Results

1,25(OH)₂D₃ inhibits the proliferation of keratinocytes and increases their differentiation in vitro and in vivo (^{Bikle et al, 2003}). To elucidate the molecular mechanisms underlying vitamin D-mediated differentiation and anti-proliferative activity, a global picture of 1,25(OH)₂D₃-mediated gene expression in proliferating keratinocytes was determined by interrogating the Affymetrix GeneChips containing 12,600 genes with mock-treated and 1,25(OH)₂D₃-treated keratinocytes. KerTr cells were treated for 0, 6, 24, and 48 h with 1,25(OH)₂D₃ (10^-7 M) in three independent experiments. The response to 1,25(OH)₂D₃ was verified by Taqman quantitative PCR (Q-PCR) analysis of 24-hydroxylase gene expression. For microarray screening, RNA from each independent experiment at a given time point was interrogated with duplicate gene chips. Therefore, six gene chips were used for expression profiling at each time point. Differentially expressed genes were identified based on a combination of fold changes (>2.0) and t test (p<0.05), and data were filtered to eliminate genes because of inconsistent expression across the chip replicates. We identified 98 genes (82 upregulated and 16 downregulated) that were reproducibly regulated by 1,25(OH)₂D₃ (Supplemental data). Q-PCR on a limited number of these genes confirmed the changes in their expression in independent proliferating keratinocyte cultures. The results indicate that the VDR signaling in keratinocytes affects many cell functions that ultimately explain at least in part, the differentiation, anti-proliferative, and immunomodulatory activities of 1,25(OH)₂D₃.

Identification of PADI family members as vitamin D-responsive genes

PADI is a family of Ca²⁺-dependent enzymes that catalyze the post-translational deimination of arginine residues to citrullines. The terminally differentiated cornified layer of the epidermis contains deiminated keratins (K1, K10, K14, and K5) and filaggrin (^{Ohsawa et al, 1999;Ishida-Yamamoto et al, 2000}), thus suggesting a role for protein deimination during the final stages of epidermal differentiation. The degree of modification of arginines to citrulline residues directly correlates with the structural order of substrate (^{Tarcsa et al, 1996}). Therefore, the PADI family of enzymes is proposed in protein unfolding as disordered structure of cornified envelope component proteins is required for optimal interaction with intermediary filaments and transglutaminase I (TGase I) (^{Tarcsa et al, 1996}). There are four PADI genes in humans, namely PADI 1, PADI 2, PADI 3, and PADI 4, and of these PADI 1 and PADI 3 have shown to be expressed in the epidermis (^{Nakashima et al, 2002}). The expression of PADI 3 was strongly induced by 1,25(OH)₂D₃ treatment at 24 and 48 h time points (Supplemental data). The regulation of PADI 3 was confirmed by Q-PCR, and its expression was found to be upregulated by 6, 24, and 48 h treatment of keratinocytes with the VDR ligand (Figure 1a). As the PADI family of genes is clustered on chromosome 1p36 in humans, we next analyzed the expression of PADI 1 and PADI 2 in 1,25(OH)₂D₃-treated keratinocytes by Q-PCR. PADI 1 and PADI 2 gene expression was also induced after 6, 24, and 48 h treatment of keratinocytes with 1,25(OH)₂D₃ (Figure 1a). In NHEK, induction of the expression of PADI 1, 2, and 3 genes was observed after 6 h treatment with 1,25(OH)₂D₃ (Figure 1a).

Figure 1.

1, 25-dihydroxyvitamin D₃ (1,25(OH)₂D₃)-dependent regulation of the expression of peptidylarginine deiminase (PADI), kallikrein (KLK), and serine proteinase inhibitor (SERPIN B) family members. (A–C). Taqman quantitative PCR was performed on total RNA prepared from vehicle-treated KerTr cells (red bars) and cells treated for 6, 24, and 48 h with 1,25(OH)₂D₃ (blue bars). RNA was also prepared from vehicle-treated normal human epidermal keratinocyte (NHEK) cells (red bars) and cells treated 6 h (blue bars) with 1,25(OH)₂D₃. The amount of PADI 1, PADI 2, and PADI 3 (A), KLK 5, KLK6, KLK7, KLK8, KLK10, and KLK13 (B), and SERPIN B1, SERPIN B6, and SERPIN B9 (C) gene transcripts relative to the glyceraldehyde-3-phosphate dehydrogenase transcript is shown as meanSE of triplicate experiments.

