BAG-1L promotes keratinocyte differentiation in organotypic culture models and changes in relative BAG-1 isoform abundance may lead to defective stratification |
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Authors: | Hinitt C A M Benn T M Threadgold S Wood J Williams A C Hague A |
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Institution: | aUniversity of Bristol, School of Oral and Dental Sciences, Lower Maudlin Street, Bristol, BS1 2LY, UK;bUniversity of Bristol, School of Cellular and Molecular Medicine, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK |
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Abstract: | In keratinocytes the human Bag-1 gene produces three different protein isoforms from a single messenger RNA, BAG-1L, BAG-1M and BAG-1S. In this study we questioned whether BAG-1L or the shorter isoforms would promote keratinocyte differentiation in organotypic cultures of HaCaT. HaCaT parental and vector cells showed stratification, but terminal differentiation was not complete. Cultures overexpressing BAG-1L isoform-specifically were of increased thickness, demonstrated pronounced expression of basal cytokeratin 5 and β1-integrin, suprabasal involucrin, cytokeratin 1 and plasma membrane-localised filaggrin, and a marked keratinized layer of cells at the surface. We were unable to overexpress BAG-1S and BAG-1M isoform-specifically. Overexpression of BAG-1M gave rise to organotypic cultures intermediate in differentiation to controls and those overexpressing BAG-1L. Cells overexpressing BAG-1S also exhibited elevated endogenous BAG-1. These produced slow growing cultures with high levels of basal cytokeratin 5, but little involucrin or cytokeratin 1. Suprabasal β1-integrin and Ki67 positive cells indicated defective stratification. The results suggest that BAG-1L potentiates epidermal differentiation, but disruption in the relative balance of isoforms towards overexpression of BAG-1S can lead to defective tissue patterning. Hence, a delicate balance of BAG-1 isoforms may be required to regulate normal epidermal stratification and differentiation, with important implications for aberrant differentiation in cancer. |
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Keywords: | Abbreviations: BAG-1 Bcl-2-associated Athanogene 1 CK1 Cytokeratin 1 CK5 Cytokeratin 5 DMEM Dulbecco's Modified Eagle's Medium FBS foetal bovine serum HGF hepatocyte growth factor PBS Phosphate buffered saline |
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