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  2. KLF6 activates Sp1-mediated prolidase transcription during TGF-β1 signaling

KLF6 activates Sp1-mediated prolidase transcription during TGF-β1 signaling

  • J Biol Chem. 2023 Dec 28:105605. doi: 10.1016/j.jbc.2023.105605.
Ireti Eni-Aganga 1 Zeljka Miletic Lanaghan 2 Farah Ismail 3 Olga Korolkova 4 Jeffery Shawn Goodwin 4 Muthukumar Balasubramaniam 4 Chandravanu Dash 5 Jui Pandhare 6
Affiliations

Affiliations

  • 1 Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, TN 37208, USA; School of Graduate Studies, Meharry Medical College, Nashville, TN 37208, USA; Department of Microbiology, Immunology and Physiology, Meharry Medical College, Nashville, TN 37208, USA.
  • 2 Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, TN 37208, USA; Neuroscience Graduate Program, Vanderbilt University, Nashville, TN.
  • 3 Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, TN 37208, USA.
  • 4 Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, TN 37208, USA; Department of Biochemistry, Cancer Biology, Pharmacology and Neuroscience, Meharry Medical College, Nashville, TN 37208, USA.
  • 5 Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, TN 37208, USA; Department of Microbiology, Immunology and Physiology, Meharry Medical College, Nashville, TN 37208, USA; Department of Biochemistry, Cancer Biology, Pharmacology and Neuroscience, Meharry Medical College, Nashville, TN 37208, USA. Electronic address: cdash@mmc.edu.
  • 6 Center for AIDS Health Disparities Research, Meharry Medical College, Nashville, TN 37208, USA; School of Graduate Studies, Meharry Medical College, Nashville, TN 37208, USA; Department of Microbiology, Immunology and Physiology, Meharry Medical College, Nashville, TN 37208, USA. Electronic address: jpandhare@mmc.edu.
Abstract

Prolidase (PEPD) is the only hydrolase that cleaves the Dipeptides containing C-terminal proline or hydroxyproline- the rate-limiting step in collagen biosynthesis. However, the molecular regulation of prolidase expression remains largely unknown. In this study, we have identified overlapping binding sites for the transcription factors- Krüppel-like factor 6 (KLF6) and Specificity protein 1 (Sp1) in the PEPD promoter and demonstrate that KLF6/Sp1 transcriptionally regulate prolidase expression. By cloning the PEPD promoter into a luciferase reporter and through site-directed deletion, we pinpointed the minimal sequences required for KLF6 and Sp1-mediated PEPD promoter-driven transcription. Interestingly, Sp1 inhibition abrogated KLF6-mediated PEPD promoter activity, suggesting that Sp1 is required for the basal expression of prolidase. We further studied the regulation of PEPD by KLF6 and Sp1 during transforming growth factor β1 (TGF-β1) signaling, since both KLF6 and Sp1 are key players in TGF-β1 mediated collagen biosynthesis. Mouse and human fibroblasts exposed to TGF-β1 resulted in the induction of PEPD transcription and prolidase expression. Inhibition of TGF-β1 signaling abrogated PEPD promoter-driven transcriptional activity of KLF6 and Sp1. Knock-down of KLF6 as well as Sp1 inhibition also reduced prolidase expression. Chromatin immunoprecipitation assay supported direct binding of KLF6 and Sp1 to the PEPD promoter and this binding was enriched by TGF-β1 treatment. Finally, immunofluorescence studies showed that KLF6 co-operates with Sp1 in the nucleus to activate prolidase expression and enhance collagen biosynthesis. Collectively, our results identify functional elements of the PEPD promoter for KLF6 and Sp1-mediated transcriptional activation and describe the molecular mechanism of prolidase expression.

Keywords

Collagen; Kruppel‐like factor 6 (KLF6); Prolidase; Specificity protein 1 (Sp1); Transcriptional Regulation; Transforming growth factor beta (TGF‐β).

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