Non-canonical Telomerase Reverse Transcriptase Controls Osteogenic Differentiation of Aortic Valve Cells Through STAT5

Abstract

BackgroundCalcific aortic valve disease (CAVD) is the pathological remodeling of valve leaflets. The initial steps in valve leaflet osteogenic reprogramming are not fully understood. As telomerase reverse transcriptase (TERT) overexpression primes mesenchymal stem cells to differentiate into osteoblasts, we investigated whether TERT contributes to the osteogenic reprogramming of valve interstitial cells (VICs). MethodsHuman 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. Calcification was assessed in Tert+/+ and Tert-/- mice ex vivo and in vivo. In silico modeling, proximity ligation and co-immunoprecipitation assays defined novel TERT interacting partners. Chromatin immunoprecipitation and CUT&TAG sequencing defined protein-DNA interactions. ResultsTERT 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 or inflammatory stimuli, and knock-down or genetic deletion of TERT prevented calcification in vitro and in vivo. Mechanistically, TERT was upregulated via NF-{kappa}B and required to initiate osteogenic reprogramming, independent of its canonical reverse transcriptase activity and the lncRNA TERC. TERT exerts non-canonical osteogenic functions via binding with Signal Transducer and Activator of Transcription 5 (STAT5). Depletion or 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. Lastly, we demonstrate that TERT and STAT5 are upregulated and colocalized in CAVD tissue compared to control tissue. ConclusionsTERTs non-canonical activity is required to initiate calcification. TERT is upregulated via inflammatory signaling pathways and partners with STAT5 to bind the RUNX2 gene promoter. These data identify a novel mechanism and potential therapeutic target to decrease vascular calcification. Novelty and SignificanceWhat is known? Calcific aortic valve disease (CAVD) is the most prevalent form of aortic valve pathology. CAVD strongly correlates with age and leads to heart failure and a high risk of stroke. Currently, the only therapeutic option is valve replacement, which comes with significant healthcare costs and additional risks to patients. Runt-related transcription factor 2 (RUNX2) is the master transcription factor required for osteogenic differentiation of stem cells to osteoblasts and osteogenic reprogramming of cardiovascular cells. Yet, the early events driving its activity in aortic valve cells are poorly defined. In addition to its reverse transcriptase enzymatic activity, TERT exhibits non-canonical transcriptional regulatory functions and overexpression of TERT primes mesenchymal stem cells to differentiate down the osteoblast lineage. What new information does this article contribute? TERT protein levels in calcified aortic leaflets and valve interstitial cells, and its non-canonical osteogenic activity are independent of changes in telomere length and cell senescence. Genetic loss or depletion of TERT prevented calcification in valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells in vitro and the vasculature in vivo. Early in the osteogenic reprogramming inflammatory signaling promotes TERT to co-localize with SMARCA4 and STAT5, and this TERT-tethered STAT5 binds to the RUNX2 gene promoter, the master regulator of osteogenic transcriptional programs. STAT5 depletion and pharmacological inhibition prevent calcification of human valve interstitial cells, coronary smooth muscle cells, and mesenchymal stem cells. What are the clinical implications? We have identified TERT-STAT5 as a novel signaling axis that orchestrates the early steps in the osteogenic reprogramming of aortic valve cells. Inhibiting TERT/STAT5 interaction or their activity may be leveraged for the development of therapeutic strategies to halt or prevent calcification in the aortic valve, bioprosthetic valves, and or perhaps other cardiovascular tissues. Invasive and expensive surgical procedures are currently the only treatment option for patients with CAVD. The discovery and defining of the early events driving vascular calcification identifies novel and druggable targets for developing non-surgical therapies.