title: Autophagy in human keratinocytes: an early step of the differentiation?
abstract: Studies have established that autophagy constitutes an efficient process to recycle cellular components and certain proteins. The phenomenon was demonstrated primarily in response to nutrient starvation, and there are increasing evidences that it is implied in differentiation. Keratinocyte differentiation was going along an activation of lysosomal enzymes and organelle clearance, and terminal steps are sometimes described as a specialized form of cell death leading to corneocytes. We examined whether initiation of the process in human keratinocyte HaCaT involves autophagy. The KSFM™ culture medium was substituted by M199, which contains a low glucose concentration but a high calcium level (known to induce differentiation). Metabolic stress reduced enhanced cell number in G(1) phase, without apoptotic features (ΔΨmt and membrane integrity are unchanged). Morphological changes were associated with a lower integrin ß1 expression and modifications of protein levels involved in keratinocyte differentiation (involucrin, keratin K10 and ΔNp63α). Whereas autophagic signalling was supported by SIRT1 and pAMPK (T172) increase according to time kinetic, which led to the disappearance of mTOR phosphorylated on S2448 residue. The significant Bcl-X(L) level reduction with stress promoted autophagy, by the release of Beclin-1, whereas ATG5-ATG12 and LC3-II that are involved in autophagosome formation were enhanced significantly. Then, the level of lysosomal protein cathepsin B rose to execute autophagy. Kinetic studies established that autophagy would constitute an early signalling process required for keratinocyte commitment in differentiation pathway.
authors.affiliation: EA 3842 Homeostasie Cellulaire & Pathologies - Faculte de Medecine, Rue du Dr Marcland, Limoges Cedex, France R&D Department, SILAB, BP 213, Brive Cedex, France.
authors.forename: Elodie
authors.initials: E
authors.lastname: Aymard
authors.forename: Vincent
authors.initials: V
authors.lastname: Barruche
authors.forename: Thomas
authors.initials: T
authors.lastname: Naves
authors.forename: Sylvie
authors.initials: S
authors.lastname: Bordes
authors.forename: Brigitte
authors.initials: B
authors.lastname: Closs
authors.forename: Mireille
authors.initials: M
authors.lastname: Verdier
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Ratinaud
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000263:Adenylate Kinase / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D000071192:Autophagy-Related Protein 12; D000071187:Autophagy-Related Protein 5; D000071190:Autophagy-Related Protein 8 Family; D000071186:Beclin-1; D002118:Calcium / Q000494:pharmacology; D002401:Cathepsin B / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002461:Cell Line, Transformed; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism; D053550:Keratin-10 / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011498:Protein Precursors / Q000378:metabolism; D056564:Sirtuin 1 / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D013312:Stress, Physiological / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism
chemical_list: C466624:ATG12 protein, human; C502778:ATG5 protein, human; D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D000071192:Autophagy-Related Protein 12; D000071187:Autophagy-Related Protein 5; D000071190:Autophagy-Related Protein 8 Family; C494811:BCL2L1 protein, human; C491997:BECN1 protein, human; D000071186:Beclin-1; D016895:Culture Media, Serum-Free; C498698:GABARAPL2 protein, human; D019012:Integrin beta1; C507493:KRT10 protein, human; D008565:Membrane Proteins; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D011498:Protein Precursors; D025841:Small Ubiquitin-Related Modifier Proteins; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D051020:bcl-X Protein; D053550:Keratin-10; C025946:3-methyladenine; C032253:involucrin; D000263:Adenylate Kinase; C533912:CTSB protein, human; D002401:Cathepsin B; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D000225:Adenine; D002118:Calcium