Colonocyte keratins stabilize mitochondria and contribute to mitochondrial energy metabolism

Abstract

<p>Keratin intermediate filaments form dynamic filamentous networks, which provide mechanical stability, scaffolding and protection against stress to epithelial cells. Keratins and other intermediate filaments have been increasingly linked to the regulation of mitochondrial function and homeostasis in different tissues and cell types. While deletion of keratin 8 (K8^‒/‒) in mouse colon elicits a colitis-like phenotype, epithelial hyperproliferation and blunted mitochondrial ketogenesis, the role for K8 in colonocyte mitochondrial function and energy metabolism is unknown. We used two K8 knockout mouse models and CRISPR/Cas9 K8^‒/‒ colorectal adenocarcinoma Caco-2 cells to answer this question. The results show that K8^‒/‒ colonocyte mitochondria <em>in vivo</em> are smaller and rounder, and that mitochondrial motility is increased in K8^‒/‒ Caco-2 cells. Furthermore, K8^-/- Caco-2 cells displayed diminished mitochondrial respiration and decreased mitochondrial membrane potential compared to controls, whereas glycolysis was not affected. The levels of mitochondrial respiratory chain complex proteins and mitochondrial regulatory proteins mitofusin-2 and prohibitin were decreased both <em>in vitro</em> in K8^‒/‒ Caco-2 cells and <em>in vivo</em> in K8^‒/‒ mouse colonocytes, and re-expression of K8 into K8^‒/‒ Caco-2 cells normalizes the mitofusin-2 levels. Mitochondrial Ca²⁺ is an important regulator of mitochondrial energy metabolism and homeostasis, and Caco-2 cells lacking K8 displayed decreased levels and altered dynamics of mitochondrial matrix and cytoplasmic Ca²⁺. In summary, these novel findings attribute an important role for colonocyte K8 in stabilizing mitochondrial shape and movement and maintaining mitochondrial respiration and Ca²⁺ signaling. Further, how these metabolically compromised colonocytes are capable of hyperproliferating presents an intriguing question for future studies.<br></p>