Non-canonical Telomerase Reverse Transcriptase Controls Osteogenic Reprogramming of Aortic Valve Cells Through STAT5 [CUT&Run]
Background: Calcific aortic valve disease (CAVD) is the pathological remodeling of valve leaflets. The initial steps in the osteogenic reprogramming of the leaflet are not fully understood. Studies have shown that TERT overexpression primes mesenchymal stem cells to differentiate into osteoblasts, and we investigated whether TERT contributes to the osteogenic reprogramming of valve interstitial cells (VICs). Methods: Human control and CAVD aortic valve leaflets and patient-specific hVICs were used in in vivo and in vitro calcification assays. Loss of function experiments in hVICs and cells isolated from Tert-/- and Terc-/- mice were used for mechanistic studies. In silico modeling, proximity ligation and co-immunoprecipitation assays defined novel TERT interacting partners. Chromatin immunoprecipitation and CUT&TAG sequencing defined protein-DNA interactions. Results: TERT protein was highly expressed in calcified valve leaflets without changes in telomere length, DNA damage, or senescence markers, and these features were retained in isolated primary hVICs. TERT expression increased with osteogenic stimuli, while knock-down or genetic deletion of TERT prevented calcification. Mechanistically, TERT was required to initiate osteogenic reprogramming independent of its canonical telomere-extending activity and the lncRNA TERC. TERT's osteogenic functions via binding with Signal Transducer and Activator of Transcription 5 (STAT5). Depletion and inhibition of STAT5 prevented calcification. STAT5 was found to bind the promoter region of Runt-Related Transcription Factor 2 (RUNX2), the master regulator of osteogenic reprogramming. Finally, we demonstrate that TERT and STAT5 are upregulated and colocalized in CAVD tissue. Conclusions: TERT's non-canonical activity is required to initiate VIC calcification. TERT partners with STAT5 to bind the RUNX2 gene promoter. These data identify a novel mechanism and potential therapeutic target to decrease vascular calcification. Overall design: Cleavage Under Targets & Release Using Nuclease (CUT&RUN) coupled with DNA sequencing for the Histone marker H3K4me3 in human valve interstitial cells after seven days of osteogenic treatment in vitro.