Skin samples were obtained from normal donors and from patients with a variety of skin diseases including psoriasis, atopic dermatitis, actinic keratosis, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). All specimens were obtained after informed consent was obtained, in accordance with the ethics committee approval process of Chungnam National University, College of Medicine, Daejeon, Korea.

Normal human skin samples were briefly sterilized in 70% ethanol, minced, and then treated with dispase overnight at 4℃. The epidermis was separated and placed in a solution containing 0.05% trypsin and 0.025% EDTA at 37℃ for 15 min. After vigorous pipetting, the cells were pelleted and resuspended in serum-free keratinocyte growth medium (KGM) supplemented with bovine pituitary extract, recombinant human epidermal growth factor, insulin, and hydrocortisone (Clonetics, Walkersville, MD, USA). The cells were added to 100 mm collagen-coated dishes, incubated at 37℃ in 5% CO₂. At 70~80% confluence, the cells were passaged.

Total RNA was isolated using the easy-BLUE™ Total RNA Isolation system kit (INTRON) following the manufacture's protocols. The quantity and quality of RNA were assessed by spectrometer and agarose gel electrophoresis. Two µg of total RNAs were reverse transcribed with M-MLV reverse transcriptase (Promega, Madison, WI, USA). Aliquots of RT mixture were subjected to PCR cycles with specific primer pairs derived from the corresponding GenBank sequences as follows: 94℃ for 30 s, 57℃ for 30 s, and 72℃ for 1 min for 30 cycles. Primers amplifying a control fragment from cyclophilin were included in each reaction.

Semi-confluent cells growing in serum-free growth medium were suspended in trypsin/EDTA and centrifuged. The cell pellet was washed in ice-cold PBS, suspended in PRO-PREP™ protein extraction solution (iNtRON, Korea), and boiled briefly. The supernatant was collected and the protein concentration was determined with the BioRad assay (BioRad, Hercules, CA, USA). Typically, 20 or 30 µg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to Pall corporation nitrocellulose membranes (Life sciences, FL, USA), and blocked by PBST with 5% non-fat milk. Protein was detected with peptide polyclonal anti-Nkx2.5 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anti-species horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and visual detection was performed using the enhanced chemoluminescence method (iNtRON, Korea).

Paraffin sections of normal and affected human skin were de-waxed and re-hydrated, washed three times with phosphate-buffered saline, and treated with proteinase K (DAKO ready to use kit) for 5 min at 37℃, followed by the DAKO LSAV 2 System peroxidase kit. Briefly, sections were washed with phosphate-buffered saline 0.1% Tween 20 and treated with H₂O₂ for 10 min at room temperature (RT), blocked in phosphate-buffered saline with 0.1% Tween 20 and 1% bovine serum albumin for 20 min at RT, and washed three times with phosphate-buffered saline with 0.1% Tween 20, followed by reaction with anti-Nkx2.5 antibodies (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4℃. After washing and color detection, the sections were counter stained with hematoxylin and mounted. Immunostaining was visualized and photographed under the microscope (Leica DMR). Sections without primary antibody were used as negative controls.

We used primary cultured human epidermal keratinocytes as a model system for investigating calcium-induced differentiation of keratinocytes. In the cultured keratinocytes, total RNA was isolated at four different times (1, 3, 7 and 14 days) after treatment with 1.2 mM calcium. We analyzed the mRNA level of Nkx2.5 during keratinocyte differentiation using RT-PCR. Involucrin was used as a terminal differentiation marker. Cyclophilin was used to compare the total amounts as an internal control (Fig. 1). Nkx2.5 mRNA was detected at 7 and 14 days after treatment with calcium of the NHK cultured in vitro. Therefore, Nkx2.5 appeared to increase in the upper layer of epidermis. However, it was expressed earlier than involucrin, a well known marker of early differentiation, suggesting that Nkx2.5 might be associated with early differentiation in the epidermis.

To investigate Nkx2.5 expression at the protein level, we performed immune blot analysis. Western blot analysis showed that the Nkx2.5 protein began to be expressed 1-day after calcium treatment and reached maximum levels at 3-days; then, its expression was slightly decreased but maintained until 14 days (Fig. 2). Involucrin used as a control marker for differentiation was also increased with calcium treatment. Actin was used as a loading control. These results indicated that this protein was involved in the early events of keratinocyte differentiation and in maintaining their differentiation before death to become the cornified layer.

To verify expression of Nkx2.5 protein in the epidermis, we performed immunohistochemical staining in skin tissues including normal skin, inflammatory skin diseases and malignant tumors (Fig. 3). Nkx2.5 expression was generally observed in the tissues with various skin diseases as well as normal skin. In normal tissue, the nuclei were shown to be stained entirely in almost all layers of the epidermal keratinocytes except the basal layer. For the inflammatory diseases of the skin, two representative diseases including psoriasis and atopic dermatitis showed opposite patterns of Nkx2.5 expression in the epidermal keratinocytes. In psoriasis, not only the nuclei but also the cytoplasm was stained strongly in the epidermis, and these results were more significant in the upper layer. However, for atopic dermatitis, there was almost no staining in the epidermal keratinocytes except for a mild staining in the cytoplasm of keratinocytes in the focal spinous layer. For skin tumors, BCC, not only the nuclei but also the cytoplasm were mildly stained in the tumor components of the dermis compared to the staining of the normal epidermis. Although the cytoplasm stained mildly in the focal spinous layer of actinic keratosis, the nuclei and cytoplasm stained strongly in the tumor components of the dermis in SCC. The results of the immunohistochemical staining in normal skin were similar to the RT-PCR and Western blot results from cultured keratinocytes and provided more clues about the role of Nkx2.5 in keratinocyte differentiation.