UC San Diego

HEG1 (Heart of glass1), a transmembrane protein, directly binds to and recruits KRIT1 (Krev interaction trapped protein 1) to endothelial junctions to form the HEG1-KRIT1 protein complex that establishes and maintains junctional integrity. Genetic inactivation or knockdown of endothelial HEG1 or KRIT1 leads to the upregulation of transcription factors Krüppel-like Factors 4 and 2 (KLF4 and KLF2), which are factors implicated in endothelial vascular homeostasis. However, the effect of acute inhibition of the HEG1-KRIT1 interaction remains incompletely understood. Through structure-based design, we have established the feasibility of pharmacologically disrupting the KRIT1-HEG1 interaction to uncover acute changes in signaling pathways downstream of the HEG1-KRIT1 protein complex disruption. Previous work in the lab shows that the HEG1-KRIT1 inhibitor 3 (HKi3) is a bona fide interaction inhibitor by competing orthosterically with HEG1 for binding to the KRIT1 band 4.1, ezrin, radixin, and moesin (FERM) domain. In this study, we observed that acute inhibition of the HEG1-KRIT1 interaction by HKi3 triggers PI3K/Akt signaling in endothelial cells. We also observed that inhibition of KRIT1-HEG1 interaction using HKi3 also increase KLF4 and KLF2 mRNA expression levels. In addition, we developed an inducible shRNA expression system to the time- controlled knockdown of genes associated with the HEG1-KRIT1 protein complex that we denominated TRMPV-PDCD10 (tetracycline-responsive element (TRE)-dsRed-miR30-against- PDCD10-PGK-Venus). Using this RNAi system, we generated stable hCMEC/D3 cell lines, each expressing the TRMPV-PDCD10 construct. We observed that TRMPVIR-PDCD10 doxycycline-treated cells show marked disruption of VE-cadherin staining and upregulation in KLF2 and KLF4 mRNA gene expression. Thus, our results demonstrate that acute inhibition of the HEG1-KRIT1 interaction activates PI3K/Akt activity and elevates KLF4 and KLF2 gene expression. Thus, HKi3 provides a new pharmacologic tool to study acute inhibition of the HEG1-KRIT1 protein complex. We also adopted an inducible shRNA expression system to regulate genes in endothelial cells in a time-controlled manner.