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. Author manuscript; available in PMC: 2006 Apr 11.
Published in final edited form as: J Invest Dermatol. 2006 Jan;126(1):49–60. doi: 10.1038/sj.jid.5700018

Regulatory role for Krüppel-like Zinc-finger Protein Gli-similar 1 (Glis1) in PMA-treated and psoriatic epidermis

Gen Nakanishi 1,, Yong-Sik Kim 1, Takeshi Nakajima 1, Anton M Jetten 1,*,✉
PMCID: PMC1435652  NIHMSID: NIHMS6414  PMID: 16417217

Abstract

In this study, we analyze the expression and potential function of the Krüppel-like zinc-finger protein Gli-similar protein 1 (Glis1) in normal and inflammatory skin and in the differentiation of epidermal keratinocytes. Glis1 mRNA is not expressed in normal human epidermis but is significantly induced in psoriatic epidermis and in mouse skin upon treatment with the tumor promoter phorbol-12-myristate-13-acetate (PMA). The expression of Glis1 is restricted to the suprabasal layers. These observations suggest that Glis1 expression is associated with hyperplastic, inflammatory epidermis. Consistent with these findings, Glis1 mRNA is not expressed in undifferentiated or differentiated normal human epidermal keratinocytes (NHEK) in culture but is dramatically induced after the addition of PMA or interferon γ. A similar induction of Glis1 mRNA by PMA treatment was observed in the immortalized epidermal keratinocyte cell line NHEK-HPV whereas PMA did not induce Glis1 in HaCaT cells or in several squamous cell carcinoma cell lines. To obtain insight into its function, Glis1 and a C-terminal deletion mutant Glis1 ΔC were expressed in NHEK-HPV cells and changes in epidermal differentiation and gene expression examined. Microarray analysis revealed that Glis1 ΔC promoted PMA-induced epidermal differentiation as indicated by increased expression of many differentiation-specific genes. This, in association with its induction in psoriasis, suggests that transcriptional factor Glis1 is involved in the regulation of aberrant differentiation observed in psoriatic epidermis.

Keywords: Gli, zinc finger protein, psoriasis, phorbol ester, differentiation, epidermal keratinocyte

Introduction

Hedgehog signaling plays a critical role in embryonic development and various physiological processes in the adult and has been implicated in a number of human diseases (Bale, 2002; Ruiz i Altaba et al, 2002; Toftgard, 2000; Villavicencio et al, 2000; Wicking et al, 2001). Sonic hedgehog (Shh) signaling involves binding of Shh to the transmembrane receptor patched (Ptch). This relieves the inhibition of downstream transcriptional events by the transmembrane protein smoothened (Smo). The Gli1-3 proteins constitute a subfamily of Krüppel-like zinc finger transcription factors that mediate the regulation of gene expression downstream of Shh/Ptch/Smo (Ruiz i Altaba et al, 2002; Villavicencio et al, 2000).

Recent studies have demonstrated that Shh/Ptch/Gli signaling plays an important role in skin biology and skin carcinogenesis (Chiang et al, 1999; Dahmane et al, 1997; Gailani et al, 1997; Grachtchouk et al, 2000; Green et al, 1998; Ikram et al, 2004; Mill et al, 2003; Oro et al, 2003; St-Jacques et al, 1998). The Shh signaling cascade, largely through the activation of Gli2, is a key regulatory pathway in hair follicle development (Chiang et al, 1999; Ellis et al, 2003; Mill et al, 2003; St-Jacques et al, 1998; Wang et al, 2000). Shh signaling also has an important function in the regulation of cellular proliferation. Inappropriate activation of Shh/Ptch/Gli cascade has been linked to the development of various human cancers, including basal cell carcinoma (BCC) (Dahmane et al, 1997; GailaniBale, 1997; Grachtchouk et al, 2000; Matise et al, 1999; Nilsson et al, 2000; Ruiz i Altaba et al, 2002).

Recently, we identified three novel proteins, referred to as Gli-similar 1-3 (Glis1-3), that are closely related to Gli proteins (Kim et al, 2002; Kim et al, 2003; Lamar et al, 2001; Nakashima et al, 2002; Zhang et al, 2001a; Zhang et al, 2001b). Glis1-3 contain five Cys2-His2-type zinc finger motifs that exhibit high homology with those of Gli. However, Glis and Gli proteins exhibit little sequence homology outside their zinc finger domain. Deletion analysis demonstrated that Glis proteins contain both repressor and activation domains suggesting that they may function as positive as well as negative regulators of gene transcription (Kim et al, 2002; Kim et al, 2003; Zhang et al, 2001b). During embryonic development Glis1-3 are expressed in a spatial and temporal manner suggesting roles in the regulation of different steps in organogenesis (Kim et al, 2003; Lamar et al, 2001; Nakashima et al, 2002; Zhang et al, 2001b). During embryonic development, Glis1 (also named GliH1) expression was most prominent in craniofacial regions, branchial arches, somites, and kidney and was also detected in the dermal papilla (Kim et al, 2002; Nakashima et al, 2002).

In this study, we began to examine the potential role of Glis1 in the epidermis. We show that in contrast to normal epidermis, Glis1 mRNA is expressed in the epidermis of human psoriatic and PMA-treated mouse skin. In addition, Glis1 expression is induced in normal human epidermal keratinocytes (NHEK) and immortalized NHEK-HPV cells by two inflammatory mediators interferon gamma (IFNγ) and PMA, both of which are strong inducers of epidermal differentiation (Hennings et al, 1987; Saunders et al, 1994). To obtain insight into the role of Glis1 in epidermal differentiation, the effect of Glis1 on gene expression was examined by microarray analysis. These findings indicated that Glis1 promotes the expression of a number of differentiation-specific genes. Our observations provide evidence for a regulatory role for Glis1 in the aberrant differentiation observed in inflammatory skin.

Results

Glis1 is induced in psoriatic epidermis. To study whether Glis1 has a regulatory function in the epidermis we examined the expression of Glis1 mRNA in normal human skin, skin from psoriatic patients, and basal cell carcinomas. As shown in Fig. 1A, in situ hybridization analysis was unable to detect any Glis1 mRNA in normal human epidermis. In addition, Glis1 mRNA was undetectable in sections from basal cell carcinomas (not shown). In contrast, Glis1 was highly induced in the epidermis of psoriatic patients. Glis1 mRNA was undetectable in the dermal layer of either normal or psoriatic skin. Psoriatic epidermis is hyperplastic and is characterized by an infiltration of neutrophilic inflammatory cells (Fujimoto et al, 1997; Peters et al, 2000; Van de Kerkhof et al, 1996). The thickness of the stratum spinosum and expression of a number of early differentiation markers, including involucrin, cornifin, and transglutaminase I, are greatly increased (de Rie et al, 2004; Fujimoto et al, 1997; Michel et al, 1992). In psoriatic epidermis, Glis1 mRNA was expressed in the suprabasal layers and was absent in the basal cell layers (Fig. 1G), suggesting that undifferentiated keratinocytes do not express Glis1 mRNA. Thus, Glis1 mRNA is induced when cells transit from the basal into the suprabasal layers and begin to differentiate. The expression of Glis1 was compared with that of the differentiation marker involucrin, that is expressed in the upper stratum spinosum and stratum granulosum of normal epidermis and is highly induced in the suprabasal layers of psoriatic epidermis (Fig. 1F). These observations demonstrate that the expression of Glis1 mRNA is greatly enhanced in psoriatic skin but that, unlike involucrin and many other differentiation markers, its expression appears to be restricted to hyperplastic, inflammatory skin. The pattern of Glis1 expression and its association with hyperplastic, inflammatory skin suggests that Glis1 does not play a role normal differentiation but has might be implicated in the regulation of aberrant differentiation observed in psoriatic epidermis.

Figure 1.

