P72Krueppel-like factor 15 regulates Wnt/beta-catenin transcription and controls cardiac progenitor cell fate in the postnatal heart

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Abstract

Wnt/β-catenin signaling controls cardiac progenitor cells (CPC) in early development and the postnatal heart. We identified and characterized a β-catenin interaction partner, the Krüppel like factor 15 (KLF15)concerning CPC biology. A reporter assay showed KLF15 to significant repress β-catenin/TCF-dependent transcription in a concentration-dependent manner. Mutagenesis and co-immunoprecipitation analysis showed both, the KLF15 N- and C-terminus to be necessary for transcription repression. KLF15 specifically interacted with TCF4, a β-catenin co-factor that activates target gene expression and target it to ubiquitination. Moreover, KLF15 interacted with the NEMO-like kinase (NLK), which phosphorylates TCF4 to target it for degradation. Thus, KLF15 may recruit NLK to the β-catenin/TCF-complex to prevent activation of transcription via targeting TCF to protein degradation. The analysis of Klf15 knock-out (KO) mice showed a cardiac de-repression of β-catenin transcription demonstrated by increase of its target genes TCF4 and c-Myc in KO mice. KLF15 KO mice showed impaired fractional shortening (n = 10). Flow cytometry analysis revealed a significant reduction of cardiogenic-committed CPC identified as Sca1/aMHC (WT 1.75 ± 0.08% vs KO 0.83 ± 0.19%) and Tbx5 (WT 5.23 ± 0.48% vs KO 3.47 ± 0.26%). In contrast, endothelial Sca1/CD31 cells were increased in KO mice (WT 8.60 ± 0.36% vs KO 11.25 ± 0.39% n≥9. Under the hypothesis that β-catenin transcriptional activation promotes an endothelial cell fate of CPCs, we analyzed the CPCs fraction in mice with a cardiac β-catenin inactivation, which indeed showed a reduction of CD31pos (WT 18.01 ± 0.84% vs KO 15.06 ± 0.76% n≥6) as well as CD31pos/Ki67pos CPCs in comparison to controls. In favour of an endothelial fate of CPCs in klf15 KO Sca-1 isolated cells showed increased RNA expression of endothelial markers: von Willebrand Factor and Flk-1 as well as upregulation of the β-catenin target genes c-myc and TCF4. Increased endothelial Flk1 cells and reduction of aMHC and Hand1 cardiogenic cells was observed in co-cultured KO CPCs at day 10 of culture (n = 6). Treating the co-cultures with Quercetin, an inhibitor of β-catenin transcription, resulted in abrogation of Flk1 cell increase and aMHC and Hand1 cell reduction. Our data allows us to conclude that KLF15-dependent β-catenin repression favors endothelial differentiation and antagonizes cardiogenic cell differentiation similar to what has been observed in embryonic development. This work may have identified a novel molecular switch to therapeutically modulate CPC fate and unlock the regenerative potential of the adult heart.

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