Mitochondrial MAP4 regulates keratinocyte autophagy, apoptosis, and migration upon wounding through BCL2/SIRT5 functional coupling under hypoxia: an experimental in vitro study

Microtubule-associated protein 4 (MAP4) phosphorylates and translocates to the outer mitochondrial membrane, thus promoting keratinocyte migration under hypoxia; however, the underlying mechanism remains unclear. Given that a BCL2 homology 3-like (BH3-like) domain has been previously identified in MAP4, the mitochondrial outer membrane protein BCL2 is a potential molecule for elucidating the underlying pathway of MAP4 mitochondrial translocation. HaCaT keratinocytes were subjected to hypoxia and transfected with Ad-MAP4(Glu), Ad-MAP4(Ala), or si-SIRT5 to modulate MAP4 phosphorylation and SIRT5 expression, respectively, with or without the BCL2 inhibitor ABT-199. Keratinocyte migration, autophagy, and apoptosis were assessed using scratch wound healing assays, immunoblotting, transmission electron microscopy, and transferase dUTP nick end labeling staining. The BCL2–SIRT5 interaction was evaluated using molecular docking, yeast two-hybrid screening, and confocal immunofluorescence co-localization analysis. ABT-199, a BCL2-selective inhibitor, decreased the mitochondrial content of MAP4 under hypoxia, resulting in the inhibition of keratinocyte migration, autophagy, and apoptosis. These findings suggest that phosphorylated MAP4 translocates to the mitochondria mainly via BCL2. Sirtuin 5 (SIRT5), a mitochondrial matrix protein, is a potential effector molecule because of its importance in maintaining mitochondrial homeostasis and regulating autophagy and apoptosis. Unexpectedly, BCL2 was found to interact with SIRT5 and form a functional coupling complex on mitochondria. SIRT5 knockdown greatly influenced phosphorylated MAP4-induced keratinocyte autophagy, apoptosis, and migration. MAP4 triggers the formation of the BCL2/SIRT5 functional coupling complex after its mitochondrial translocation via BCL2, thereby inducing keratinocyte autophagy, apoptosis, and migration under hypoxia. The mechanistic pathways uncovered in this study provide strategic insights into wound treatment.