Figure 1

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Glis1 mRNA is not expressed in normal epidermis but induced in the suprabasal layers of psoriatic skin. Paraffin sections of normal human skin were hybridized with a Glis1 antisense probe (A) and adjacent sections were stained with H&E (B) or an anti-involucrin antibody (C). Sections of psoriatic skin were hybridized with an antisense (D, G) or sense (I) probe for Glis1 and adjacent sections were stained with H&E (E, H) or an anti-involucrin antibody (F). (G) and (H) are higher magnifications of D and E, respectively. Bars indicate 50 □m.

Induction of Glis1 expression by PMA in mouse skin. As in psoriatic epidermis, PMA-treated mouse skin exhibits epidermal hyperplasia and neutrophil-dominant, inflammatory cell infiltration (Stanley et al, 1991). As in normal human skin, in situ hybridization analysis showed that Glis1 was not detectable in normal mouse epidermis (Fig 2B). However, Glis1 mRNA expression was induced in PMA-treated mouse skin (Fig. 2D). Expression of Glis1 mRNA was associated largely with the suprabasal layers of the epidermis. The sense probe did not detect any signal in PMA-treated skin (Fig. 2E). The induction of Glis1 mRNA by PMA was supported by RT-PCR analysis. RT-PCR using RNA extracted from normal mouse skin and mouse skin treated for 1 to 7 days with PMA confirmed that Glis1 mRNA was not expressed in untreated mouse skin, but induced in PMA-treated mouse skin (Fig. 2F).

Figure 2.

Figure 2

Figure 2

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Induction of Glis1 mRNA expression in PMA-treated mouse skin. Mouse skin was treated with PMA or vehicle (control) once a day during a 7-day period as described in Materials and Methods. A-E. Paraffin sections of skin were obtained and subsequently examined by in situ hybridization analysis with sense or antisense Glis1 probes. (A) H&E staining of a section of control mouse skin; (B) control skin section hybridized with a Glis1 antisense probe; (C) H&E staining of a representative section of PMA-treated skin; (D) a representative section of PMA-treated skin hybridized with a Glis1 antisense probes; (E) section of PMA-treated skin hybridized with a Glis1 sense probe. Bar indicates 50 □m. (F) At the times indicated skin was isolated, RNA extracted and the level of Glis1 and β-actin (control) mRNA determined by RT-PCR as described in Materials and Methods.

PMA and IFNγ induce Glis1 mRNA expression in NHEK cells. We next examined the expression of Glis1 mRNA during differentiation of cultured human epidermal keratinocytes (NHEK). NHEK cells growing in the exponential phase do not express or express low levels of differentiation markers. When cultures reach confluence, cells become committed to terminal differentiation and expression of various differentiation markers are induced (Jetten et al, 1997). Glis1 was undetectable in exponential phase cultures of NHEK cells as well as in confluent cultures maintained in medium with high calcium (Fig. 3A, lanes 1 and 2). Differentiated NHEK cells grown at air-liquid interface also did not express Glis1 (Fig. 4A, lane 3). The tumor promoter PMA and IFNγ are strong inducers of differentiation in NHEK cells (Hennings et al, 1987; Saunders et al, 1996). As shown in Fig. 3A (lanes 4 and 5), Glis1 mRNA expression was increased significantly in NHEK cells treated with PMA or IFNγ. The lack of Glis1 expression during normal (confluence-induced) epidermal differentiation and the induction of Glis1 during the differentiation process initiated by PMA and IFNγtwo proinflammatory agents, appear to mirror the pattern of Glis1 expression observed in psoriatic human skin and PMA-treated mouse skin (Reynolds et al, 1998).

Figure 3.

Figure 3

Figure 3

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(A) Induction of Glis1 mRNA expression in NHEK cells in response to PMA and IFNγ. NHEK cells grown under different conditions were harvested and RNA isolated. Glis1 and β-actin (control) mRNA levels were determined by RT-PCR as described in Materials and Methods. RNA from exponential phase culture of NHEK cells; lane 2: RNA from confluent NHEK cells grown in the presence of 1.5 mM CaCl2; lane 3: RNA from cells grown at an air-liquid interface; lane 4: RNA from cells treated for 24 h with PMA (20 nM) and 1.5 mM calcium; lane 5: RNA from cells treated for 24 h with IFNγ (100 U/ml). (B) PMA induces Glis1 mRNA expression in NHEK-HPV but not in HaCaT and squamous cell carcinoma cell lines SCC13 and SQCC/Y1. Cells were treated with 20 nM PMA in the presence of 1.5 mM CaCl2. After 24 h, RNA was isolated and the level of Glis1 and β-actin (control) mRNA determined by semi-quantitative RT-PCR. (D) Time course of the induction of Glis1 mRNA in NHEK-HPV cells by PMA. Cells were treated with 20 nM PMA in the presence of 1.5 mM CaCl2. At the times indicated, cells were harvested and RNA and protein isolated. Glis1 and β-actin (control) mRNA levels were determined by RT-PCR. Protein samples were used for Western blot analysis to determine the level of cornifin expression. (D) Induction of Glis1 mRNA expression by PMA and IFNγ in NHEK and NHEK-HPV cells. Real-time QRT-PCR analysis was performed as described in Materials and Methods.

Figure 4.

Figure 4

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A. Analysis of Glis1 protein in NHEK-HPV and HaCaT cells expressing Flag-Glis1 and Flag-Glis1△C. NHEK-HPV and HaCaT cells were infected with retroviruses containing pLXIN(empty), pLXIN-Flag-Glis1, or pLXIN-Flag-Glis1△C and stable cell lines established as described in Materials and Methods. Protein extracts (30 μg) were examined by Western blot analysis using anti-Flag M2 antibody. Lane 1: NHEK-HPV(empty); lane 2: NHEK-HPV-Flag-Glis1; lane 3: NHEK-HPV-Flag-Glis1△C; lane 4: HaCaT-Flag-Glis1FL; lane 5: HaCaT-Flag-Glis1△C; lane 6: NHEK-HPV cells transiently transfected with pCMV-3xFlag-Glis1 construct. A schematic view of the two fusion proteins is shown above the Western blot. B-D. Comparison of the cell morphology of NHEK-HPV(empty) (B), NHEK-HPV-Flag-Glis1 (C), and NHEK-HPV-Flag-Glis1△C (D) cells.

We then compared Glis1 expression in two immortalized epidermal keratinocyte cell lines NHEK-HPV and HaCaT, and in the squamous cell carcinoma cell lines SCC13 and SQCC/Y1. NHEK-HPV are human epidermal keratinocytes immortalized by HPV-E6 (Harvat et al, 2001; Saunders and Jetten, 1994) and retain the ability to undergo differentiation upon PMA treatment and at confluence. In the exponential phase NHEK-HPV cells did not express Glis1 mRNA; however, Glis1 expression was induced upon PMA treatment (Fig. 3B). In contrast, PMA did not induce Glis1 mRNA expression in HaCaT, SQCC/Y1, and SCC13 cells which do not undergo differentiation after PMA treatment. The induction of Glis1 mRNA by PMA in NHEK-HPV cells occurred in a time-dependent manner (Fig. 3C). An increase in Glis1 mRNA expression was observed as early as 16 h after the addition of PMA. This induction paralleled that of the increase in the level of cornifin protein, a marker of epidermal differentiation (Marvin et al, 1992). The induction of Glis1 mRNA expression by PMA and IFNγin NHEK and NHEK-HPV cells was confirmed by Real Time QRT-PCR analysis (Fig. 3D).

Expression of Glis1 and Glis1ΔC in NHEK-HPV cells. To study the role of Glis1 in epidermal differentiation, the effect of Glis1 and Glis1ΔC on the differentiation of NHEK-HPV cells was examined. Using pLXIN retroviral vectors NHEK-HPV cell lines were established that stably expressed Flag-Glis1(full-length) fusion protein (Flag-Glis1) and a mutant Glis1 containing a C-terminal deletion (Flag-Glis1ΔC) (Fig. 4A). These cell lines are referred to as NHEK-HPV-Flag-Glis1 and NHEK-HPV-Flag-Glis1ΔC, respectively. The cell line NHEK-HPV(empty) containing the empty vector pLXIN-Flag, was used as control. In addition, three corresponding HaCaT cell lines were established using the same retroviruses.

