Differential expression of CRABP-II in fibroblasts derived from dermis and subcutaneous fat

https://doi.org/10.1016/j.bbrc.2004.01.069Get rights and content

Abstract

We have shown previously that fibroblasts derived from fat or dermal tissue differ in their functional properties, such as proliferation rate and contractile properties. To study these differences further, two-dimensional electrophoresis (2D PAGE) was performed on proteins isolated from cultured subcutaneous fat and dermal fibroblasts. The 2D gels were screened for proteins that were differentially expressed in all donors (n=5). Five protein spots were subjected to further analysis by mass spectrometry. Two proteins could be identified: brain acid soluble protein 1 (BASP1) and cellular retinoic acid binding protein-II (CRABP-II). CRABP-II is of interest in terms of re-epithelialisation and was clearly expressed in dermal fibroblasts but not in fat fibroblasts. Real time PCR was performed to confirm the 2D data on CRABP-II. The CRABP-II mRNA level was significantly increased in dermal tissue and cultured dermal fibroblasts compared to fat tissue and cultured fat-derived fibroblasts, respectively. The mode of action of CRABP-II in skin is to mediate retinoic acid activity. Retinoic acid is known to inhibit migration and to stimulate differentiation of keratinocytes. The expression of CRABP-II by dermal fibroblasts implicates a role for these fibroblasts in wound re-epithelialisation, in contrast to subcutaneous fat-derived fibroblasts.

Section snippets

Materials and methods

Tissue handling and cell culture. Dermal and subcutaneous fat tissues were obtained from healthy donors during abdominal dermolipectomy. A 0.3 mm split skin biopsy was taken using a dermatome (Aesculap AG & Co. KG, Tuttlingen, Germany). Pieces of subcutaneous fat were harvested using a scalpel. Large blood vessels, glandular tissue, and fascia were omitted. Part of the tissue was frozen in liquid nitrogen for RNA isolation. Dermis and fat were treated for cell isolation as described earlier [6].

Identification of differentially expressed proteins by 2D PAGE

The 2D-gel electrophoresis approach has been chosen to obtain information on the overall protein spectrum of cultured dermal and fat fibroblasts. The protein samples isolated from these cells were run on parallel 2D-gels and visualised using silver staining. Low inter-individual differences in profiles of proteins extracted from cultured cells derived from five different skin donors have been observed. Different spots were found to vary in intensity between images of dermal and fat fibroblast

Discussion

Wound healing in skin comprises a cascade of events during which the neo-dermis is formed and the wound surface is re-epithelialised. During re-epithelialisation keratinocytes migrate, proliferate, and differentiate on the formed neo-dermis. These processes are thought to be regulated amongst others by retinoids (e.g., retinoic acid and retinol). This specific group of vitamin A metabolites is reported to be vital for vision, (embryonic) growth, and reproduction [10]. They are essential in

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