Two-dimensional electrophoretic profiling of atopic dermatitis in primary cultured keratinocytes from patients.

Recently, we reported altered protein expression in primary cultured fibroblasts from atopic dermatitis (AD) patients. As a sequential study, we conducted proteomic analysis of primary keratinocytes derived from AD patients to further identify AD-related proteins. Three pH ranges, 4-7, 6-9, and 7-11, were used to profile the altered protein expression in AD. We obtained 46 candidate spots from the 2-D gel image analysis: 18 proteins were up-regulated and 27 proteins were down-regulated. Among the several important candidate proteins, NCC27 showed the same profile of a defect in PTM in both AD-derived keratinocytes and fibroblasts. On the basis of current and previous reports, real-time PCR was performed on select candidate genes to compare RNA and protein expression levels in AD-derived keratinocytes and fibroblasts. Our results provide new clues to aid in understanding the mechanism of atopic alterations in keratinocytes and suggest new AD-associated proteins that are important in AD pathogenesis.

Identification of parathyroid hormone-regulated proteins in mouse bone marrow cells by proteomics.

The ability of parathyroid hormone (PTH) to enhance bone formation has recently been exploited in the treatment of osteoporosis. Several studies have suggested that the activation of bone marrow stromal cells could be preceded to show the anabolic effect of PTH on bone formation, but little is known of PTH-regulated proteins in bone marrow cells. Therefore, protein profiling in the intermittent PTH-treated bone marrow cells was evaluated using proteomics. Daily treatment for 5 days consisting of subcutaneous injection of either 150 microg/kg per day of mouse PTH (1-84) or vehicle (0.9% normal saline) was performed on the ICR mouse. At the end of the treatment period, bone marrow cells were separated and used in proteomics. The expression levels of seven proteins including vimentin were decreased, but those of four proteins including calreticulin and thioredoxin domain containing 7 protein (Txnde7) were increased. Among these, the decrease of vimentin and the increase of both calreticulin Txnde7 in mRNA levels were confirmed by semi-quantitative RT-PCR. In PTH-treated mouse MC3T3-E1 osteoblast cells, mRNA expression levels were not totally consistent with the results observed in proteomics. In conclusion, the differentially expressed proteins in bone marrow cells depending on PTH could be highly linked to the differentiation of osteoprogenitor cells in the bone marrow into preosteoblast cells.

Towards a proteomic analysis of atopic dermatitis: a two-dimensional-polyacrylamide gel electrophoresis/mass spectrometric analysis of cultured patient-derived fibroblasts.

Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease typically characterized by a distribution of eczematous skin lesions with lichenification, pruritic excoriations, and dry skin with wide varieties of pathophysiologic aspects. Recently, AD was divided into extrinsic and intrinsic forms according to the presence or absence of an allergy. We investigated alterations in protein expression in primary cultured AD cells from the patient biopsy samples by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight. In the primary cultured fibroblasts, we obtained 31 candidate proteins from the two-dimensional gel image analysis in which 18 proteins were up-regulated, eight proteins were down-regulated and five proteins were post-translationally modified. From these 2-DE results, we found several candidate genes matched proteomic expression patterns by semiquantitative reverse transcription PCR. Since the exact mechanism of atopic alterations in fibroblasts remains unknown, our results may provide new clues to aid in understanding AD.