The expression of Flag-Glis1 fusion protein was examined by Western blot analysis using anti-Flag M2 antibody. In protein extracts from NHEK-HPV-Flag-Glis1 and HaCaT-Flag-Glis1 cells the anti-Flag antibody recognized the Flag-Glis1 protein with the expected size of 100 kD. However, the antibody reacted with several other smaller proteins (Fig. 4A; lane 2 and 4, respectively). These smaller Flag-Glis1 proteins may be generated by proteolytic processing of Glis1 at two different sites at the carboxyl terminus. Protease inhibitors included during the isolation of cellular protein did not prevent this cleavage. We believe that Glis1 might be specifically processed within the cell. Transient expression of Flag-Glis1 in NHEK-HPV cells showed similar processing of Glis1 (Fig. 4A, lane 6). In extracts from NHEK-HPV-Flag-Glis1ΔC and HaCaT-Flag-Glis1ΔC cells the antibody recognized only the Flag-Glis1ΔC fusion protein of the expected size of 50 kD suggesting that it is not further cleaved. The latter is in agreement with the conclusion that the processing of full-length Glis1 involves the carboxyl terminal half of Glis1.

The morphology of the NHEK-HPV-Flag-Glis1 cells was very similar to that of the parental NHEK-HPV or NHEK-HPV(empty) cells (Fig. 4B, C). Cultures consisted of migratory cells that formed more loose colonies. NHEK-HPV-Flag-Glis1ΔC cells formed more compact colonies with fewer migrating cells (Fig. 4D). These cells were also more adhesive than parental NHEK-HPV cells (not shown). No differences in cellular morphology were observed between HaCaT-Flag-Glis1ΔC, HaCaT-Flag-Glis1, and HaCaT(empty) cells (not shown).

Subcellular localization of Glis1. The activity of Gli and Ci proteins is greatly dependent on their nuclear localization and controlled by suppressor-of-fused (SUFU), proteolytic processing, protein kinases, and nuclear export signals (Dunaeva et al, 2003; Kogerman et al, 1999; Ruiz i Altaba et al, 2002; Villavicencio et al, 2000). We, therefore, examined the subcellular localization of Glis1 in stably-transfected cells. In more than 90% of NHEK-HPV-Flag-Glis1 cells, Glis1 protein localized predominantly to cytoplasm (Fig. 5A). This cytoplasmic staining likely represents full-length Glis1 and proteolytically cleaved Glis1 based on the results shown in Fig. 4. In contrast, in NHEK-HPV-Flag-Glis1ΔC cells Glis1ΔC was predominantly localized in the nucleus (Fig. 5C). These results suggest that the C-terminal region of Glis1 contains a domain that retains Glis1 in the cytoplasm.

Figure 5.

Figure 5

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Subcellular localization of Glis1 and Glis1△C in NHEK-HPV cells. Cells were stained by mouse anti-Flag M2 antibody and Alexa 488-conjugated anti-mouse IgG and subsequently examined by confocal microscopy (A,C). Right panels are phase contrast images. A,B. NHEK-HPV-Flag-Glis1; C,D. NHEK-HPV-Flag-Glis1△C.

Identification of Glis1-regulated genes. To investigate changes in gene expression resulting from the expression of Glis1 and Glis1ΔC, we performed microarray analysis using a human oligo-chip representing approximately 17,000 human genes. No significant differences in gene expression were detected between PMA-treated NHEK-HPV(empty) and PMA-treated NHEK-HPV-Flag-Glis1 cells (not shown). In contrast, microarray analysis with PMA-treated NHEK-HPV(empty) and PMA-treated NHEK-HPV-Flag-Glis1ΔC cells identified more than 54 genes that were down-regulated and 23 genes that were up-regulated in NHEK-HPV-Flag-Glis1ΔC cells (Table I). The expression of insulin-like growth factor binding protein 2 (IGFBP2), which is involved in growth inhibition and induction of apoptosis, showed the largest increase in NHEK-HPV-Glis1ΔC cells. Interestingly, among the genes that were significantly increased in NHEK-HPV-Flag-Glis1ΔC compared to NHEK-HPV(empty) cells included many well-established markers of epidermal differentiation, including S100A9, KLK7, SPRR (cornifin), involucrin, and transglutaminase 1. These observations are consistent with the interpretation that Glis1 enhances epidermal differentiation. Genes down-regulated by Glis1 include the notch-1 receptor Jagged 1, the Wnt receptor Frizzled-10, the TGFβ family member inhibin βA (activin A), and the activin receptor ACVR1.

Table I.

Gene Genbank # Description Fold-increase Function
IGFBP2 P18065 Insulin-like growth factor binding protein 2 13.59 Proliferation/apoptosis
KLK6 Q92876 Kallikrein 6 8.66 Serine proteinase
I_929176 P22466 Preprogalanin 6.42 Neuropeptide*
KLK10 O43240 Kallikrein 10 6.33 Serine proteinase
S100A9 P06702 S100 calcium-binding protein A9 6.29 Ca2+-activated signaling protein*
KLK7 P49862 Kallikrein 7 5.89 Stratum corneum serine protease*
KLK8 NM_144507.1 Kallikrein 8 5.59 Serine protease*
IVL P07476 Involucrin 5.38 Structural component of stratum corneum*
KLK5 Q9Y337 Kallikrein 5 5.43 Stratum corneum serine protease
G1P2 P05161 Interferon-inducible protein 15 4.77 Ubiquitin-like protein
TXNIP BAB18859.1 Vitamin D-3 upregulated protein-1 4.29 Inhibitor of thioredoxin
NMU P48645 Neuromedin U 3.89 Agonist of G protein-coupled receptors
TGM1 P22735 Transglutaminase 1 3.78 Crosslinking enzyme*
SLPI P03973 Secretory leukocyte protease inhibitor 3.7 Antileukoproteinase*
CST6 Q15828 Cystatin E (cystatin M) 3.66 Protease inhibitor*
KRT13 P13646 Keratin 13 3.59 Intermediate filament protein
IFI27 P40305 IFNa-inducible protein 27 3.44 Membrane protein*
S100A8 NM_002964.3 S100 calcium binding protein A8 3.38 Ca2+-activated signaling protein*
AK3L1 Q9UIJ7 Adenylate kinase 3 like 1 3.28 Unknown
NID2 NM_007361.1 Nidogen 2 3.22 Basement membrane protein/cell adhesion
HSPA1A NM_005345.4 Heat shock 70 kDa protein 1A 3.16 Protein folding*
CLIC3 NM_004669.1 Chloride intracellular channel 3 3.09 Chloride ion transport
HSPA8 P11142 Heat shock 70kD protein 8 3.08 Protein folding*
TIMP3 P35625 Tissue inhibitor of MP3 3 Protease inhibitor*
RUVBL1 Q9Y265 ATP-dependent helicase 2.88 Nuclear protein with ATPase-helicase motifs
CDH22 Q9UJ99 Cadherin-like 2 2.8 Ca2+-dependent cell adhesion
SPRR1B NM_003125.1 Small proline-rich protein 1B (cornifin) 2.63 Structural component of stratum corneum*
CXCL14 AAH03513.1 Chemokine C-X-C motif ligand 14 2.62 Chemokine/senescence
KRT6B NM_005555.2 Keratin 6B 2.53 Epidermal differentiation and wound response*
FGFR3 NM_000142.2 Fibroblast growth factor receptor 3 2.52 Receptor for FGF
HAIK1 Q9C075 Intermediate filament protein 2.42 Intermediate filament protein
DDX9 NM_001357.2 DEAD box protein 9 2.42 RNA helicase A
EXO1 AAC69879.1 Exonuclease 1 2.4 Nuclease
FABP5 Q01469 Epidermal fatty acid binding protein 2.36 Transport fatty acids/keratinocyte differentiation*
EPN3 NM_017957.1 Epsin 3 2.35 Involved in wound healing
DEFB1 Q09753 Defensin beta 1 2.19 Host defense*
WDR3 Q9UNX4 WD repeat domain 3 2.34 Unknown
MKI67 P46013 Ki-67 antigen 2.17 Nuclear protein/proliferation*
TUBA6 NM_032704.1 Keratinocyte alpha-tubulin 2.14 Tubulin family member
ID3 CAA01342.1 Inhibitor of DNA binding 3 2.02 Regulation of proliferation
PHB P35232 Prohibitin 2.09 Suppressor of cell proliferation
BIRC5 O15392 Survivin 1.98 Inhibitor of apoptosis/involved in G2-M transition
CTSC P53634 Cathepsin C 1.98 Cysteine proteinase
CGREF1 Q99674 Cell growth regulator with EF-hand domain 1 1.97 Negatively regulator of cell proliferation
KREMEN2 NM_172229.1 Kringle containing transmembrane protein 2 1.91 Receptor for Dkk1
GBA P04062 Beta-glucocerebrosidase 1.77 Hydrolysis of glucosylceramide*
SULT2B1 AAC78554.1 Sulfotransferase 2B1 1.59 Cholesterol sulfonation*
S100A4 P26447 S100 calcium-binding protein A4 1.53 Ca2+-activated signaling protein
S100A7 P31151 S100 calcium-binding protein A7 (psoriasin) 1.53 Ca2+-activated signaling protein*
ANGPTL4 NM_016109.1 ANGPTL4 1.5 Lipid metabolism
EPPK1 P58107 Epiplakin 1 1.49 Cytolinker
SPRR2C NM_006518.1 Small proline-rich protein (cornifin) 1.49 Structural component of stratum corneum
PPL O60437 Periplakin 1.42 Component of (hemi)desmosomes
Gene Genbank # Description Fold-Decrease Function
ACVR1 Q04771 Activin A receptor type 1 −2.23 Receptor for activin
DDIT3 NM_004083.2 DNA damage inducible transcript 3 −2.27 Transcription factor
ZFP36L1 Q07352 Zinc finger protein −2.35 Tristetraprolin family member
ALDH1A3 P47895 Aldehyde dehydrogenase 6 −2.37 Retinoic acid metabolism
FOXA2 NM_021784.3 Forkhead box A2 −2.55 Transcription factor
HIPK2 AAL37371.1 Homeodomain interacting protein kinase 2 −2.58 Signaling
FZD10 Q9ULW2 Frizzled-10 −2.6 Receptor for Wnt
SERPINE1 P05121 Plasminogen activator inhibitor 1 −2.77 Protease
VEGFC P49767 Vascular endothelial growth factor C −2.79 Angiogenesis
NDRG1 Q92597 N-myc downstream regulated gene 1 −2.79 Proliferation
PCK2 Q16822 Phosphoenolpyruvate carboxykinase 2 −3.06 Gluconeogenesis
HSPA5 P11021 Heat shock 70kD protein 5 −3.07 Protein folding
JAG1 NM_000214.1 Jagged 1 −3.32 Receptor for notch
CDKN1A P38936 Cyclin-dependent kinase inhibitor 1A (p21) −3.33 Cell cycle control
NR2F1 NM_005654.2 Nuclear receptor COUPTF1 −3.35 Transcription factor
TNFRSF6B NM_032957.1 TNF receptor superfamily member 6b −3.38 Receptor for TNF
TRA1 P14625 Tumor rejection antigen 1 −3.69 Chaperone/protein folding
NUCB2 P80303 Nucleobindin 2 −3.78 Ca2+-binding protein
MMP1 P03956 Matrix metalloproteinase 1 −3.97 Protease
LAMB3 Q13751 Laminin beta 3 −4.52 Extracellular matrix
HNT Q9P121 Neurotrimin −5.02 Adhesion
PLAB Q99988 Macrophage inhibitory cytokine 1 −5.18 Apoptosis/cancer
INHBA P08476 Inhibin beta A (activin A) −11.48 Proliferation/differentiation
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The differential expression of several genes was validated by Northern blot analysis. This analysis confirmed the enhanced expression of the differentiation markers S100A9, KLK7, and transglutaminase type I and the down-regulation of Jagged-1 and inhibin βA (Fig. 6).

Figure 6.

Figure 6

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Validation of several differentially expressed genes identified by microarray analysis. RNA isolated from PMA-treated NHEK-HPV(empty), NHEK-HPV-Flag-Glis1, and NHEK-HPV-Flag-Glis1△C cells were examined by Northern blot analysis using radiolabeled cDNA probes for S100A9, transglutaminase type I (TGM1), kallikrein 7 (KLK7), Jagged-1 (JAG1), and inhibin βA (INHBA) as described in Materials and Methods.

Glis1 expression promotes epidermal differentiation. To analyze the action of Glis1 further, we compared the induction of differentiation in NHEK-HPV(empty), NHEK-HPV-Flag-Glis1, and NHEK-HPV-Flag-Glis1ΔC cells upon treatment with PMA. The expression of several proteins known to be differentially regulated during epidermal differentiation were examined by Western blot analysis (Fig. 7A). Previous studies reported that the p53-family member p63 is down-regulated during squamous differentiation in vivo and in vitro (Koster et al, 2002; Pellegrini et al, 2001). The basal level of p63 protein in undifferentiated NHEK-HPV(empty), NHEK-HPV-Flag-Glis1, and NHEK-HPV-Flag-Glis1ΔC cells was not significantly different in the three cell lines. In addition, PMA decreased p63 expression in all three cell lines in a similar way (Fig. 7A). The differentiation markers involucrin and cornifin are up-regulated by PMA in all three NHEK-HPV cell lines; however, their level is increased to a significant greater extent in NHEK-HPV-Flag-Glis1ΔC than in NHEK-HPV(empty) or NHEK-HPV-Flag-Glis1 cells. These findings are in agreement with the results obtained by microarray analysis (Table I) and are consistent with the hypothesis that Glis1 promotes epidermal differentiation. Treatment of HaCaT(empty), HaCaT-Flag-Glis1□□ HaCaT-Flag-Glis1ΔC cells with PMA caused a small reduction in p63 while cornifin expression was very low in all three cell lines and remained unchanged after PM treatment (Fig. 7B) indicating that Glis1 expression has little effect on the induction of differentiation in these cells.

Figure 7.

Figure 7

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A. Glis1△C enhances PMA-induced differentiation in NHEK-HPV cells. NHEK-HPV(empty), NHEK-HPV-Glis1, and NHEK-HPV-Glis1△C cells were treated with vehicle (control; lanes 1, 4, 7), 2 nM PMA (lanes 2, 5, 8) or 20 nM PMA in the presence of 1.5 mM CaCl2 (lanes 3, 6, 9). After 24 h treatment, cells were collected and proteins examined by Western blot analysis using antibodies for p63, involucrin and cornifin. B. Glis1△C does not induce differentiation in HaCaT cells. Cells were treated for 24 h with vehicle or 50 nM PMA and 1.5 mM CaCl2. Proteins were examined by Western blot analysis for p63 and cornifin expression.

