Lukas Nollet, Matthias Van Gils, Olivier M. Vanakker
Center For Medical Genetics, Ghent University Hospital, Belgium - Department Of Biomolecular Medicine, Ghent University, Belgium - Ectopic Mineralization Research Group Ghent, Belgium
Aim: Pseudoxanthoma elasticum (PXE) is a multisystemic ectopic calcification disorder characterized by deposition of calcium crystals in the skin, eyes and blood vessels, resulting in significant morbidity and occasional mortality. PXE is caused by bi-allelic pathogenic variants in the ABCC6 gene, encoding a transmembrane transporter whose substrate is still unknown. Recently, excessive activation of the DNA damage response (DDR), in particular poly(ADP-ribose) polymerase 1 (PARP1), was shown to be involved in aberrant mineralization raising the hypothesis that dysfunctional DDR/PARP1 signaling also contributes to PXE pathogenesis.
Methods: Differential expression analysis of primary DDR targets (PARP1, PARP2, ATM, p21, p53, SIRT1), downstream PARP1 signaling effectors (STAT1/3, IL-6, TET1, RUNX2) and ectopic calcification markers (ALPL, MSX2, BMP2, CTGF, MMP2) was performed on PXE patient-derived (n=6) and age- and sex-matched healthy control (n=6) fibroblasts using quantitative real-time reverse transcription PCR (qPCR), western blot and enzyme activity assays. Expression levels of the RUNX2-antagonist microRNA-204 was evaluated using a miR-204-specific qPCR assay. Then, PXE fibroblasts were treated for 7 days with the PARP1 inhibitor minocycline and gene and protein expression profiles were re-assessed and compared to untreated PXE fibroblasts. Effect of minocycline treatment (3 µM) on calcium crystal deposition was evaluated using a ß-glycerophosphate-induced in vitro calcification assay followed by Alizarin Red staining. Finally, anti-calcifying properties of minocycline (10 µM) in vivo were investigated using the abcc6a-/- zebrafish (danio rerio) PXE model.
Results: Significantly increased expression of the primary DDR targets PARP1, p21, p53 and ATM was found in PXE fibroblasts compared to controls, while the DDR inhibitor SIRT1 was shown to be suppressed. Downstream PARP1 signaling effectors including STAT1/3, IL-6, TET1 and RUNX2 were also upregulated in PXE. Additionally, the RUNX2-antagonist miR-204 was found to be decreased in PXE compared to controls, correlating with higher RUNX2 levels. Treatment with the PARP1 inhibitor minocycline significantly attenuated expression of the DDR targets ATM, p21 and p53, and of the PARP1-STAT1/3-TET1-RUNX2 axis, leading to reduced levels of pro-calcifying mediators such as ALPL, MSX2, CTGF, MMP2 and BMP2. Minocycline increased miR-204 levels in PXE fibroblasts, thereby further antagonizing RUNX2 expression. Additionally, minocycline significantly reduced aberrant mineralization of the extracellular matrix of PXE fibroblasts cultured in calcification medium. In vivo, minocycline treatment of abcc6a-/- zebrafish larvae for 7 days resulted in a 60% reduction in ectopic calcification compared to untreated controls, without negatively affecting physiological calcification of the bony skull structures.
Conclusion: We demonstrated for the first time the involvement of dysfunctional DDR and PARP1 signaling in PXE, showing that excessive activation of primary DDR targets and PARP1 evokes a STAT-driven cascade resulting in upregulation of the epigenetic modifier TET1 and pro-calcifying transcription factor RUNX2. Minocycline treatment attenuated this deleterious molecular mechanism and reduced ectopic calcification both in vitro and in vivo, fueling the exciting prospect of a novel therapeutic compound for PXE.