds to its high affinity TGFβ receptor, a receptor serine/threonine kinase, inducing phosphorylation of Smad2/3 which subsequently forms a complex with Smad4 to translocate to the nucleus where it interacts with multiple co-activators and repressors generating distinct transcriptional responses.Indeed, TGFβ signaling shows a remarkable cellular context dependency and apparent multifunctionality: e.g. TGFβ is able to inhibit cell proliferation in many epithelial cells but can also enhance proliferation in fibroblasts and cell growth in endothelial cells (Guasch et al., 2007; Xiao et al., 2012); it enhances stem cell pluripotency, but promotes differentiation in other cells (Park, 2011); in cancer development it suppresses pre-malignant cell proliferation, but at the same time promotes conversion to a metastatic phenotype (Chaudhury and Howe, 2009). The TGFβ stimulation assay monitors the responsiveness of cells to TGFβ. Upon TGFβ stimulation short-term effects such as Smad2 phosphorylation and long-term effects such as cell proliferation can be analyzed. The assay will be described for murine keratinocytes, where TGFβ strongly inhibits cell proliferation, but both assays are applicable for other cell types as well.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. A. Representative FACS histogram of murine kerationcytes using Click-iT EdU Alexa Fluor 488 assay kit. Keratinocytes were incubated for 6 h with indicated TGFβ1 concentrations before addition of 10 µM EdU. Cells were analyzed after 8 h of EdU incorporation to detect proliferating cells. Note the decrease of proliferating cells with increasing TGFβ1 concentration. B. Representative Western bot for pSmad2 detection in murine kerationcytes after stimulation with 5 ng/ml TGFβ1 for indicated time points.
Recipes
Acknowledgments
This work was funded by the Max Planck Society.
References
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