Journal: bioRxiv
Article Title: ARHGEF17 Deficiency Induces Endothelial Dysfunction and Intracranial Aneurysm Formation via RhoA/ROCK2/MLC Signaling Pathway
doi: 10.1101/2025.10.14.682476
Figure Lengend Snippet: ARHGEF17 Deficiency Activates the RhoA/ROCK2/MLC Signaling Pathway in Endothelial Cells via a ROCK2-Dependent Mechanism (A–C) G-LISA assays measuring RhoA activity in control and ARHGEF17 knockdown HUVECs. Lentiviral knockdown of ARHGEF17 significantly increased the level of active GTP-bound RhoA and the ratio of active-to-total RhoA compared with control cells, with the strongest activation observed in shRNA-76396 cells. (D) Western blot analysis showing protein expression of key components in the RhoA/Rock2/MLC signaling cascade, including Rock1, Rock2, MYPT1, phosphorylated MYPT1 (Thr853), MLC, phosphorylated MLC (Thr18/Ser19), and MLCK. (E-F) Rock1 levels showed a mild increase after ARHGEF17 knockdown, Y-27632 treatment partially suppressed this elevation. ROCK2 expression was significantly increased by approximately 2.4-fold in shRNA-76396 HUVECs compared with controls, and Y-27632 treatment markedly suppressed ROCK2 upregulation, restoring its expression to near-baseline levels. (G-H) Total MYPT1 expression was unaffected by either ARHGEF17 knockdown or Y-27632, confirming that the observed activation occurs via phosphorylation rather than altered protein abundance. p-MYPT1 (Thr853) levels were elevated almost 2.2-fold in shRNA-76396 HUVECs and normalized by Y-27632. (I-K) Total MLC2 levels were unaffected by either treatment. Phosphorylated MLC2 (Thr18/Ser19) was markedly elevated and effectively restored by Y-27632, indicating ROCK2-dependent MLC activation. MLCK expression was significantly upregulated, suggesting cooperative regulation of MLC phosphorylation through both ROCK2 and MLCK pathways. Data are presented as mean ± SEM (n = 6 independent experiments). Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc test (*P < 0.05, **P < 0.01, ***P, ****P < 0.001).
Article Snippet: RhoA activity in HUVECs was detected by G-LISA (BK124, Cytoskeleton, US) according to the manufacturer’s instructions, and normalized to total RhoA levels, which were measured using the Total RhoA ELISA kit (BK150, Cytoskeleton) according to the manufacturer’s instructions.
Techniques: Activity Assay, Control, Knockdown, Activation Assay, shRNA, Western Blot, Expressing, Phospho-proteomics, Quantitative Proteomics
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