IADR Abstract Archives
Regulation of Profibrotic TGF-β Signaling by CD26 in Skin Fibroblasts
Objectives: A skin fibroblast (SFBLs) subpopulation which expresses high levels of CD26, a multifunctional cell surface protein that regulates ATP-signaling, may drive skin fibrosis and scarring. In the gingiva where wound healing results in minimal scarring, this distinct population of CD26+ fibroblasts is largely absent. We have shown that siRNA-induced downregulation of CD26 in SFBLs modulates expression of profibrotic TGF-β signaling pathway target genes. Thus, we hypothesized that downregulation of CD26 regulates the profibrotic phenotype of SFBLs by ATP and TGF-β signaling. Our objective was to elucidate CD26 regulation of this pathway.
Methods: Human SFBLs were transfected with or without control or CD26 siRNA, cultured in three-dimensional in vivo-like cultures for up to 6 days, and then treated for 24 h with or without inhibitors targeting ATP signaling pathways or catalytic functions of CD26. Gene and protein expression were analyzed by real-time RT-PCR and Western blotting, respectively.
Results: Treatments with siRNA potently suppressed CD26 mRNA (>90%) and protein levels (>70%), and resulted in concomitant phosphorylation of SMAD3, a key effector of TGF-β signaling, at both S423/425 activation and S208 inhibitory sites, and upregulation of TGIF, a transcriptional repressor of TGF-β signaling. Pharmacological inhibition of CD26 catalytic functions or a set of molecules involved in ATP-adenosine signaling did not alter CD26-regulated gene expression or TGF-β signaling in SFBLs. However, CD26 siRNA treatments lead to significant upregulation of mRNA and protein levels of Cx43, a cell surface molecule that regulates cell-to-cell communication, ATP release, and TGF-β signaling.
Conclusions: CD26 downregulation in SFBLs associates with auto/paracrine TGF-β-induced phosphorylation of SMAD3 at activating S423/425 and inhibitory S208 sites, and an increase in TGIF. These changes were independent of CD26 catalytic activity or adenosine signaling but associated with upregulation of Cx43. This pathway may provide a novel target to modulate the profibrotic phenotype of SFBLs.
Methods: Human SFBLs were transfected with or without control or CD26 siRNA, cultured in three-dimensional in vivo-like cultures for up to 6 days, and then treated for 24 h with or without inhibitors targeting ATP signaling pathways or catalytic functions of CD26. Gene and protein expression were analyzed by real-time RT-PCR and Western blotting, respectively.
Results: Treatments with siRNA potently suppressed CD26 mRNA (>90%) and protein levels (>70%), and resulted in concomitant phosphorylation of SMAD3, a key effector of TGF-β signaling, at both S423/425 activation and S208 inhibitory sites, and upregulation of TGIF, a transcriptional repressor of TGF-β signaling. Pharmacological inhibition of CD26 catalytic functions or a set of molecules involved in ATP-adenosine signaling did not alter CD26-regulated gene expression or TGF-β signaling in SFBLs. However, CD26 siRNA treatments lead to significant upregulation of mRNA and protein levels of Cx43, a cell surface molecule that regulates cell-to-cell communication, ATP release, and TGF-β signaling.
Conclusions: CD26 downregulation in SFBLs associates with auto/paracrine TGF-β-induced phosphorylation of SMAD3 at activating S423/425 and inhibitory S208 sites, and an increase in TGIF. These changes were independent of CD26 catalytic activity or adenosine signaling but associated with upregulation of Cx43. This pathway may provide a novel target to modulate the profibrotic phenotype of SFBLs.