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Identification of KLK family members as vitamin D-responsive genes

Proliferation of keratinocytes to terminally differentiated corneocytes is balanced by the shedding of old corneocytes at the stratum corneum. This process of desquamation is achieved physiologically by tissue KLK, which constitute a group of serine proteases encoded by clusters of 15 genes on chromosome 19q13.3–4 (^{Komatsu et al, 2003}). Microarrays identified KLK5, KLK6, and KLK10 as vitamin D-responsive genes (Supplemental data). The expression of another KLK family member, KLK8, was also induced by 1,25(OH)₂D₃ at 48 h (1.8-fold induction), but it fell through the filtering process because of the 2-fold cut-off for retaining differentially expressed genes. 1,25(OH)₂D₃ induced the expression of KLK5, KLK6, KLK7, KLK8, KLK10, and KLK13 in KerTr as well as in NHEK cells (Figure 1b). The expression of KLK5, KLK6, KLK10, and KLK13 was induced after 6, 24, and 48 h of treatment in KerTr cells, whereas that of KLK8 was induced at 24 and 48 h but not at 6 h. Significant vitamin D-mediated expression of KLK7 was observed only at 24 h time point in KerTr cells (Figure 1b). But in NHEK, 1,25(OH)₂D₃ induced the expression of KLK5, KLK6, KLK7, KLK8, KLK10, and KLK13 after 6 h of ligand treatment (Figure 1b). KLK11 expression was not regulated by the VDR ligand (data not shown). The expression of another serine protease, HtrA1 (encoded by protease serine (PRSS) 11 gene), was also induced by 1,25(OH)₂D₃ (Supplemental data).

Vitamin D-mediated induction of the expression of serine proteinase inhibitor gene family members

Microarray analysis showed that the expression of serine proteinase inhibitors SERPIN B1 and SERPIN B6 was induced in keratinocytes by 1,25(OH)₂D₃ treatment (Supplemental data). SERPIN B1 expression was induced by 6-, 19-, and 19-fold, respectively, by 6, 24, and 48 h treatment with the VDR ligand. SERPIN B6 induction (3-fold) was observed only at 48 h time point (Supplemental data). Serine proteinase inhibitor activity in the corneocytes has been proposed to be important for negative feedback regulation of stratum corneum serine protease activity (^{Komatsu et al, 2003}). The 13 human ov-serpin genes are clustered at 6p25 (three genes, namely SERPIN B1, SERPIN B6, and SERPIN B9) and 18q21 (10 genes). By Q-PCR, the expression of SERPIN B1, SERPIN B6, and SERPIN B9 was found to be increased in KerTr cells by 6, 24, and 48 h of treatment by the VDR ligand (Figure 1c). The expression of the SERPIN genes was also upregulated by 6 h exposure of NHEK to 1,25(OH)₂D₃ (Figure 1c).

Identification of vitamin D-regulated genes involved in keratinocyte terminal differentiation

Expression of a number of genes that are involved in terminal differentiation of keratinocytes was modulated by 1,25(OH)₂D₃ treatment. These genes include involucrin, cystatin EM, small-proline-rich protein 1B (SPRR1B), TGase I, Kruppel-like factor 4 (KLF4), and c-fos (Supplemental data and Figure 2). In microarrays, involucrin expression was increased by 1.5-fold after 48 h of 1,25(OH)₂D₃ treatment (data not shown). Q-PCR results, however, showed significant induction of involucrin expression after 6, 24, and 48 h of 1,25(OH)₂D₃ treatment in KerTr and 6 h in NHEK cells (Figure 2a). Another differentiation-specific protein, cystatin EM, a component of the cornified envelope (^{Zeeuwen et al, 2001}), showed a vitamin D-dependent induction in its expression at all the three time points analyzed in KerTr cells and after 6 h of treatment in NHEK (Supplemental data and Figure 2a). Microarray results indicated downregulation of SPRR1B after 6 h treatment of keratinocytes with 1,25(OH)₂D₃ (Supplemental data). This observation was confirmed by Q-PCR, where 6 h treatment of KerTr cells with 1,25(OH)₂D₃ led to a suppression of SPRR1B expression (Figure 2b). Epithelial enriched transcription factor, KLF4, is highly expressed in the differentiating layers of epidermis and it is required for establishing epidermal permeability barrier (^{Segre et al, 1999;Jaubert et al, 2003}). KLF4 showed 1,25(OH)₂D₃-dependent induction in its expression in KerTr and NHEK cells (Figure 2b). In microarrays, the expression of fos gene was induced 10-, 10-, and 4-fold, respectively, after 6, 24, and 48 h treatment (Supplemental data). Vitamin D-mediated regulation of c-fos expression was confirmed by Q-PCR, where 6 h treatment of KerTr and NHEK showed 3–5-fold induction in its expression. Its expression was also induced at 24 and 48 h time points in KerTr cells (Figure 2c).

