Ann Dermatol Vol. 21, No. 4, 2009 ORIGINAL ARTICLE The Role of Nkx2.5 in Keratinocyte Differentiation Chul Hwang, M.D., Sunhyae Jang, M.S., Dae Kyoung Choi, M.S., Sujong Kim, Ph.D.1, Joong Hwa Lee, M.D., Ph.D.2, Young Lee, M.D., Ph.D., Chang Deok Kim, Ph.D., Jeung Hoon Lee, M.D., Ph.D. Department of Dermatology, College of Medicine, Chungnam National University, Daejeon, 1Skin Research Institute, Amorepacific R&D Center, Yongin, 2Lee Joong Hwa Urologic Clinic, Daejeon, Korea Background: Nkx2.5 is a homeodomain-containing nuclear -Keywords- transcription protein that has been associated with acute Keratinocyte differentiation, Nkx2.5, Transcription factor T-lymphoblastic leukemia. In addition, Nkx2.5 has an essential role in cardiomyogenesis. However, the expression of Nkx2.5 in the skin has not been investigated. Objective: INTRODUCTION In an attempt to screen the differentially regulated genes involved in keratinocyte differentiation, using a cDNA Keratinocytes provide the rigid stratified structure of the microarray, we identified Nkx2.5 as one of the transcription skin through a highly complicated and tightly regulated factors controlling the expression of proteins associated with process of differentiation1. Many differentiation-related keratinocyte differentiation. Methods: To investigate the genes, including those encoding transglutaminase 1 and 3 expression of Nkx2.5 during keratinocyte differentiation, we (TGase 1 and 3), involucrin, cornifin, loricrin, filaggrin, used a calcium-induced keratinocyte differentiation model. and small proline-rich proteins (SPRs), have been shown Results: RT-PCR and Western blot analysis revealed that the to be expressed in a temporally regulated manner during expression of Nkx2.5, in cultured human epidermal kerati- keratinocyte differentiation2-6. Despite intensive investiga- nocytes, increased with calcium treatment in a time-depend- tion to identify and determine the regulation of numerous ent manner. In normal skin tissue, the expression of Nkx2.5 candidate genes, the precise gene expression profile that was detected in the nuclei of the keratinocytes in all layers of governs the keratinocyte differentiation process remains to the epidermis except the basal layer by immunohistoche- be determined. Recently, several molecular techniques mistry. In addition, the expression of Nkx2.5 was sig- such as cDNA microarray have enabled investigators to nificantly increased in psoriasis and squamous cell carcino- analyze global gene expression profiles. Using this ma, but was barely detected in atopic dermatitis and basal technique, we attempted to identify the genes related to cell carcinoma. Conclusion: These results suggest that keratinocyte differentiation. Nkx2.5 may play a role in the change from proliferation to After we isolated hundreds of genes that may play a role differentiation of keratinocytes and in the pathogenesis of during keratinocyte differentiation, we analyzed the skin disease with aberrant keratinocyte differentiation. (Ann promoter sequences of some of the differentially-regulated Dermatol 21(4) 376∼381, 2009) genes, and identified several candidate transcription factors for gene regulation during calcium-induced keratinocyte Received March 26, 2009, Revised May 22, 2009, Accepted for differentiation. Among them, we focused our attention on publication June 29, 2009 one interesting transcription factor, Nkx2.5, which has *This work was supported by the Korea Research Foundation Grant previously been designated as a cardiac specific homeo- funded by the Korean Government (KRF-2008-314-E00152). This box protein (Csx) belonging to the gene family of Nkx2 of study was also supported by a research fund from the Amore-Pacific 7,8 Corporation, Yongin, Korea. homeodomain-containing nuclear transcription factors . Nkx-2.5 is a homolog of the Tinman protein expressed in Reprint request to: Jeung Hoon Lee, M.D., Department of Dermatology, School of Medicine, Chungnam National University, 33, Munhwaro, Drosophila, and is essential for normal cardiomyogenesis; Daejeon 301-040, Korea. Tel: 82-42-280-7707, Fax: 82-42-280-8459, it is required for cardiac septation in which a single atrium E-mail: [email protected] and ventricle are separated into four chambers. Mutations 376 Ann Dermatol The Role of Nkx2.5 in Keratinocyte Differentiation that disrupt Nkx2.5 can result in atrial-septal defects, control fragment from cyclophilin were included in each embryonic lethality and a variety of congenital heart reaction. abnormalities9-11. Nkx2.5 has also been associated with Western blot analysis acute T-lymphoblastic leukemia12-14. However, the expre- ssion and putative role of Nkx2.5 has not been previously Semi-confluent cells growing in serum-free growth studied in skin