A study of protein synthesis in subpopulations of cultured human epidermal keratinocytes using two‐dimensional gel electrophoresis

Abstract

A study of protein synthesis in subpopulations of cultured human epidermal keratinocytes using two-dimensional gel electrophoresis A procedure for the isolation of protein markers of epidermal differentiation in vitro is described. Human epidermal keratinocytes were cultured and radiolabelled in vitro. Fractionation was performed according to buoyant density (which reflects the degree of terminal differentiation) using Percoll density gradient centrifugation. Subpopulations of keratinocytes were characterised using light and electron microscopy, and proteins fractionated using high resolution two-dimensional gel electrophoresis. Radio-labelled proteins were detected using autoradiography and fluorography. Integral membrane proteins were characterised using Triton X-114 phase shift extraction. Data from this in vitro study were compared to silver stainedgels ofsamplesfrom intact epidermis (in vivo). We report quantitative differences between 14 specific protein moieties expressed in subpopulations of keratinocytes and identify some of these proteins. The differential expression of these protein markers and their possible use in the interpretation of the keratinocyte maturation pathway in cultured cells from patients with skin diseases are discussed.

A study of protein synthesis in cells cultured from involved psoriatic skin

“…A comparison of data for psoriatic ( n = 3) and normal ( n = 5 ) keratinocytes, each sample run at least twice, indicated that protein synthesis was similar. The pattern of keratin separation and previously described markers of keratinocyte differentiation [8,9] were expressed in psoriatic cells to the same extent as in normal samples (Fig. 1).…”

“…Plating efficiency was about 1 Yo of plated basal cells for primary cultures. Nonadherent cells were removed at the first washlfeed at 24 h. Primary cultures of psoriatic keratinocytes were grown on feeder cells as described previously [8]. Flasks were treated with EDTA to remove fibroblasts and fed 2 or 3 times per week until confluence.…”

“…The level of incorporation of radioactivity into the samples was determined by trichloroacetic acid (TCA) precipitation of an aliquot followed by scintillation counting. A keratinocyte suspension was prepared from noncultured surgical specimen of psoriatic skin using the trypsin flotation method [15] and a fraction enriched for basal cells isolated by Percoll density centrifugation as described previously [8]. Cell fractions were monitored by immunocytochemistry with monoclonal antibody HCl specific for keratinocytes juxtaposed to the basement membrane in normal [16] and psoriatic skin (data not shown).…”

“…First-dimensional IEF gels (4%T, 4O/oC) containing SM urea, 0.5% w/v CHAPS, and a mixture of synthetic carrier ampholytes (0.92 O/ o w/v Pharmalyte 3-10,0.92% w/v Servalyt pH 2-11,0.92% w/v Ampholine pH 3.5-10,0.24% w/v Pharmalyte pH 5-7,0.12% w/v Servalyt pH 4-6). Gels (0.5 mm thick, 14 cm interelectrode distance) were cast on GelBond PAG film using a cassette technique as previously described [8]. Gels were run on a horizontal flat-bed IEF apparatus cooled to 15°C and samples (lo6 cpm or 25 pg unlabelled protein) were focused for 15000 Vh.…”

“…Thus, our approach to identifying new markers of epidermal differentiation has been to isolate subpopulations of keratinocytes from skin and analyse these by 2-D PAGE. Putative markers of keratinocyte maturation have been identified using differential adhesion [7] and density gradient centrifugation [8] to isolate basal and suprabasal fractions from the epidermis. On this basis, it has been possible to establish a catalogue of markers for epidermal differentiation [9, 101, and the hope is that this can be used to delineate the biochemical lesion present in various histologically defined skin diseases.…”