Figure 2.

Taqman quantitative PCR (Q-PCR) analysis of , 25-dihydroxyvitamin D₃ (1,25(OH)₂D₃)-regulated genes in immortalized and normal keratinocytes. (A–D) Q-PCR was performed on total RNA prepared from vehicle-treated KerTr cells (red bars) and cells treated for 6, 24, and 48 h with 1,25(OH)₂D₃ (blue bars). RNA was also prepared from vehicle-treated normal human epidermal keratinocytes (NHEK) cells (red bars) and cells treated for 6 h (blue bars) with 1,25(OH)₂D₃. The amount of involucrin and Cystatin EM (A), small-proline proline-rich protein 1B (SPRR1B), transglutaminase I (TGase I) (two independent RNA samples after 6 h 1,25(OH)₂D₃ treatment) and Kruppel-like factor 4 (KLF4) (B), c-fos and T1/ST2 (C) and tazarotene-induced gene 1 (TIG1), insulin-like growth factor binding protein-3 (IGFBP-3) and dual specificity phosphatase-10 (DUSP-10) gene transcripts relative to the glyceraldehyde-3-phosphate dehydrogenase transcript is shown as meanSE of triplicate experiments. (E). Schematic representation of vitamin D-responsive chromosomal loci. A diagrammatic representation of peptidylarginine deiminase (PADI), kallikrein (KLK), and serine proteinase inhibitor family members at 1p36, 19q13, and 6p25 chromosomal loci, respectively, in humans is shown. Arrows indicate the direction of transcription of genes.

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Regulation of class II tumor suppressor/growth-regulatory and immunomodulatory genes by 1,25(OH)₂D₃

VDR ligand induced the expression of class II tumor suppressors PRSS 11, cystatin EM, KLK10, lysyl oxidase (LOX), thrombomodulin, semaphorin 3B, thrombospondin, and tazarotene-induced gene 1 (TIG1)/retinoic acid receptor responsive gene 1 (RARRES1) in keratinocytes (Supplemental data, Figure 1b and Figure 2d). 1,25(OH)₂D₃ also induced the expression of growth-regulatory/cell cycle control genes dual specificity phosphatase 10 (DUSP-10), insulin-like growth factor binding protein-3 (IGFBP)-3, and IGFBP-6 in keratinocytes (Supplemental data and Figure 2d).

Keratinocytes, the first line of defense against invading microbes, are considered to be part of the immune system. 1,25(OH)₂D₃ induced the expression of monocyte/macrophage differentiation marker CD14, and lipopolysaccharide-binding protein CAP18, highlighting the role of keratinocytes in innate immunity. CD14 has previously been described as a vitamin D-responsive gene in HL-60 promyelocytic leukemic and SCC25 cells (^{Rots et al, 1998;Lin et al, 2002}). The expression of IL1 receptor like 1 (IL1RL1; T1/ST2) gene was induced by 1,25(OH)₂D₃ in keratinocytes (Supplemental data and Figure 2c) and SCC 25 cells (^{Lin et al, 2002}). As IL1RL1 gene disruption leads to a defect in T-helper type 2 (Th2) differentiation (^{Townsend et al, 2000}), vitamin D-mediated induction of its expression suggests that the shift of balance from pathogenic Th1 (IL-2-, IFN--, and TNF--secreting T cells) to non-pathogenic Th2 (IL-4-, IL-5-, and IL-10-producing T cells) phenotype in psoriasis by VDR ligands may involve a direct role of keratinocytes in Th2 differentiation.