keratinocytes. medium were suspended in trypsin/EDTA and centrifuged. In this study, we investigated the expression of Nkx2.5 The cell pellet was washed in ice-cold PBS, suspended in during calcium-induced keratinocyte differentiation under PRO-PREPTM protein extraction solution (iNtRON, Korea), in vitro culture conditions and in vivo with several skin and boiled briefly. The supernatant was collected and the diseases. The results suggest that Nkx2.5 may play a role protein concentration was determined with the BioRad in the change from proliferation to differentiation in assay (BioRad, Hercules, CA, USA). Typically, 20 or 30μg keratinocytes and in the pathogenesis of skin diseases with of protein was separated by sodium dodecyl sulfate-po- aberrant keratinocyte differentiation. lyacrylamide gel electrophoresis (SDS-PAGE), transferred to Pall corporation nitrocellulose membranes (Life MATERIALS AND METHODS sciences, FL, USA), and blocked by PBST with 5% non-fat milk. Protein was detected with peptide polyclonal Patients and tissue specimens anti-Nkx2.5 antibody (Santa Cruz Biotechnology, Santa Skin samples were obtained from normal donors and from Cruz, CA, USA). Anti-species horseradish peroxidase-con- patients with a variety of skin diseases including psoriasis, jugated secondary antibodies were obtained from Santa atopic dermatitis, actinic keratosis, basal cell carcinoma Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (BCC) and squamous cell carcinoma (SCC). All specimens and visual detection was performed using the enhanced were obtained after informed consent was obtained, in chemoluminescence method (iNtRON, Korea) accordance with the ethics committee approval process of Immunohistochemical staining Chungnam National University, College of Medicine, Daejeon, Korea. Paraffin sections of normal and affected human skin were de-waxed and re-hydrated, washed three times with Cell culture phosphate-buffered saline, and treated with proteinase K Normal human skin samples were briefly sterilized in (DAKO ready to use kit) for 5 min at 37oC, followed by 70% ethanol, minced, and then treated with dispase the DAKO LSAV 2 System peroxidase kit. Briefly, sections overnight at 4oC. The epidermis was separated and placed were washed with phosphate-buffered saline 0.1% Tween in a solution containing 0.05% trypsin and 0.025% EDTA 20 and treated with H2O2 for 10 min at room temperature at 37oC for 15 min. After vigorous pipetting, the cells were (RT), blocked in phosphate-buffered saline with 0.1% pelleted and resuspended in serum-free keratinocyte Tween 20 and 1% bovine serum albumin for 20 min at growth medium (KGM) supplemented with bovine pitui- RT, and washed three times with phosphate-buffered tary extract, recombinant human epidermal growth factor, saline with 0.1% Tween 20, followed by reaction with insulin, and hydrocortisone (Clonetics, Walkersville, MD, anti-Nkx2.5 antibodies (1:200 dilution, Santa Cruz Biote- USA). The cells were added to 100 mm collagen-coated chnology, Santa Cruz, CA, USA) overnight at 4oC. After o dishes, incubated at 37 C in 5% CO2. At 70∼80% washing and color detection, the sections were counter confluence, the cells were passaged. stained with hematoxylin and mounted. Immunostaining was visualized and photographed under the microscope Reverse transcription-polymerase chain reaction (RT-PCR) (Leica DMR). Sections without primary antibody were Total RNA was isolated using the easy-BLUETM Total RNA used as negative controls. Isolation system kit (INTRON) following the manufacture’s protocols. The quantity and quality of RNA were assessed RESULTS by spectrometer and agarose gel electrophoresis. Two μg Reverse transcription-polymerase chain reaction (RT- of total RNAs were reverse transcribed with M-MLV PCR) reverse transcriptase (Promega, Madison, WI, USA). Aliquots of RT mixture were subjected to PCR cycles with We used primary cultured human epidermal keratinocytes specific primer pairs derived from the corresponding as a model system for investigating calcium-induced GenBank sequences as follows: 94oC for 30 s, 57oC for 30 differentiation of keratinocytes. In the cultured keratino- s, and 72oC for 1 min for 30 cycles. Primers amplifying a cytes, total RNA was isolated at four different times (1, 3, Vol. 21, No. 4, 2009 377 C Hwang, et al Fig. 1. mRNA level of Nkx2.5 during calcium-induced kerati- nocyte differentiation. Two micrograms of total RNA were reverse transcribed with M-MLV reverse transcriptase and used Fig. 2. Nkx2.5 expression in calcium-induced keratinocyte for PCR amplification. Involucrin used as a control marker for differentiation. The protein from cultured keratinocyte extracts μ differentiation was also increased with calcium treatment. of 20 or 30 g was loaded for SDS-PAGE,