Discussion

The Shh/Gli signaling pathway has been reported to play an important role in skin biology and in skin disease (Chiang et al, 1999; Dahmane et al, 1997; Gailani and Bale, 1997; Grachtchouk et al, 2000; Green et al, 1998; Ikram et al, 2004; Mill et al, 2003; Oro and Higgins, 2003; St-Jacques et al, 1998). Gli1 and Gli2 are frequently highly induced in basal cell carcinomas and critical in the development of these tumors (Grachtchouk et al, 2000; Green et al, 1998; Nilsson et al, 2000). In this study, we demonstrate that Glis1 mRNA is not expressed in normal human epidermis nor in basal cell carcinomas. These observations suggest that Glis1 does not play a role in the development of this type of skin cancer. In contrast to normal skin, Glis1 mRNA was induced in the epidermis of human psoriatic skin. Psoriasis is one of the most common chronic inflammatory skin diseases and is characterized by local infiltration of T lymphocytes, neutrophils, and monocytes, and epidermal hyperproliferation (de Rie et al, 2004; Peters et al, 2000). The latter results in a significant increase in the thickness of the epidermis, the stratum spinosum in particular, and a greatly enhanced and aberrant expression of many differentiation-specific genes. In psoriatic epidermis, Glis1 mRNA is not detectable in the most basal layers but only in the suprabasal layers, particularly throughout the lower stratum spinosum. Comparison of Glis1 mRNA expression with that of the early differentiation marker involucrin shows that its induction precedes the appearance of involucrin protein. PMA-treated skin exhibits several similarities with psoriatic skin (Marks et al, 1993; Stanley et al, 1991). Treatment of mouse skin with PMA causes inflammation, enhances the thickness of the epidermis, and causes an increase in the expression of many differentiation markers. As in the epidermis of psoriatic patients, expression of Glis1 mRNA is induced in PMA-treated mouse skin as shown by in situ hybridization and confirmed by RT-PCR. In PMA-treated mouse skin Glis1 mRNA was predominantly localized to the suprabasal layers as observed for psoriatic epidermis.

The expression pattern of Glis1 in cultured human epidermal keratinocytes mirrors that seen in normal and PMA-treated skin. Glis1 mRNA was undetectable in NHEK cells growing in the exponential phase or in cells grown at an air-liquid interface. High Ca2+, which is not a very effective inducer of differentiation in NHEK, did not induce Glis1 expression. In contrast, treatment with PMA and IFNγ, both of which are proinflammatory mediators and strong inducers of growth arrest and differentiation in NHEK cells (Hennings et al, 1987; Saunders et al, 1996), significantly induced Glis1 mRNA levels. PMA treatment did not induce Glis1 expression in squamous carcinoma cells that have lost the ability to differentiate.

Epidermal differentiation is a multi-step process that starts with the transition of epidermal stem cells into transient amplifying cells, keratinocytes with a diminished lifespan (Watt, 2001). These cells subsequently undergo terminal differentiation that is associated with loss of proliferative potential and the expression of differentiation-specific genes. The expression of differentiation markers is carefully controlled and induced at specific steps during differentiation. The pattern of Glis1 expression observed in skin and cultured NHEK cells indicates that Glis1 is not required for normal epidermal differentiation. Instead, Glis1 appears to have a specific regulatory function in aberrant epidermal differentiation observed psoriatic and PMA-treated epidermis where it might be involved in the up-regulation of the expression of a number of squamous differentiation-specific markers or inflammatory genes. Previous studies have provided indications for additional functions of Glis1 in skin. During embryonic development, Glis1 mRNA is expressed in dermal papilla cells (Nakashima et al, 2002; Zhang et al, 2001b). These cells have been reported to play a critical role in hair follicle development (Oro and Higgins, 2003; St-Jacques et al, 1998). However, hair follicle development in mice deficient in Glis1 appears undisturbed suggesting that Glis1 is not required for this process (Nakashima et al, 2002). What the function is of Glis1 in dermal papilla cells has yet to be established.

To study the potential role of Glis1 in the regulation of epidermal differentiation, we examined the effects of Glis1 and Glis1ΔC expression in an immortalized epidermal keratinocyte cell line NHEK-HPV. Treatment of NHEK-HPV(empty), NHEK-HPV-Glis1, and NHEK-HPV-Glis1ΔC cells with PMA induces differentiation in all three cell lines and no significant differences were observed in the repression of p63, an early event in epidermal differentiation (Koster et al, 2002; Pellegrini et al, 2001). However, the induction of the differentiation markers involucrin and cornifin was significantly elevated in NHEK-HPV-Glis1ΔC cells compared to NHEK-HPV(empty) and NHEK-HPV-Glis1. These observations are consistent with the hypothesis that Glis1 does not act at the level of the commitment to differentiation but promotes the expression of the differentiated phenotype.

Analysis of the expression of Glis1 protein showed that Glis1 was proteolytically cleaved, that Glis1 and Glis1ΔC have a different subcellular localization. Previous studies have shown that the regulation of Gli protein activity involves activation by Shh, proteolytic processing, and translocation to the nucleus (Dunaeva et al, 2003; Kogerman et al, 1999; Ruiz i Altaba, 1999; Villavicencio et al, 2000). Mouse Gli2 contains a repressor domain near its amino-terminus that is cleaved off to generate a transcriptionally active form and overexpression of the latter induces tumor formation in mouse skin (Grachtchouk et al, 2000; Sheng et al, 2002). Transient transfection of Glis1 or Glis1ΔC inhibits Gli1-mediated transcriptional activation suggesting that both Glis1 proteins function as repressors (unpublished observations). The lack of a cellular response in NHEK-HPV-Glis1 cells might be due to the observation that Glis1 is largely localized to the cytoplasm. Removal of the carboxyl-terminus results in the translocation of Glis1ΔC to the nucleus and enables it to induce changes in gene transcription.

To obtain greater insight into the possible role of Glis1 in the regulation of gene expression during differentiation of epidermal keratinocytes, we compared the pattern of gene expression between PMA-treated NHEK-HPV(empty) and NHEK-HPV-Glis1ΔC cells by microarray analysis. This analysis identified more than 22 genes that were down-regulated and 54 genes that were up-regulated in NHEK-HPV-Glis1ΔC compared to NHEK-HPV(empty) cells (Table I). Many of these genes encode proteins for which specific functions in epidermal differentiation have been reported. The notch-1 receptor Jagged 1 and the TGFβ family member inhibin βA (activin A) belong to the group of genes down-regulated in NHEK-HPV-Glis1ΔC cells. Jagged 1 has been reported to be expressed in the basal layer of the epidermis and down-regulated in psoriatic epidermis (Thelu et al, 2002). In contrast, inhibin βA which plays a positive role in cell proliferation, is increased in psoriasis (Hubner et al, 1996). Genes expressed at higher levels in NHEK-HPV-Glis1ΔC cells include the cross-linked envelope precursors involucrin and small proline-rich proteins (SPRRs or cornifins), the crosslinking enzyme transglutaminase I that catalyzes the crosslinking between these structural proteins, and sulfotransferase Sult2B1b which catalyzes the formation of cholesterol sulfate. These observations are in agreement with the hypothesis that expression of Glis1ΔC promotes differentiation.

It is interesting to note that many of these genes, including involucrin, transglutaminase 1, several of the kallikrein serine proteases, the protease inhibitor cystatin E, epidermal fatty acid binding protein, beta-glucocerebrosidase, and S100 calcium-activated signaling proteins have been reported to be significantly induced in psoriatic epidermis (Eckert et al, 2004; Fujimoto et al, 1997; Kuwae et al, 2002; Michel et al, 1992; Peters et al, 2000; Zeeuwen et al, 2002)(see * in Table I). S100A9 and S100A8 are not or minimally expressed in normal epidermis but are highly expressed in psoriatic epidermis (Eckert et al, 2004). In addition, many of these genes are induced in PMA-treated epidermis (Michel et al, 1992; Owens et al, 1996). The enhanced expression of several differentiation-specific genes in epidermal keratinocytes overexpressing Glis1□C, in combination with the observations that Glis1 is induced in psoriatic and PMA-treated epidermis, suggests that there may be a link between these events. Since Glis1 functions as a transcriptional regulator (Kim et al, 2002), it is tempting to hypothesize that the expression of at least a subset of genes induced in psoriasis might be regulated by Glis1. Glis1 regulates gene transcription by binding to Glis DNA response elements (Glis-RE; consensus sequence ACCACCCA) in the promoter regulatory region of target genes (Kim et al, 2002). Although Glis1ΔC appears to act as a repressor, it might enhance the expression of differentiation-specific genes by inhibiting the expression of another transcriptional repressor. Further study is needed to determine whether Glis1 regulates these genes directly or indirectly.

