Glycosylphosphatidylinositol-anchored proteins regulate transforming growth factor-beta signaling in human keratinocytes.
Glycosylphosphatidylinositol (GPI)-anchored proteins have been demonstrated to bind transforming growth factor-beta (TGF-beta) in certain cell lines. However, the identity of these GPI-anchored proteins and the role they may play in TGF-beta signaling remain unknown. We have previously reported the presence of GPI-anchored TGF-beta-binding proteins on human skin fibroblasts and keratinocytes (Tam, B. Y. Y., and Philip, A. (1998) J. Cell. Physiol. 176, 553-564; Tam, B. Y. Y., Germain, L., and Philip, A. (1998) J. Cell. Biochem. 70, 573-586). On human keratinocytes, we identified a 150-kDa GPI-anchored TGF-beta1-binding protein (r150) and demonstrated that it can form a heteromeric complex with the type I and II TGF-beta signaling receptors. To explore whether GPI-anchored proteins modulate TGF-beta signaling in keratinocytes, we created keratinocytes defective in GPI anchor biosynthesis (GPI mutant cells) by chemical mutagenesis of HaCaT cells. Mutant clones were selected by fluorescence-activated cell sorting analysis based on the loss of a CD59 marker. In comparison with parental HaCaT cells, GPI mutant cells demonstrated a significant loss of r150 expression. In contrast, the levels of the type I and II TGF-beta receptors and their ligand affinities, cell morphology, and doubling time remained unchanged. Importantly, GPI mutant cells displayed enhanced gene transcriptional activity and Smad2 and Smad3 activation in response to TGF-beta1 treatment in a dose-dependent manner. Taken together, our results indicate that GPI-anchored protein(s) inhibit TGF-beta signaling and implicate r150 as the GPI-anchored protein responsible for this inhibition in human keratinocytes.
D002455:Cell Division; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis / Q000502:physiology*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction / Q000502:physiology*; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D004268:DNA-Binding Proteins; D017261:Glycosylphosphatidylinositols; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta