BMP-2 and TGF-β1 mediate biglycan-induced pro-osteogenic reprogramming in aortic valve interstitial cells

Biglycan accumulates in aortic valves affected by calcific aortic valve disease (CAVD), and soluble biglycan upregulates BMP-2 expression in human aortic valve interstitial cells (AVICs) via Toll-like receptor (TLR) 2 and induces AVIC pro-osteogenic reprogramming, characterized by elevated pro-osteogenic activities. We sought to identify the factors responsible for biglycan-induced pro-osteogenic reprogramming in human AVICs. Treatment of AVICs with recombinant biglycan induced the secretion of BMP-2 and TGF-beta 1, but not BMP-4 or BMP-7. Biglycan upregulated TGF-beta 1 expression in a TLR4-dependent fashion. Neutralization of BMP-2 or TGF-beta 1 attenuated the expression of alkaline phosphatase (ALP), osteopontin, and runt-related transcription factor 2 (Runx2) in cells exposed to biglycan. However, neutralization of both BMP-2 and TGF-beta 1 abolished the expression of these osteogenic biomarkers and calcium deposition. Phosphorylated Smad1 and Smad3 were detected in cells exposed to biglycan, and knockdown of Smad1 or Smad3 attenuated the effect of biglycan on the expression of osteogenic biomarkers. While BMP-2 and TGF-beta 1 each upregulated the expression of osteogenic biomarkers, an exposure to BMP-2 plus TGF-beta 1 induced a greater upregulation and results in calcium deposition. We conclude that concurrent upregulation of BMP-2 and TGF-beta 1 is responsible for biglycan-induced pro-osteogenic reprogramming in human AVICs. The Smad 1/3 pathways are involved in the mechanism of AVIC pro-osteogenic reprogramming. Biglycan upregulates BMP-2 and TGF-beta 1 in human aortic valve cells through TLRs. Both BMP-2 and TGF-beta 1 are required for aortic valve cell pro-osteogenic reprogramming. Smad signaling pathways are involved in mediating the pro-osteogenic effects of biglycan.