In summary, in this study we demonstrate that Glis1 is not expressed in normal epidermis but is induced in psoriatic and PMA-treated skin and in PMA- and IFNγ-treated epidermal keratinocytes. Expression of Glis1ΔC in epidermal keratinocytes NHEK-HPV results in enhanced induction of a great number of differentiation-specific genes after treatment with PMA. Our observations indicate that expression of Glis1 promotes epidermal differentiation and suggest a regulatory role for Glis1 in aberrant epidermal differentiation and remodeling in psoriatic and PMA-treated skin.

Materials and Methods

Cell lines and cell culture. Normal human epidermal keratinocytes (NHEK) were obtained from Cambrex (Walkersville, MD). NHEK-HPV cells is an immortalized cell line derived by stable integration of the plasmid pHPV-18 (Harvat and Jetten, 2001). NHEK, SCC13, SQCC/Y1 cells, HaCaT cells, and NHEK-HPV cells were grown in KGM (Gibco BRL, Gaithersburg, MD) as previously described (Saunders and Jetten, 1994).

Retroviral vectors. The entire coding sequence of mouse Glis1 and a C-terminal truncated mouse Glis1, in which the region immediately following the zinc finger domain (518S) up to the C-terminus was deleted, were amplified by polymerase chain reaction (PCR) and subcloned into pCMV3xFlag 7.1 (Sigma, St. Louis, MO) generating pCMV-Flag-Glis1 and pCMV-Flag-Glis1ΔC, respectively. Flag-Glis1 and Flag-Glis1ΔC were re-amplified and then subcloned into the retroviral vector pLXIN (BD Biosciences Clontech, Palo Alto, CA). Retroviral infection and neomycin selection was carried out as described (Stapleton et al, 2005). The established cell lines are referred to as NHEK-HPV(empty), NHEK-HPV-Flag-Glis1, NHEK-HPV-Flag-Glis1ΔC, HaCaT(empty), HaCaT-Flag-Glis1, and HaCaT-Flag-Glis1ΔC.

Western blot analysis. Cells were lysed in lysis buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 10 mM DTT, 1 mM phenylmethylsulfonyl fluoride, aprotinin and leupeptin). The proteins were analysed by Western blot analysis using ECL Western Blotting Kit (Amersham Pharmacia Biotech., Piscataway, NJ) as described. Rabbit anti-cornifin antibody, mouse anti-involucrin antibody (Sigma), and mouse anti-p63 antibody (Santa Cruz Biotech., Santa Cruz, CA) were used as previously described (Daniely et al, 2004; Fujimoto et al, 1997). Peroxidase-conjugated anti-mouse and anti-rabbit IgG were purchased from Chemicon (Temecula, CA). Antibodies were diluted in PBS containing 5% milk and 0.01% Tween 20.

Reverse Transcriptase-PCR Analysis. Total RNA was prepared with TriReagent (Sigma) or RNeasy mini Kit (Qiagen, Valencia, CA). cDNA was synthesized using the SuperScriptIII first strand cDNA synthesis system for RT-PCR (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Semi-quantitative PCR was carried out using the following primer pair for mouse and human Glis1: forward primer 5'-TCAGCAACTCCAGCGACCGC and reverse primer 5'-GGAGCCAGGA-TACACACCTG. Primers for β-actin were used in controls.

Northern Blot Analysis. Total RNA (20 μg) was isolated using TriReagent (Sigma, St. Louis, MO) and examined by Northern blot analysis as described previously (Nakajima et al, 2004) with radiolabeled probes for transglutaminase I, kallikrein 7, S100A9, Jagged-1, and inhibin βA.

In situ hybridization analysis. Adult mouse skin and human skin were fixed in 4% paraformaldehyde, embedded in paraffin, and sections (5 μm thick) prepared for in situ hybridization analysis as described (Fujimoto et al, 1997). In situ hybridization analyses with antisense and sense Glis1 probes were carried out as described previously. Sections were stained with hematoxylin and eosin (H&E).

Microarray analysis. Microarray analysis was carried out by the NIEHS Microarray Group (NMG). Gene expression analysis was conducted using Agilent human oligo arrays (Agilent Technologies, Palo Alto, CA), the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol, the Agilent In Situ Hybridization Kit protocol, and the Agilent Feature Extraction software (v7.1). Images and GEML files were deposited into Rosetta Resolver (version 3.2, build 3.2.2.0.33) (Rosetta Biosoftware, Kirkland, WA). The resultant ratio profiles were combined into ratio experiments as described (Stoughton, 2002). Intensity plots were generated for each ratio experiment and genes were considered “signature genes” if the p value was less than 0.001. RNA preparations from two independent experiments were used and microarray hybridizations were performed in duplicate.

Subcellular localization. Cells overexpressing 3XFlag-Glis1 or 3XFlag-Glis1ΔC were fixed in 4% paraformaldehyde/PBS for 15 min and then treated with 0.3% Triton X-100/PBS for 15 min. Cells were subsequently incubated with anti-Flag M2 monoclonal Antibody (Sigma) for 1h. After washing, cells were incubated with Alexa Fluor 488-conjugated anti-mouse antibody (Molecular Probes, OR) for 1h. After several washings, Alexa Fluor 488 fluorescence was examined in a Zeiss confocal microscope LSM 510 (Zeiss, Thornwood, NY) at excitation and emission frequencies of 488 and 505 nm, respectively.

Immunohistochemical staining. Paraffin-embedded sections of normal and psoriatic skin were stained immunohistochemically with an anti-involucrin antibody using a streptavidin-biotin-peroxidase staining kit (Biomedical Technologies Inc., Stoughton, MA) following the manufacturer's instructions.

Animal and human skin samples. PMA-treated mouse skin samples were kindly provided by Dr. Sung-Jen Wei (NIEHS, NIH). Animal studies were carried out as previously reported (Wei et al, 2003). Normal human skin samples were obtained as excess tissue from unrelated surgical procedures. Psoriasis samples used were obtained from the pathology archives, Department of Dermatology, Okayama University Medical School.

Real Time QRT-PCR analyses. The Real Time QRT-PCR reactions were carried out in triplicate on a 7300 Real Time PCR system using the TaqMan® One-Step RT-PCR mix and pre-designed Assays-on-Demand™ primers/probe set for human Glis1 (Hs00377690_m1) from Applied Biosystems. All results were normalized to an internal control, the 18S transcript.

Acknowledgments

The authors would like to thank to Jennifer Collins and Sherry Grissom from the Microarray Group, NIEHS, and Dr. Xiao-Ping Yang and Martin Angers for their assistance and advise with this study.

Abbreviations:

