Transcriptional and post-translational regulation of beta sub(1) integrin expression during keratinocyte terminal differentiation

During suspension-induced terminal differentiation of human epidermal keratinocytes, the alpha sub(5) beta sub(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 sub(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 sub(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 sub(5) beta sub(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 sub(5) and beta sub(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 sub(5) and beta sub(1) mRNAs were similar in adherent and suspended cells. Thus, loss of alpha sub(5) beta sub(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.