Colonocyte keratins stabilize mitochondria and contribute to mitochondrial energy metabolism

Abstract

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 of 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 in vivo 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 with 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 in vitro in K8^-/- Caco-2 cells and in vivo in K8^-/- mouse colonocytes, and reexpression 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.NEW & NOTEWORTHY In this study, we show that colonocyte intermediate filament protein keratin 8 is important for stabilizing mitochondria and maintaining mitochondrial energy metabolism, as keratin 8-deficient colonocytes display smaller, rounder, and more motile mitochondria, diminished mitochondrial respiration, and altered Ca²⁺ dynamics. Changes in fusion-regulating proteins are rescued with reexpression of keratin 8. These alterations in colonocyte mitochondrial homeostasis contribute to keratin 8-associated colitis pathophysiology.

Keywords: colon; energy metabolism; inflammatory bowel diseases; keratins; mitochondria.