Glis

Gli-similar

PMA

phorbol-12-myristate-13-acetate

NHEK

normal human epidermal keratinocytes

BCC

basal cell carcinoma

Shh

sonic hedgehog

ptch

patched

IFN

interferon

References

  1. Bale AE. Hedgehog signaling and human disease. Ann Rev Genomics & Hum Genet. 2002;3:47–65. doi: 10.1146/annurev.genom.3.022502.103031. [DOI] [PubMed] [Google Scholar]
  2. Chiang C, Swan RZ, Grachtchouk M, Bolinger M, Litingtung Y, Robertson EK, Cooper MK, Gaffield W, Westphal H, Beachy PA, Dlugosz AA. Essential role for Sonic hedgehog during hair follicle morphogenesis. Dev Biol. 1999;205:1–9. doi: 10.1006/dbio.1998.9103. [DOI] [PubMed] [Google Scholar]
  3. Dahmane N, Lee J, Robins P, Heller P, Ruiz i Altaba A. Activation of the transcription factor Gli1 and the Sonic hedgehog signalling pathway in skin tumours. Nature. 1997;389:876–881. doi: 10.1038/39918. [published erratum appears in Nature 1997 Dec 4;390(6659):536] [DOI] [PubMed] [Google Scholar]
  4. Daniely Y, Liao G, Dixon D, Linnoila RI, Lori A, Randell SH, Oren M, Jetten AM. Critical role of p63 in the development of a normal esophageal and tracheobronchial epithelium. Am J Physiol Cell Physiol. 2004;287:C171–181. doi: 10.1152/ajpcell.00226.2003. [DOI] [PubMed] [Google Scholar]
  5. de Rie MA, Goedkoop AY, Bos JD. Overview of psoriasis. Dermatol Ther. 2004;17:341–349. doi: 10.1111/j.1396-0296.2004.04037.x. [DOI] [PubMed] [Google Scholar]
  6. Dunaeva M, Michelson P, Kogerman P, Toftgard R. Characterization of the physical interaction of Gli proteins with SUFU proteins. J Biol Chem. 2003;278:5116–5122. doi: 10.1074/jbc.M209492200. [DOI] [PubMed] [Google Scholar]
  7. Eckert RL, Broome AM, Ruse M, Robinson N, Ryan D, Lee K. S100 proteins in the epidermis. J Invest Dermatol. 2004;123:23–33. doi: 10.1111/j.0022-202X.2004.22719.x. [DOI] [PubMed] [Google Scholar]
  8. Ellis T, Smyth I, Riley E, Bowles J, Adolphe C, Rothnagel JA, Wicking C, Wainwright BJ. Overexpression of Sonic Hedgehog suppresses embryonic hair follicle morphogenesis. Develop Biol. 2003;263:203–215. doi: 10.1016/s0012-1606(03)00394-4. [DOI] [PubMed] [Google Scholar]
  9. Fujimoto W, Nakanishi G, Arata J, Jetten AM. Differential expression of human cornifin alpha and beta in squamous differentiating epithelial tissues and several skin lesions. J Invest Dermatol. 1997;108:200–204. doi: 10.1111/1523-1747.ep12334240. [DOI] [PubMed] [Google Scholar]
  10. Gailani MR, Bale AE. Developmental genes and cancer: role of patched in basal cell carcinoma of the skin. J Natl Cancer Inst. 1997;89:1103–1109. doi: 10.1093/jnci/89.15.1103. [DOI] [PubMed] [Google Scholar]
  11. Grachtchouk M, Mo R, Yu S, Zhang X, Sasaki H, Hui CC, Dlugosz AA. Basal cell carcinomas in mice overexpressing Gli2 in skin. Nat Genet. 2000;24:216–217. doi: 10.1038/73417. [DOI] [PubMed] [Google Scholar]
  12. Green J, Leigh IM, Poulsom R, Quinn AG. Basal cell carcinoma development is associated with induction of the expression of the transcription factor Gli-1. Br J Dermatol. 1998;139:911–915. doi: 10.1046/j.1365-2133.1998.02598.x. [DOI] [PubMed] [Google Scholar]
  13. Harvat BL, Jetten AM. Decreased growth inhibitory responses of squamous carcinoma cells to interferon-gamma involve failure to recruit cki proteins into cdk2 complexes. J Invest Dermatol. 2001;117:1274–1281. doi: 10.1046/j.0022-202x.2001.01495.x. [DOI] [PubMed] [Google Scholar]
  14. Hennings H, Michael D, Lichti U, Yuspa SH. Response of carcinogen-altered mouse epidermal cells to phorbol ester tumor promoters and calcium. J Invest Dermatol. 1987;88:60–65. doi: 10.1111/1523-1747.ep12465014. [DOI] [PubMed] [Google Scholar]
  15. Hubner G, Hu Q, Smola H, Werner S. Strong induction of activin expression after injury suggests an important role of activin in wound repair. Dev Biol. 1996;173:490–498. doi: 10.1006/dbio.1996.0042. [DOI] [PubMed] [Google Scholar]
  16. Ikram MS, Neill GW, Regl G, Eichberger T, Frischauf AM, Aberger F, Quinn A, Philpott M. GLI2 is expressed in normal human epidermis and BCC and induces GLI1 expression by binding to its promoter. J Invest Dermatol. 2004;122:1503–1509. doi: 10.1111/j.0022-202X.2004.22612.x. [DOI] [PubMed] [Google Scholar]
  17. Jetten AM, Harvat BL. Epidermal differentiation and squamous metaplasia: from stem cell to cell death. J Dermatol. 1997;24:711–725. doi: 10.1111/j.1346-8138.1997.tb02523.x. [DOI] [PubMed] [Google Scholar]
  18. Kim YS, Lewandoski M, Perantoni AO, Kurebayashi S, Nakanishi G, Jetten AM. Identification of Glis1, a novel Gli-related, Kruppel-like zinc finger protein containing transactivation and repressor functions. J Biol Chem. 2002;277:30901–30913. doi: 10.1074/jbc.M203563200. [DOI] [PubMed] [Google Scholar]
  19. Kim YS, Nakanishi G, Lewandoski M, Jetten AM. GLIS3, a novel member of the GLIS subfamily of Kruppel-like zinc finger proteins with repressor and activation functions. Nucl Acids Res. 2003;31:5513–5525. doi: 10.1093/nar/gkg776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kogerman P, Grimm T, Kogerman L, Krause D, Unden AB, Sandstedt B, Toftgard R, Zaphiropoulos PG. Mammalian suppressor-of-fused modulates nuclear-cytoplasmic shuttling of Gli-1. Nat Cell Biol. 1999;1:312–319. doi: 10.1038/13031. [DOI] [PubMed] [Google Scholar]
  21. Koster MI, Huntzinger KA, Roop DR. Epidermal differentiation: transgenic/knockout mouse models reveal genes involved in stem cell fate decisions and commitment to differentiation. J Investig Dermatol Symp Proc. 2002;7:41–45. doi: 10.1046/j.1523-1747.2002.19639.x. [DOI] [PubMed] [Google Scholar]
  22. Kuwae K, Matsumoto-Miyai K, Yoshida S, Sadayama T, Yoshikawa K, Hosokawa K, Shiosaka S. Epidermal expression of serine protease, neuropsin (KLK8) in normal and pathological skin samples. Mol Pathol. 2002;55:235–241. doi: 10.1136/mp.55.4.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lamar E, Kintner C, Goulding M. Identification of NKL, a novel Gli-Kruppel zinc-finger protein that promotes neuronal differentiation. Development. 2001;128:1335–1346. doi: 10.1242/dev.128.8.1335. [DOI] [PubMed] [Google Scholar]
  24. Marks F, Furstenberger G. Proliferative responses of the skin to external stimuli. Environ Health Perspect. 1993;101(Suppl 5):95–101. doi: 10.1289/ehp.93101s595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Marvin KW, George MD, Fujimoto W, Saunders NA, Bernacki SH, Jetten AM. Cornifin, a cross-linked envelope precursor in keratinocytes that is down-regulated by retinoids. Proc Natl Acad Sci USA. 1992;89:11026–11030. doi: 10.1073/pnas.89.22.11026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matise MP, Joyner AL. Gli genes in development and cancer. Oncogene. 1999;18:7852–7859. doi: 10.1038/sj.onc.1203243. [DOI] [PubMed] [Google Scholar]
  27. Michel S, Bernerd F, Jetten AM, Floyd EE, Shroot B, Reichert U. Expression of keratinocyte transglutamine mRNA revealed by in situ hybridization. J Invest Dermatol. 1992;98:364–368. doi: 10.1111/1523-1747.ep12499806. [DOI] [PubMed] [Google Scholar]