Discussion

In this study, by gene expression profiling of 1,25(OH)₂D₃-treated keratinocytes, we demonstrate that PADI, KLK, and SERPIN B family members are vitamin D-responsive genes. We further identify a vitamin D-regulated differentiation network that provides a plausible mechanism of therapeutic action of VDR ligands in psoriasis and squamous cell carcinoma.

Identification of a vitamin D-regulated keratinocyte terminal differentiation network

Expression profiling and Q-PCR results could be distilled into a vitamin D-mediated keratinocyte/epidermal differentiation network, wherein many of the components were identified for the first time as vitamin D-responsive genes (Figure 3). The network involves seven major nodes or components that are directly regulated by the VDR ligand. These components are: (1) cross-linked proteins that give rise to the cornified envelope; (2) PADI family of enzymes; (3) TGase I, the enzyme that cross-links substrates to form the cornified envelope; (4) KLK family of serine proteases; (5) SERPIN B family members; (6) KLF4; and (7) c-fos (Figure 3). The first component (node) of this network comprises cross-linked proteins, namely involucrin, cystatin EM, and SPRR1B (Figure 2a and b, Figure 3 and Supplemental data). Involucrin, a major component of the cross-linked envelope (^{Eckert et al, 2004}), is vitamin D-responsive and contains a VDRE in its promoter (^{Bikle et al, 2003}). Q-PCR results showed significant induction of involucrin expression after 1,25(OH)₂D₃ treatment in keratinocytes (Figure 2a). Another differentiation-specific protein, cystatin EM, also a component of the cornified envelope that is highly expressed in differentiated epidermis (^{Zeeuwen et al, 2001}), showed a vitamin D-dependent induction in its expression at all the three time points analyzed (Supplemental data and Figure 2a). Expression profiling of an HNSCC cell line also showed induction of cystatin EM by a synthetic vitamin D analog EB1089 (^{Lin et al, 2002}). Importantly, microarray results indicated downregulation of small proline-rich protein, SPRR1B expression (Table S1 and Figure 3b). The expression of SPRR1B protein is increased significantly in psoriasis (^{Koizumi et al, 1996}) and it may contribute to abnormal keratinocyte differentiation in psoriatic lesional skin. Therefore, potent inhibition of SPRRIB expression by 1,25(OH)₂D₃ may contribute toward normalization of keratinocyte differentiation observed by VDR ligands in psoriatic skin.

Figure 3.

Vitamin D-regulated differentiation network. 1, 25-dihydroxyvitamin D₃ (1,25(OH)₂D₃)-regulated genes identified by microarrays and Taqman quantitative-PCR analysis were arranged in the form of a network based upon their function and action on keratinocyte/epidermal differentiation in normal and/or psoriatic states. The network consists of seven nodes (shown as colored circles), where each node is represented by a group of vitamin D-regulated genes. The seven nodes are as follows: cornified envelope proteins, peptidylarginine deiminase (PADI) family members, transglutaminase I (TGase I), kallikrein (KLK) family members, serine proteinase inhibitor (SERPIN) B family members, Kruppel-like factor (KLF)4, and c-fos.

Full figure and legend (57K)

The expression of PADI family members, the second node of the differentiation network, was upregulated by the VDR ligand in KerTr and NHEK cells (Figure 1a). PADI family members facilitate unfolding of ordered -helical structure of proteins that are then cross-linked by TGase I (Figure 3). The induction of the expression of PADI gene family members by 1,25(OH)₂D₃ appears to be therapeutically relevant in psoriasis, because immunostaining based on citrulline residues revealed that the normal and uninvolved psoriatic epidermis contained deiminated proteins in the terminally differentiated cornified layers, whereas lesional psoriatic epidermis presented with strikingly reduced levels of deiminated proteins (^{Ishida-Yamamoto et al, 2000}). Thus, VDR ligands may improve the underlying pathology of psoriasis by inducing the expression of PADI gene family members. Basal-level expression of PADI genes was higher in NHEK than KerTr cells (Figure 1a), suggesting that immortalization leads to suppression of PADI gene expression. Further, another anti-psoriatic agent, retinoic acid, which normalizes keratinocyte differentiation, induced PADI enzymatic activity in primary rat keratinocytes (^{Ishigami et al, 1996}).

