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

Eni-Aganga et al., Journal of Biological Chemistry, 2024 set out to explain how prolidase, the only enzyme that completes collagen breakdown by cleaving dipeptides ending in proline, is switched on at the level of transcription. Sequence analysis of the PEPD promoter found overlapping binding sites for two transcription factors, KLF6 and Sp1, conserved across mammals and clustered near the transcription start site. Luciferase reporters, a deletion series and chromatin immunoprecipitation then showed that both factors bind the promoter directly, that Sp1 sets the basal level, and that KLF6 raises it further but only while Sp1 can still bind.

The genetic test of that model used AUMsilence sdASO from AUM BioTech against mouse KLF6. In NIH3T3 mouse embryonic fibroblasts the oligonucleotide was delivered gymnotically, meaning it was added straight to the culture with no transfection reagent, twice at a 24 hour interval at a final concentration of 5 μM, against a scrambled control oligonucleotide from the same source. KLF6 protein fell by about 50% and prolidase protein fell with it. The parallel attempt to knock down the partner factor Sp1 with a small interfering RNA did not work, so Sp1 was blocked instead with the small molecule mithramycin A, which lowered both prolidase messenger RNA and protein.

The knockdown was then used to close the loop on transforming growth factor beta 1. Treating the fibroblasts with the cytokine raised KLF6 by about 2.5-fold at 10 ng/ml and prolidase by about 2 to 2.5-fold, and increased the binding of both KLF6 and Sp1 to the PEPD promoter. In cells where KLF6 had been silenced with the AUMsilence sdASO, prolidase stayed low even after the cytokine was added, which places KLF6 on the path between the cytokine and the enzyme. Higher prolidase tracked with higher collagen type I alpha 1, and the same pattern of promoter binding and induction held in primary human dermal fibroblasts, although the oligonucleotide itself was used only in the mouse line.