  28. Mill P, Mo R, Fu H, Grachtchouk M, Kim PC, Dlugosz AA, Hui CC. Sonic hedgehog-dependent activation of Gli2 is essential for embryonic hair follicle development. Genes & Development. 2003;17:282–294. doi: 10.1101/gad.1038103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nakajima T, Fujino S, Nakanishi G, Kim YS, Jetten AM. TIP27: a novel repressor of the nuclear orphan receptor TAK1/TR4. Nucl Acids Res. 2004;32:4194–4204. doi: 10.1093/nar/gkh741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nakashima M, Tanese N, Ito M, Auerbach W, Bai C, Furukawa T, Toyono T, Akamine A, Joyner AL. A novel gene, GliH1, with homology to the Gli zinc finger domain not required for mouse development. Mech Dev. 2002;119:21. doi: 10.1016/s0925-4773(02)00291-5. [DOI] [PubMed] [Google Scholar]
  31. Nilsson M, Unden AB, Krause D, Malmqwist U, Raza K, Zaphiropoulos PG, Toftgard R. Induction of basal cell carcinomas and trichoepitheliomas in mice overexpressing GLI-1. Proc Natl Acad Sci USA. 2000;97:3438–3443. doi: 10.1073/pnas.050467397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Oro AE, Higgins K. Hair cycle regulation of Hedgehog signal reception. Develop Biol. 2003;255:238–248. doi: 10.1016/s0012-1606(02)00042-8. [DOI] [PubMed] [Google Scholar]
  33. Owens DM, Zainal TA, Jetten AM, Smart RC. Localization and expression of cornifin-alpha/SPRR1 in mouse epidermis, anagen hair follicles, and skin neoplasms. J Invest Dermatol. 1996;106:647–654. doi: 10.1111/1523-1747.ep12345463. [DOI] [PubMed] [Google Scholar]
  34. Pellegrini G, Dellambra E, Golisano O, Martinelli E, Fantozzi I, Bondanza S, Ponzin D, McKeon F, De Luca M. p63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA. 2001;98:3156–3161. doi: 10.1073/pnas.061032098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Peters BP, Weissman FG, Gill MA. Pathophysiology and treatment of psoriasis. Am J Health-System Pharm. 2000;57:645–659. doi: 10.1093/ajhp/57.7.645. [DOI] [PubMed] [Google Scholar]
  36. Reynolds NJ, Voorhees JJ, Fisher GJ. Cyclosporin A inhibits 12-O-tetradecanoyl-phorbol-13-acetate-induced cutaneous inflammation in severe combined immunodeficient mice that lack functional lymphocytes. Brit J Dermatol. 1998;139:16–22. doi: 10.1046/j.1365-2133.1998.02307.x. [DOI] [PubMed] [Google Scholar]
  37. Ruiz i Altaba A. Gli proteins encode context-dependent positive and negative functions: implications for development and disease. Development. 1999;126:3205–3216. doi: 10.1242/dev.126.14.3205. [DOI] [PubMed] [Google Scholar]
  38. Ruiz i Altaba A, Sanchez P, Dahmane N. GLI and hedgehog in cancer: tumours, embryos and stem cells. Nature Rev. 2002;2:361–372. doi: 10.1038/nrc796. [DOI] [PubMed] [Google Scholar]
  39. Saunders N, Dahler A, Jones S, Smith R, Jetten A. Interferon-gamma as a regulator of squamous differentiation. J Dermatol Sci. 1996;13:98–106. doi: 10.1016/s0923-1811(96)00535-x. [DOI] [PubMed] [Google Scholar]
  40. Saunders NA, Jetten AM. Control of growth regulatory and differentiation-specific genes in human epidermal keratinocytes by interferon gamma. Antagonism by retinoic acid and transforming growth factor beta 1. J Biol Chem. 1994;269:2016–2022. [PubMed] [Google Scholar]
  41. Sheng H, Goich S, Wang A, Grachtchouk M, Lowe L, Mo R, Lin K, de Sauvage FJ, Sasaki H, Hui CC, Dlugosz AA. Dissecting the oncogenic potential of Gli2: deletion of an NH(2)-terminal fragment alters skin tumor phenotype. Cancer Res. 2002;62:5308–5316. [PubMed] [Google Scholar]
  42. St-Jacques B, Dassule HR, Karavanova I, Botchkarev VA, Li J, Danielian PS, McMahon JA, Lewis PM, Paus R, McMahon AP. Sonic hedgehog signaling is essential for hair development. Curr Biol. 1998;8:1058–1068. doi: 10.1016/s0960-9822(98)70443-9. [DOI] [PubMed] [Google Scholar]
  43. Stanley PL, Steiner S, Havens M, Tramposch KM. Mouse skin inflammation induced by multiple topical applications of 12-O-tetradecanoylphorbol-13-acetate. Skin Pharmacol. 1991;4:262–271. doi: 10.1159/000210960. [DOI] [PubMed] [Google Scholar]
  44. Stapleton CM, Jaradat M, Dixon D, Kang HS, Kim SC, Liao G, Carey MA, Cristiano J, Moorman MP, Jetten AM. Enhanced susceptibility of staggerer (RORalphasg/sg) mice to lipopolysaccharide-induced lung inflammation. Am J Physiol Lung Cell Mol Physiol. 2005;289:L144–152. doi: 10.1152/ajplung.00348.2004. [DOI] [PubMed] [Google Scholar]
  45. Thelu J, Rossio P, Favier B. Notch signalling is linked to epidermal cell differentiation level in basal cell carcinoma, psoriasis and wound healing. BMC Dermatol. 2002;2:7. doi: 10.1186/1471-5945-2-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Toftgard R. Hedgehog signalling in cancer. Cell Mol Life Sci. 2000;57:1720–1731. doi: 10.1007/PL00000654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Van de Kerkhof PC, Van Erp PE. The role of epidermal proliferation in the pathogenesis of psoriasis. Skin Pharmacology. 1996;9:343–354. doi: 10.1159/000211445. [DOI] [PubMed] [Google Scholar]
  48. Villavicencio EH, Walterhouse DO, Iannaccone PM. The Sonic Hedgehog-Patched-Gli Pathway in Human Development and Disease. Am J Hum Genet. 2000;67:1047–1054. doi: 10.1016/s0002-9297(07)62934-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wang LC, Liu ZY, Gambardella L, Delacour A, Shapiro R, Yang J, Sizing I, Rayhorn P, Garber EA, Benjamin CD, Williams KP, Taylor FR, Barrandon Y, Ling L, Burkly LC. Conditional disruption of hedgehog signaling pathway defines its critical role in hair development and regeneration. J Invest Dermatol. 2000;114:901–908. doi: 10.1046/j.1523-1747.2000.00951.x. [DOI] [PubMed] [Google Scholar]
  50. Watt FM. Stem cell fate and patterning in mammalian epidermis. Curr Opin Genet Dev. 2001;11:410–417. doi: 10.1016/s0959-437x(00)00211-2. [DOI] [PubMed] [Google Scholar]
  51. Wei SJ, Trempus CS, Cannon RE, Bortner CD, Tennant RW. Identification of Dss1 as a 12-O-tetradecanoylphorbol-13-acetate-responsive gene expressed in keratinocyte progenitor cells, with possible involvement in early skin tumorigenesis. J Biol Chem. 2003;278:1758–1768. doi: 10.1074/jbc.M206328200. [DOI] [PubMed] [Google Scholar]
  52. Wicking C, McGlinn E. The role of hedgehog signaling in tumorigenesis. Cancer Letters. 2001;173:1–7. doi: 10.1016/s0304-3835(01)00676-0. [DOI] [PubMed] [Google Scholar]
  53. Zeeuwen PL, van Vlijmen-Willems IM, Egami H, Schalkwijk J. Cystatin M/E expression in inflammatory and neoplastic skin disorders. Br J Dermatol. 2002;147:87–94. doi: 10.1046/j.1365-2133.2002.04785.x. [DOI] [PubMed] [Google Scholar]
  54. Zhang F, Jetten AM. Genomic structure of the gene encoding the human GLI-related, Kruppel- like zinc finger protein GLIS2. Gene. 2001a;280:49–57. doi: 10.1016/s0378-1119(01)00764-8. [DOI] [PubMed] [Google Scholar]
  55. Zhang F, Nakanishi G, Kurebayashi S, Yoshino K, Perantoni A, Kim YS, Jetten AM. Characterization of Glis2, a novel gene encoding a Gli-related, Kruppel-like transcriptional factor with transactivation and repressor functions. Roles in kidney development and neurogenesis. J Biol Chem. 2001b;12:10139–10149. doi: 10.1074/jbc.M108062200. [DOI] [PubMed] [Google Scholar]

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