The expression of TGase I, the third component of the network, is increased in psoriatic lesional skin (^{Nonomura et al, 1993}). TGase I mediates cross-linking of structural proteins into the cornified envelope by promoting the formation of interprotein -glutamyl-lysine isopeptide bonds. Therefore, increased TGase I expression may result in abnormal/psoriatic differentiation and may contribute to the formation of excess cornified scales characteristics of psoriasis. It may actively cross-link abnormally expressed proteins (e.g., SPRR family members, myeloid-related protein-8 (MRP-8), skin-derived anti-leukoproteinase (SKALP), psoriasin, etc.) at the expense of normal cornified envelope components in psoriatic epidermis, which may affect the quality of the epidermis (^{Nagpal et al, 1996b}). Treatment of proliferating keratinocytes with the VDR ligand decreased the expression of TGase I (Figure 2b). It is noteworthy that another keratinocyte differentiation-promoting agent, tazarotene, a retinoic acid receptor /-selective retinoid that is used in clinic for the treatment of psoriasis, inhibited TGase I expression in psoriatic lesions (^{Nagpal et al, 1996b}).

The fourth node of the differentiation network is the KLK family of enzymes, whose expression is induced by the VDR ligand in KerTr and NHEK cells (Figure 1b). The topmost cornified layers of skin are constantly removed by the action of the KLK family of serine proteases. Desquamation by KLK present in the stratum corneum results by their ability to degrade corneodesmosin and desmoglein-1, components of corneodesmosomes (^{Suzuki et al, 1996;Simon et al, 2001;Komatsu et al, 2003}). KLK5, KLK6, and KLK8 have been reported to be expressed in corneocytes (^{Komatsu et al, 2003}). Abnormal differentiation, hyperproliferation, and low rate of desquamation is believed to be the cause for build-up of scales observed in psoriatic lesional skin. Vitamin D-mediated induction of the expression of KLK family members provides a molecular basis for the clinical action of topically applied VDR ligand calcitriol (1,25(OH)₂D₃) in plaque-type psoriasis, as the drug treatment results in rapid desquamation leading to decreased plaque thickness (^{Smith et al, 1988}).

The fifth component of this network is the family of serine protease inhibitors (SERPIN B1, SERPIN B6, and SERPIN B9), whose expression was induced by 1,25(OH)₂D₃ probably to balance the equilibrium between terminal differentiation and desquamation, by virtue of their ability to inhibit the activity of KLK (^{Komatsu et al, 2003}). Genetic defect of a serine protease inhibitor, serine protease inhibitor Kazal-type 5, presents with overdesquamation, aberrant hair growth, and temperature instability in the Netherton syndrome (^{Chavanas et al, 2000}).

The sixth component, KLF4, which showed vitamin D-dependent regulation in keratinocytes (Figure 2b), inhibits cell proliferation by blocking G1/S transition of the cell cycle (^{Chen et al, 2001}). KLF4 gene ablation studies have underscored its importance in epidermal differentiation and for establishing barrier function of the skin (^{Segre et al, 1999}). Transcriptional profiling of KLF4 revealed that a large number of genes encoded keratins, further indicating the importance of KLF4 in epithelial differentiation (^{Chen et al, 2003}).

The expression of c-fos, the seventh node in this network, was induced by 1,25(OH)₂D₃ treatment in keratinocytes (Figure 3c). Activator protein 1 (AP1), a complex of transcription factors c-jun and c-fos, regulates the transcription of a number of genes that are involved in keratinocyte differentiation. In addition, all the treatments that induce AP1 activity and c-fos expression also result in keratinocyte differentiation (^{Johansen et al, 2003;Eckert et al, 2004;Schmuth et al, 2004}). Further, c-fos expression is reduced in psoriatic lesional skin compared with normal epidermis (^{Basset-Seguin et al, 1991}). Therefore, induction of c-fos by 1,25(OH)₂D₃ may help in normalization of differentiation observed by the treatment of psoriatic lesions by VDR ligands.

