TLNRD1-CCM2 interaction maintains endothelial actin networks through a KLF2/4-independent mechanism.

内容摘要

Actin cytoskeleton dysregulation contributes to vascular anomalies such as cerebral cavernous malformation (CCM). Talin rod domain containing-1 (TLNRD1) has been reported to interact with cerebral cavernous malformations 2 protein (CCM2), yet the downstream signaling remains debated, as previous studies have described opposite directions of Krueppel-like factor 2/4 (KLF2/4) changes after TLNRD1 depletion. Here, we combined biochemical analyses, structural modeling, transcriptomics, and single-cell network perturbation to examine the TLNRD1-CCM2 axis in endothelial cells. Coimmunoprecipitation and mass spectrometry confirmed the association between TLNRD1 and the CCM complex. Furthermore, protein docking predicted a stable TLNRD1-CCM2 interface (ΔG ≈ -50.36 kcal/mol) supported by prominent hydrogen bonds. Bulk RNA sequencing following TLNRD1 knockdown identified 677 differentially expressed genes, which were heavily enriched for actin cytoskeleton organization, with limited support for activation of the canonical MEKK3-KLF2/4 program. To assess KLF2/4 more directly, we analyzed human CCM single-cell RNA sequencing using scTenifoldKnk alongside complementary in vitro perturbations. Across these orthogonal analyses, KLF2 and KLF4 showed little to no consistent transcriptional alterations. Instead, TLNRD1 perturbation prominently altered endothelial F-actin stress fiber formation. Together, these data support a model in which TLNRD1 preferentially modulates endothelial cytoskeletal remodeling largely independent of overt KLF2/4 transcriptional shifts, helping to contextualize previous discrepancies and refining our understanding of its role in vascular biology. Current management of cerebral cavernous malformations (CCMs) remains limited by an incomplete understanding of the molecular basis of endothelial instability. This study identifies TLNRD1 as a CCM2-associated regulator of endothelial actin organization and cytoskeletal remodeling, while showing limited support for consistent KLF2/4 transcriptional changes. For clinicians, these findings add mechanistic context to a disease with few medical options and may help explain earlier conflicting experimental observations. Although direct clinical application is not immediate, the TLNRD1-CCM2 axis warrants further study as a biologically relevant pathway that may inform future translational research on cerebrovascular lesion progression.