NICD4/USP8-Positive Feedback Loop Contributes to the Development of Hypoxic Pulmonary Hypertension

Hypoxic pulmonary hypertension (HPH) is a representative vascular remodeling disease with a poor prognosis. Previous findings from our study have implicated the NICD4 (Notch4 intracellular domain) in pulmonary artery smooth muscle cells (PASMCs) in the pathogenesis of HPH. However, the underlying regulatory mechanisms remain unclear. In this study, we aimed to elucidate the potential regulatory mechanism of NICD4 in HPH.

Using coimmunoprecipitation combined with mass spectrometry, we identified USP8 (ubiquitin-specific peptidase 8) as a novel binding protein of NICD4 in PASMCs. The functional role of USP8 was investigated in vivo using smooth muscle cell-specific Usp8 knockout (Usp8^Acta2-/-) mice and in vitro using primarily cultured PASMCs, alongside pharmacological inhibition with DUB-IN-2 (deubiquitinase-inhibitor-2).

USP8 was significantly upregulated in lung tissues from patients with HPH due to interstitial lung disease or chronic obstructive pulmonary disease, HPH rodent models, as well as in hypoxic PASMCs. Usp8 deficiency in Acta2-positive mice (Usp8^Acta2-/-) or pharmacological inhibition of USP8 by DUB-IN-2 markedly attenuated HPH development. In vitro, USP8 knockdown suppressed hypoxia-induced PASMC proliferation, migration, and apoptosis resistance by modulating the NICD4-MAPK pathway. Mechanistically, USP8 was bound directly to NICD4 to maintain its stability by removing the K48-linked ubiquitin chain on NICD4 at lysine 1760, thus preventing proteasomal degradation. Furthermore, USP8 can be transcriptionally upregulated by CSL/NICD4 under hypoxia, forming a NICD4/USP8-positive feedback loop.

Our study unveils a critical NICD4/USP8-positive feedback loop that drives HPH pathogenesis, highlighting the importance of ubiquitination in pulmonary vascular remodeling. Targeted disruption of this loop represents a promising therapeutic strategy for HPH.