Vitamin D-dependent regulation of growth regulatory genes

Expression of a number of growth-regulatory or class II tumor suppressor genes was induced by 1,25(OH)₂D₃. These genes are PRSS 11, cystatin EM, KLK10, LOX, thrombomodulin, semaphorin 3B, thrombospondin, TIG1, DUSP-10, IGFBP-3, and IGFBP-6. Expression of PRSS 11 is reduced in tumors and its transfection into cancer cells inhibited their growth in vitro and in vivo (^{Baldi et al, 2002}). Cystatin EM, whose expression was induced by 1,25(OH)₂D₃ in keratinocytes (Figure 2a), also showed VDR ligand-dependent upregulation in an HNSCC cell line, SCC25 (^{Lin et al, 2002}). Cystatin EM was expressed in normal and premalignant breast epithelial cells but not in metastatic breast cancer cells (^{Sotiropoulou et al, 1997}) and its overexpression suppressed the malignant phenotype of a highly tumorigenic breast cancer cell line (^{Shridhar et al, 2004}). KLK10 functions as a class II tumor suppressor in breast and testicular tissues (^{Luo et al, 2001;Yousef et al, 2004}). The expression of KLK10 was also induced by 1,25(OH)₂D₃ and its synthetic analog EB1089 in SCC25 cells (^{Lin et al, 2002}). LOX has shown tumor suppressor functions in vitro and in vivo. Its expression was reduced in HNSCC cell lines, mucocutaneus malignant tissues, colorectal tumors and its overexpression led to reduced breast cancer cell invasion (^{Csiszar et al, 2002;Kirschmann et al, 2002;Jeay et al, 2003;Rost et al, 2003}). Thrombomodulin, the thrombin receptor, exerted growth suppressor effect and its expression inversely correlated with tumor progression in humans (^{Tezuka et al, 1995}). Semaphorin 3B, which is expressed at reduced levels in both small-cell and non-small-cell lung carcinomas, suppressed tumor formation in vivo (^{Tse et al, 2002}). TIG1/RARRES1, originally identified as a retinoid responsive gene (^{Nagpal et al, 1996a}), showed 1,25(OH)₂D₃-dependent induction in KerTr cells (Figure 2d). TIG1 was found to be a class II tumor suppressor in prostate carcinoma (^{Jing et al, 2002;Lotan, 2002}). DUSP-10 and IGFBP-3 have previously been shown to be responsive to 1,25(OH)₂D₃ in prostate cancer cells (^{Krishnan et al, 2003}). DUSP-10 inactivates mitogen-activated protein kinase (MAPK), indicating that inhibition of the growth-promoting actions of MAPK may partly explain the growth-inhibitory actions of 1,25(OH)₂D₃ in epithelial cells. Induction of IGFBP may inhibit the mitogenic effects of IGF on epithelial cells. Further, the expression of IGFBP-3 was found to be obligatory for the growth-inhibitory actions of 1,25(OH)₂D₃ in prostate cancer cells (^{Peng et al, 2004}). IGFBP-6 is highly expressed in normal prostate and it showed downregulation in malignant prostate (^{Welsh et al, 2001}). Therefore, VDR ligands may exert anti-proliferative effects by inducing the expression of the above-mentioned class II tumor suppressor and growth-regulatory genes.

