Cyclin E/CDK2 deficiency links cell cycle control to intestinal barrier integrity and tight junction regulation

Abstract

Background & Aims: The intestinal epithelium renews continuously through cell cycle-dependent turnover, yet it remains unknown whether core cell cycle regulators directly control intestinal barrier function. We investigated how loss of the Cyclin E1/Cyclin E2/CDK2 complex affects intestinal homeostasis. Based on the important role of this complex in compensatory liver regeneration, we hypothesized that its combined loss would impair epithelial renewal and barrier integrity. Methods: We generated Ccne2-/- mice and triple knockout (TKO) mice with intestinal epithelial cell (IEC)-specific deletion of Ccne1 and Cdk2 on a constitutive Ccne2-null background and characterized intestinal architecture, proliferation, and barrier function using histology, immunoblotting, qPCR, and FITC-dextran permeability assays. Mechanistic findings were validated in CRISPR/Cas9-generated Ccne2-deficient and CDK1-overexpressing MC38 colon carcinoma cells using transcriptomics and kinomics approaches. Results: Contrary to our hypothesis, TKO maintained and Ccne2-/- mice even enhanced cell cycle activity selectively in the colon, along with enhanced Cdk1 expression, had significantly lower serum FITC-dextran levels after oral administration, and increased Tjp1/ZO-1 expression, consistent with reduced intestinal permeability. Ccne2-deficient MC38 cells recapitulated the increase in ZO-1 and exhibited transcriptional enrichment of gene sets associated with tight junctions and cell cycle. CDK1 overexpression in MC38 cells increased ZO-1 protein abundance and was associated with activation of an AMPK-related kinase network. In TKO and Ccne2-/- colonic epithelium, CDK1, phosphorylated AMPK and ZO-1 showed partial colocalization at lateral epithelial cell-cell contacts. Conclusions: Loss of Cyclin E2, either alone or in combination with epithelial Cyclin E1/CDK2 deficiency, is associated with increased CDK1 expression and strengthens the colonic barrier integrity in mice. Findings from MC38 cells suggest that transcriptional reprogramming and enhanced CDK1 signaling in the context of Cyclin E2 deficiency may contribute to tight junction reinforcement and improved barrier function.