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Transcriptional and post-translational regulation of beta 1 integrin expression during keratinocyte terminal
1Keratinocyte Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom.
Abstract:
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.
Insights
Terminal differentiation of keratinocytes involves two stages of alpha 5 beta 1 integrin down-regulation. This process includes inhibited glycosylation, blocked cell surface transport, and reduced gene transcription.
Area of Science:
- Cell Biology
- Molecular Biology
- Dermatology
Background:
- Integrins are crucial cell surface receptors mediating cell adhesion and signaling.
- Alpha 5 beta 1 integrin plays a role in keratinocyte differentiation and fibronectin binding.
- Suspension culture is a model to study terminal differentiation of human epidermal keratinocytes.
Purpose of the Study:
- To investigate the regulatory mechanisms of alpha 5 beta 1 integrin down-regulation during keratinocyte terminal differentiation.
- To elucidate the specific stages and molecular events involved in integrin loss.
Main Methods:
- Pulse-chase experiments to track protein maturation and degradation.
- Endoglycosidase H digestion to assess N-linked glycosylation.
- Treatment with 1-deoxymannojirimycin to inhibit glycosylation.
- Nuclear run-on assays to measure gene transcription.
- Actinomycin D treatment to determine mRNA half-life.
Main Results:
- Suspension-induced differentiation prevented beta 1 integrin subunit maturation at the Golgi (N-linked glycosylation stage).
- Immature integrin subunits reached the cell surface in glycosylation-inhibited adherent cells but not in differentiating suspended cells.
- Transcription of alpha 5 and beta 1 genes was suppressed during differentiation.
- mRNA half-lives for alpha 5 and beta 1 were similar in adherent and suspended cells.
Conclusions:
- Terminal differentiation involves a block in both integrin glycosylation and cell surface transport.
- Down-regulation of alpha 5 beta 1 integrin function involves modulation of pre-existing receptors.
- Loss of cell surface alpha 5 beta 1 integrin results from inhibited gene transcription and impaired intracellular processing of newly synthesized subunits.