Identification of vitamin D-responsive chromosomal loci

Human chromosomal region 1q21 contains an epidermal differentiation locus that harbors genes required for keratinocyte differentiation (^{Marshall et al, 2001}). Here, we describe the identification of three vitamin D-responsive loci at 1p36 (PADI genes), 19q13 (KLK genes), and 6p25 (SERPIN B genes), that appear to be involved in both normal and vitamin D-induced epidermal differentiation (Figure 2e). To the best of our knowledge, these are the only vitamin D-responsive loci described to date. The induction of gene expression at these loci may at least in part explain the mechanism underlying the normalization of differentiation observed by the treatment of psoriatic plaques by VDR ligands. These results, along with the observations documenting common occurrence of the loss of heterozygosity at 1p36.1 in cutaneous malignancies (^{Araki et al, 2002}), highlight the therapeutic potential of VDR ligands in actinic keratosis, HNSCC, basal cell carcinoma, and melanoma. The vitamin D-dependent epidermal differentiation network may operate not only in pathogenic but also under normal conditions. VDR null animals showed increased epidermal thickness and keratinocyte proliferation beginning at 7 wk of age (^{Zinser et al, 2002}), indicating a keratinocyte differentiation defect that is not compensated by retinoic acid receptors, peroxisome proliferator-activated receptors, farnesoid X receptors, and liver X receptors. Finally, the presence of skin differentiation defect and epidermal hyperproliferation in keratinocyte-specific RXR and RXR/RXR compound null animals (^{Li et al, 2000}) further supports this notion, and suggests that apart from normalization of differentiation in pathogenic conditions, VDR may also play an important role in normal epidermal differentiation.

Materials and Methods

Cells, RNA isolation, and microarray analysis

HPV E6 and E7 immortalized keratinocyte cell line, KerTr (ATCC), was grown in serum-free keratinocyte growth medium (KGM) (Life Technologies, Rockville, Maryland) supplemented with 0.05 mg per mL bovine pituitary extract (BPE) and 35 ng per mL epidermal growth factor (EGF). NHEK (Bio Whittaker, Walkersville, Maryland) were cultured in KGM supplemented with BPE, EGF, Insulin, GA-1000 (gentamicin, amphotericin B), and hydrocortisone (KGM-BulletKit, Bio Whittaker, Walkersville, Maryland). KerTr cells were treated with DMSO (vehicle) or 1,25(OH)₂D₃ (10^-7 M) for 6, 24, and 48 h in three independent experiments. NHEK were treated with DMSO (vehicle) or 1,25(OH)₂D₃ (10^-7 M) for 6 h. Total RNA was isolated with TRIzol (Life Technologies). Complementary DNA (cDNA) was synthesized by the SuperScript First-Strand Synthesis System for RT-PCR (Life Technologies). For microarrays, 5 g of total RNA was labeled according to the Affymetrix protocol (Affymetrix, Santa Clara, California). Twenty micrograms of labeled cRNA was fragmented and mixed with hybridization controls. This hybridization cocktail was hybridized to duplicate Affymetrix HG-U133A GeneChips for 16 h at 45°C. After hybridization, the chips were washed and scanned on an Affymetrix GeneChip scanner, and analyzed with Microarray Suite version 5.0 (MAS5, Affymetrix).

Data analysis

Signal intensities were generated from Microarray Suite version 5.0 (MAS5), using the default settings, and a global scaling, which adjusts the trimmed mean signal for the entire chip to a value of 1500. MAS5 generates quantitative data for single arrays and can perform a pair-wise comparison of two arrays. But in order to apply statistical analysis to the experiment, for each probeset, the fold-change was calculated as the ratio of the two group means based on the observed signal values from MAS5 for the two treatment groups. At the same time, the hypothesis of no differential expression was tested for each individual probeset using t-statistics.

Q-PCR

Selected genes identified by microarrays were verified by Q-PCR using an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, California). Primers and probes for some genes (KLK10, c-fos, SERPIN B1, involucrin, KLK6, KLK7, KLK8, and KLK10) were designed using Primer Express software (Applied Biosystems). These sequences are available upon request. Probes were labeled with fluorescent reporter dye FAM at the 5'-end and quencher dye TAMRA (Applied Biosystems, Foster City, California) at the 3'-end. For other genes, optimized primer and probes sets were obtained commercially (Applied Biosystems). Each 20 L reaction consisted of forward and reverse primers (900 nM), TaqMan probe (200 nM), cDNA (prepared from 8 ng of total RNA), and TaqMan Universal PCR Master Mix (Roche Diagnostics, Indianapolis, Indiana). Reactions were incubated at 50°C for 2 min followed by 10 min at 95°C, and subjected to 40 cycles of PCR (95°C for 15 s followed by 60°C for 1 min). Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control and its primer and probe set were obtained commercially (Applied Biosystems).

Supplementary Material

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