Transcriptional and post-translational regulation of beta 1 integrin expression during keratinocyte terminal differentiation.
During suspension-induced terminal differentiation of human epidermal keratinocytes, the alpha 5 beta 1 integrin is down-regulated in two stages: first, the ability of the receptor to bind fibronectin is reduced; later, the receptor is lost from the cell surface, and the level of the subunit mRNAs declines. We have begun to examine the mechanisms that regulate these events. Pulse-chase experiments showed that when keratinocytes were placed in suspension to induce terminal differentiation maturation of the beta 1 subunit and its associated alpha subunits was prevented. The inhibition of maturation was at the stage of N-linked glycosylation in the Golgi, because the immature integrin subunits were sensitive to endoglycosidase H digestion and the inhibition could be mimicked in adherent cells by treatment with 1-deoxymannojirimycin. In 1-deoxymannojirimycin-treated adherent keratinocytes, immature integrin subunits reached the cell surface; however, in keratinocytes induced to differentiate in suspension, no beta 1-integrin precursors were detected on the cell surface. Thus commitment to terminal differentiation results in a block both in integrin glycosylation and transport to the cell surface; down-regulation of receptor function must therefore involve modulation of pre-existing receptor on the cell surface. Although fibronectin or rabbit antiserum to alpha 5 beta 1 can inhibit suspension-induced terminal differentiation they did not overcome the inhibition of glycosylation. Nuclear run-on assays showed that transcription of the alpha 5 and beta 1 genes was switched off during suspension-induced terminal differentiation, and treatment of adherent keratinocytes with actinomycin D suggested that the half-lives of the alpha 5 and beta 1 mRNAs were similar in adherent and suspended cells. Thus, loss of alpha 5 beta 1 from the cell surface reflects both inhibition of transcription of the subunit genes and inhibition of maturation and intracellular transport of newly synthesized subunits.
Hotchin
N A
NA
Keratinocyte Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom.
Watt
F M
FM
D017485:1-Deoxynojirimycin; D000906:Antibodies / Q000276:immunology; D015152:Blotting, Northern; D002454:Cell Differentiation; D002478:Cells, Cultured; D015536:Down-Regulation; D016521:Electrophoresis, Gel, Pulsed-Field; D005353:Fibronectins / Q000378:metabolism / Q000494:pharmacology; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D012313:RNA / Q000378:metabolism; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic / Q000276:immunology; D014158:Transcription, Genetic
D000906:Antibodies; D005353:Fibronectins; D016023:Integrins; D017469:Receptors, Fibronectin; D011971:Receptors, Immunologic; D017485:1-Deoxynojirimycin; D012313:RNA; D005944:Glucosamine