Molecular Cloning and Expression of Human Keratinocyte Proline-Rich Protein (hKPRP), an Epidermal Marker Isolated from Calcium-Induced Differentiating.

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Molecular Cloning and Expression of Human Keratinocyte Proline-Rich Protein (hKPRP), an Epidermal Marker Isolated from Calcium-Induced Differentiating Keratinocytes Woong-Hee Lee, Sunhyae Jang, Jung-Suk Lee, Young Lee, Eun-Young Seo, Kwan-Hee You, Seung-Chul Lee, Kwang-Il Nam, Jin-Man Kim, Sun-Ho Kee, Jun-Mo Yang, Young- Joon Seo, Jang-Kyu Park, Chang Deok Kim, Jeung-Hoon Lee Journal of Investigative Dermatology Volume 125, Issue 5, Pages 995-1000 (November 2005) DOI: 10.1111/j.0022-202X.2005.23887.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Map of the 2 Mb epidermal differentiation complex (EDC). The q21 region of chromosome 1 is expanded to illustrate the EDC. The gene for hKPRP is located between SPRL6A and IVL genes, indicated by arrow. S100A10, S100 calcium binding protein A10; THH, trichohyalin; FLG, filaggrin; NICE-1, NICE-1 protein; LEP17, late envelope protein 17; SPRL6A, small proline rich-like 6A; hKPRP, human keratinocyte proline-rich protein; IVL, involucrin; SPRR1B, small proline-rich protein 1B; LOR, loricrin; S100A9, S100 calcium binding protein A9; S100A8, S100 calcium binding protein A8; S100A6, S100 calcium binding protein A6; S100A4, S100 calcium binding protein A4; S100A1, S100 calcium binding protein A1. Journal of Investigative Dermatology 2005 125, 995-1000DOI: (10.1111/j.0022-202X.2005.23887.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Sequence alignment between human keratinocytes proline-rich protein (hKPRP) and rat keratinocytes proline-rich protein (rKPRP). Similarity of amino acid sequence between hKPRP and rKPRP is evaluated. The alignment shows 64% identity between these two proteins. Journal of Investigative Dermatology 2005 125, 995-1000DOI: (10.1111/j.0022-202X.2005.23887.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Detection of human keratinocytes proline-rich protein (hKPRP) mRNA. (A) Lower panel showed the result of quantitative real-time PCR analysis. Two micrograms of total RNA were reverse transcribed with M-MLV reverse transcriptase and used for PCR amplification. The data of real-time PCR was corrected according to the quantity of β-actin and expressed as a fold induction. Data are represented as mean±SE of triplicated experiments (p<0.01). (B) In situ hybridization. Fourteen micrometer sections of human normal skin and psoriatic lesion were hybridized with sense and antisense hKPRP riboprobes. The hKPRP-specific hybridization signals are strongly detected in the granular layer of the epidermis of both the normal and psoriatic skin. Arrows indicate the cells that express hKPRP. Pictures were taken under the magnification of × 40 (scale bar: 100μm). (C) Tissue distribution and developmental expression of mouse KPRP. Using mouse KPRP probe that was made by RT-PCR, northern hybridization was performed. About 3.0 kb band was seen in stomach and skin tissues (upper panel), and the expression of mouse KPRP begins at E 17.5 d (lower panel). Ethidium bromide-stained gel was photographed as a loading control. Journal of Investigative Dermatology 2005 125, 995-1000DOI: (10.1111/j.0022-202X.2005.23887.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Detection of human keratinocytes proline-rich protein (hKPRP) protein. (A) Western blot analysis. Normal human epidermal keratinocytes were cultured and differentiated by addition of 1.2 mM calcium (Ca) for the indicated time points. Proteins (20 μg per lane) were separated on 10% polyacrylamide gels. Duplicate blots were probed with anti-involucrin (Inv) antibody as a positive control, and anti-hKPRP antibody. (B) HaCaT keratinocytes were induced to differentiate by treatment with 0.3 mM calcium and ionophore A23187 (1 μM) for the indicated time points. Western blot was performed as in (A). (C) Immunohistochemical staining. Paraffin-embedded tissue sections of skin specimens were stained with anti-hKPRP antibody. a, normal skin; b, psoriatic lesion; c, epidermal cyst lesion; d, lichen planus lesion. Pictures were taken under the magnification of × 40 (scale bar: 100μm). (D) The expression of hKPRP during human fetal development. Paraffin-embedded tissue sections of human fetal skin were stained with anti-hKPRP antibody. (a) 14 wk estimated gestational age (EGA), (b) 16 wk, (c) 17 wk, (d) 18 wk, (e) 20 wk, (f) 24 wk, (g) 27 wk, and (h) 32 wk. Pictures were taken under the magnification of × 200 (scale bar: 10μm). Journal of Investigative Dermatology 2005 125, 995-1000DOI: (10.1111/j.0022-202X.2005.23887.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions