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Human Molecular Genetics Advance Access originally published online on January 3, 2008
Human Molecular Genetics 2008 17(7):1043-1051; doi:10.1093/hmg/ddm377
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The PSORS1 locus gene CCHCR1 affects keratinocyte proliferation in transgenic mice

Inkeri Tiala1, Janica Wakkinen1, Sari Suomela2, Pauli Puolakkainen3, Raija Tammi4, Sofi Forsberg5, Ola Rollman5, Kati Kainu1,2, Björn Rozell6,7,8, Juha Kere1,6,8,*, Ulpu Saarialho-Kere2,9 and Outi Elomaa1

1 Department of Medical Genetics 2 Department of Dermatology 3 Department of Surgery, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland 4 Department of Anatomy, University of Kuopio, Finland 5 Department of Medical Sciences, Dermatology and Venereology, Uppsala University Hospital, Sweden 6 Division of Clinical Research Center 7 Division of Pathology 8 Division of Biosciences and Nutrition, Department of Laboratory Medicine, Huddinge University Hospital, Stockholm, Sweden 9 Department of Dermatology, Karolinska Institutet at Stockholm Söder Hospital, Stockholm, Sweden

* To whom correspondence should be addressed at: Department of Biosciences and Nutrition, Karolinska Institutet, 14157 Huddinge, Sweden. Tel: +46 734213550; Fax: +46 87745538; Email: juha.kere{at}biosci.ki.se

Received September 11, 2007; Accepted December 21, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
The CCHCR1 gene (Coiled-Coil {alpha}-Helical Rod protein 1) within the major psoriasis susceptibility locus PSORS1 is a plausible candidate gene for the risk effect. We have previously generated transgenic mice overexpressing either the psoriasis-associated risk allele CCHCR1*WWCC or the normal allele of CCHCR1. All transgenic CCHCR1 mice appeared phenotypically normal, but exhibited altered expression of genes relevant to the pathogenesis of psoriasis, including upregulation of hyperproliferation markers keratins 6, 16 and 17. Here, we challenged the skin of CCHCR1 transgenic mice with wounding or 12-O-tetradecanoyl-13-acetate (TPA), treatments able to induce epidermal hyperplasia and proliferation that both are hallmarks of psoriasis. These experiments revealed that CCHCR1 regulates keratinocyte proliferation. Early wound healing on days 1 and 4 was delayed, and TPA-induced epidermal hyperproliferation was less pronounced in mice with the CCHCR1*WWCC risk allele than in mice with the normal allele or in wild-type animals. Finally, we demonstrated that overexpression of CCHCR1 affects basal keratinocyte proliferation in mice; CCHCR1*WWCC mice had less proliferating keratinocytes than the non-risk allele mice. Similarly, keratinocytes isolated from risk allele mice proliferated more slowly in culture than wild-type cells when measured by BrdU labeling and ELISA. Our data show that CCHCR1 may function as a negative regulator of keratinocyte proliferation. Thus, aberrant function of CCHCR1 may lead to abnormal keratinocyte proliferation which is a key feature of psoriatic epidermis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
The most important region for psoriasis predisposition is PSORS1 on chromosome 6p near the HLA-C gene (1,2). Three psoriasis-associated susceptibility alleles HLACw6, CCHCR1*WWCC and CDSN*5 (corneodesmosin) have been identified within it, but strong linkage disequilibrium has made it difficult to distinguish their individual genetic effect (3,4). In our previous studies, we have demonstrated that the CCHCR1 gene (Coiled-Coil {alpha}-Helical Rod protein 1, earlier known as HCR) may be an essential gene for the effect of PSORS1 in psoriasis. CCHCR1 protein is differentially expressed in psoriatic lesions compared with healthy skin or other hyperproliferative skin disorders, such as eczema (3,5). Furthermore, we have demonstrated that the expression of CCHCR1 is downregulated in cultured non-lesional psoriatic keratinocytes when compared with non-psoriatic cells (6). We have recently generated transgenic mice expressing either the psoriasis-associated risk allele or the normal allele of CCHCR1 under control of cytokeratin 14 (K14) promoter. Although transgenic mice were phenotypically and histologically normal, overexpression of CCHCR1 in mouse skin resulted in altered expression of several genes relevant in the pathogenesis of psoriasis (7).

Many mouse models exhibit a psoriasis-like phenotype, but none has shown all of the typical histological and immunophenotypical abnormalities of psoriatic lesions (8). The majority of engineered mice are transgenic, overexpressing genes that exhibit altered expression in psoriatic lesions. The most complete animal models to date are the K5-Stat3 (Signal transducers and activators of transcription) (9) and the Tie2 (Tyrosine kinase receptor) (10) transgenic mice and the JunB/c-Jun double knock out mouse (11) which all develop a spontaneous phenotype, but none of these genes map in human to the PSORS loci, and thus cannot explain the genetic risk. Wounding and topical application of 12-O-tetradecanoylporbol-13-acetate (TPA) have also been used to trigger psoriasis-like phenotypes in mouse models (9,12,13). Both manipulations are able to induce biological reactions persistently occurring in psoriatic lesions. These include processes such as proliferation, migration and re-stratification of keratinocytes, inflammation and neovascularization (14,15). Furthermore, wounding of healthy skin in psoriatic patients can trigger a complete psoriatic response, in a classic reaction termed the Koebner phenomenon (16). The mechanisms of TPA action leading to epidermal hypertrophy and hyperplasia are less well understood. One of the possible mechanisms involves activation of protein kinase C (17,18), which in turn regulates transcription factors, including Jun proteins that play a critical role in cell cycle control (19,20).

In the present study, we investigated the effects of TPA and wounding of skin on CCHCR1 transgenic mice. These treatments revealed that CCHCR1 affects keratinocyte proliferation in transgenic mice. Wound healing was delayed in mice with the psoriasis associated risk allele of CCHCR1 and TPA-induced epidermal hyperproliferation was less pronounced in transgenic mice than in wild-type animals. Simultaneously, the number of proliferating Ki67 positive cells was reduced in CCHCR1 mice. Finally, we demonstrated that the overexpression of CCHCR1 affects also normal keratinocyte proliferation in mice. Immunostaining for Ki67 in untreated mice revealed that CCHCR1*WWCC allele mice had less proliferating basal keratinocytes than the non-risk allele mice. Similarly keratinocytes isolated from risk allele mice proliferated more slowly than wild-type cells in culture. We propose that CCHCR1 participates in regulating keratinocyte proliferation and that disturbance of this function may contribute to the pathophysiology of psoriasis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Wound healing in transgenic mice expressing CCHCR1
The effect of CCHCR1 on wound healing was studied in full-thickness wounds (circular, 5 mm) on dorsal skin of wild-type and transgenic mice expressing either the risk or the normal allele of the CCHCR1 transgene (7). On days 1, 4, 11 and 30 post-wounding the mice were sacrificed, wound areas were measured and skin sections processed for histological studies. Eight wounds from each group were studied at each time point. Wound size measurements on day 1 and 4 suggested that early wound closure was delayed in mice with the CCHCR1*WWCC allele when compared with non-risk allele mice (day 1, P < 0.01 and day 4, P < 0.05; Fig. 1A). Also, histological evaluation and measurement of newly formed epithelium supported the macroscopic results (Fig. 1B–D). On day 4, wounds in risk allele mice were still largely devoid of the epithelial layer, whereas in non-risk mice neoepidermis was visible already on day 1 (data not shown) and complete re-epithelialization was seen in 50% of the wounds on day 4. On day 11, wounds were closed and regeneration of the epidermis was complete in all mouse groups. During the observation period of 30 days, no clinical or histological features of psoriasis developed in wounded skin.


Figure 1
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  		Figure 1. Wound size in transgenic CCHCR1 and wild-type mice on day 1 and 4 of healing (A). The remaining wound areas were largest in mice with the CCHCR1*WWCC risk allele, suggesting that early wound closure is delayed in risk allele mice. The largest difference was seen between the transgenic CCHCR1 animals (day 1, P < 0.01 and day 4, P < 0.05). Histological evaluation (B–D) and measurement of migrating epithelium on sections (B) supported the macroscopic results. On day 4, the distance (µm) between the original wound site (arrow head showing the edge of the muscular layer) and the leading edge of the epithelium (arrows) was shorter in CCHCR1 risk allele mice (D) than in non-risk (C) mice (P < 0.001, scale bars 200 µm). Immunostaining with Ki67 (E) demonstrated that risk allele mice have fewer proliferating keratinocytes in newly formed epidermis than non-risk allele or wild-type mice (P < 0.001) on day 4. Eight wounds from each mouse group were analyzed at each time point. Error bars represent mean ± SEM. Statistical comparisons between data sets were made with Student's t-test (**P < 0.01, *P < 0.05).

 
Histological evaluation of dermal components in HE-stained wounds based on the modified Greenhalg criteria (21,22) did not reveal any abnormalities in inflammation, formation of granulation tissues or neovascularization (data not shown). This suggested that the primary cause of retarded wound healing might be impaired regeneration of the epidermis which involves proliferation and migration of keratinocytes. Cytoplasmic laminin-5 staining for migrating keratinocytes (23) or TUNEL staining for apoptosis did not reveal any differences between mice groups. Immunostaining for Ki67 on day 1 demonstrated only a few proliferative keratinocytes next to the wound edges and there was no difference between the groups. On day 4, however risk allele mice had significantly fewer proliferating keratinocytes in newly formed epidermis than wild-type or non-risk allele mice (P < 0.001) (Fig. 1E), suggesting that this could be the reason for delayed wound closure in risk allele mice. On day 11, the number of Ki67-positive cells was lower in risk mice than non-risk mice (P = 0.01) and still on day 30 a similar trend was observed (data not shown). Staining for mast cells (toluidine blue) and immunohistochemical analyses with antibodies against cytokeratin 6 (hyperproliferation), {alpha}-smooth muscle actin (myofibroblasts) and von Willebrand factor (neoangiogenesis) did not reveal any abnormalities in transgenic mice.

Single-dose TPA application
To induce epidermal hyperplasia, dorsal skin was treated with TPA. Skin samples were harvested 24, 48 or 72 h after treatment and analyzed histologically. A single topical application of TPA resulted in increased thickening of epidermis in all groups already 24 h after application. However, the TPA-treatment of transgenic CCHCR1 mice showed a less well-developed epidermal hyperplasia when compared with wild-type controls. The difference was most evident between mouse groups 48 h after treatment (Fig. 2A and B) when the risk allele mice exhibited an epidermis of almost normal thickness. Immunostaining with Ki67 revealed that at that time point the number of proliferating cells was 20–30% lower in the epidermis of transgenic CCHCR1 mice than in wild-type animals. In transgenic mice, Ki67-positive cells were confined mainly to the basal layer of epidermis, whereas in wild-type mice proliferation was observed also suprabasally (Fig. 2C). Comparison between transgenic mice demonstrated that the number of Ki67 positive cells was 14% lower in risk allele mice than in non-risk mice (P < 0.05) (Fig. 2D) 48 h after treatment. TPA is known to induce an inflammatory response in the skin (17,18), but there was no difference in the number of infiltrating T-cells between mice groups (data not shown). Immunostaining for the keratinocyte differentiation marker cytokeratin 10 did not reveal any differences between mouse groups (data not shown).


Figure 2
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  		Figure 2. TPA treatment of transgenic CCHCR1 and wild-type mice. Skin samples were collected 0, 24, 48 and 72 h after single TPA treatment of dorsal skin, stained with HE and immunostained using antibodies for Ki67, and the thickness of epidermis was measured (A). TPA treatment resulted in increased thickening of epidermis in all mice groups already 24 h after application (A). However, after TPA treatment the epidermis of transgenic mice was significantly thinner than in wild-type mice. The difference was most evident between mouse groups 48 h after treatment when mice with the CCHCR1 risk allele (B) exhibited thinner epidermis than the non-risk allele mice or the wild-type mice (P < 0.01). Immunostaining for a Ki67 marker showed that the number of proliferating cells was lower in epidermis of CCHCR1 risk allele mice and non-risk mice than in wild-type animals (p < 0.01) (C and D). Comparison between transgenic animals revealed that the CCHCR1 risk allele mice had fewer Ki67 positive cells than the non-risk mice (P < 0.05) (D). In the non-risk and risk allele mice proliferative Ki67-positive cells were confined mainly to the basal layer of epidermis, whereas in wild-type mice proliferation was also observed suprabasally (C). Eight skin samples from each mouse group were analyzed at each time point. Error bars represent mean ± SEM. Statistical comparisons between data sets were made with Student's t-test (**P < 0.01, *P < 0.05). Scale bars 50 µm.

 
Normal cell proliferation in untreated skin and cultured keratinocytes
To study the effects of CCHCR1 on normal keratinocyte proliferation, untreated mice were injected intraperitoneally with bromodeoxyuridine (BrdU) and labeled cells were detected by immunohistochemistry. After 6 h labeling, the proportion of BrdU positive cells was 63% in the risk allele mice and 77 and 73% in the non-risk allele and wild-type mice, respectively, suggesting that cell proliferation was impaired in risk mice when compared with the non-risk mice (n = 4, P < 0.05) (Fig. 3A). Immunostaining for Ki67 showed a similar trend (n = 3) in cell proliferation of untreated skin; the number of proliferating cells was decreased in CCHCR1 risk allele mice when compared with non-risk mice (data not shown).


Figure 3
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  		Figure 3. Cell proliferation in untreated skin of transgenic CCHCR1 and wild-type mice. In in vivo proliferation assay, four mice from each mouse group were injected intraperitoneally with BrdU and labeled cells were detected by immunohistochemistry (A). The proportion of proliferating cells was lower in mice with the CCHCR*WWCC risk allele than mice with non-risk allele (P < 0.05) mice after 6 h labeling. Proliferation of cultured keratinocytes derived from transgenic CCHCR1 mice or from wild-type animals (B and C). Cells were labeled for 2, 4, 8 or 24 h with BrdU in the presence or absence of EGF after which the amount of incorporated BrdU was determined with ELISA. Absorbance (450 nm) values were plotted as a function of time and relative proliferation rates ({Delta}A450 nm/{Delta}h) were estimated from the slope of a line. Both in the presence (B) and absence of EGF (C), the relative proliferation rate was slower in keratinocytes expressing the risk allele of CCHCR1 when compared with wild-type keratinocytes. There was no obvious difference in proliferation rate between cells expressing the risk allele and non-risk allele of CCHCR1. Error bars represent mean ± SEM.

 
Cultured keratinocytes derived from newborn transgenic CCHCR1 mice or from wild-type animals were used to examine proliferation. Cells were labeled at different time points with BrdU in the presence or absence of EGF after which the amount of incorporated BrdU was determined with ELISA. Absorbance values were plotted as a function of time and relative proliferation rates were estimated from the slope of a line (Fig. 3B and C). Both in the presence or absence of EGF relative proliferation rate was slower in keratinocytes expressing the risk allele of CCHCR1 when compared with wild-type keratinocytes. There was no obvious difference in proliferation rate between cells expressing the risk allele and non-risk allele of CCHCR1.

CCHCR1 does not affect cell migration or skin explant outgrowths
We studied migration in keratinocytes isolated from the newborn transgenic and wild-type mice using the Transwell chambers (Costar) or in vitro scratch system (24,25). Radial outgrowth of mouse epidermis was also measured using the recently described skin explant model (26,27) in which re-epithelialization of acellular dermis is demonstrated by sequential fluorescence imaging. According to these experiments, the different alleles of CCHCR1 did not affect cell migration (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
There is unequivocal evidence that PSORS1 is by far the strongest genetic determinant of psoriasis, and it is the only one detected in all genetic mapping and association studies. However, identifying the effector gene within PSORS1 has turned out remarkably difficult. While the association of HLA-Cw6 to psoriasis has been known for over two decades, more recent association mapping results first excluded HLA-C and implicated a region including the CDSN and CCHCR1 genes (1,28–31). Most recently, however, the strongest association has again been attributed to markers near but outside the coding region of HLA-C (4,32–34). Genetic mapping alone may not answer the question about the effector gene, because long-range regulatory effects are documented for a number of loci. Such mechanisms, however, have not been studied for PSORS1. Studies on SNP or haplotype effects on gene expression are technically challenging, because only some cell types in skin express the main functional candidates HLA-C, CCHCR1 and CDSN, and cell-type-specific regulatory effects may get diluted if whole skin samples are used. As another line of investigation, it is well motivated to test the hypothesis of the impact of each of these genes through functional studies, an approach that we have chosen to understand better the functions of CCHCR1.

The first and so far only putative risk gene mapping to a confirmed locus for psoriasis susceptibility examined in a mouse model is the CCHCR1 (7). The CCHCR1 transgenic mice were phenotypically normal, but detailed analyses of gene expression in skin by Affymetrix array revealed genotype-dependent, allele-specific differences between the transgenic mice, such as upregulation of cytokeratins 6, 16 and 17, SPRRs (small proline-rich proteins) and certain matrix metalloproteinases in risk allele mice (7). These observations are compatible with a model that a susceptibility gene for psoriasis induces changes that are contributory but not sufficient alone to produce the clinical phenotype. This suggested that additional genes and/or external stimuli might be needed to trigger a phenotype in CCHCR1 mice. In the present study, wounding and TPA treatment were used to challenge the skin of transgenic CCHCR1 mice. These treatments did not lead to psoriasis-like skin phenotype. However, our experiments suggested that CCHCR1 may function as a negative regulator of keratinocyte proliferation. This function supports our previous hypothesis that downregulation of CCHCR1 may promote the pathogenesis of psoriasis (6).

In our earlier studies, we have highlighted a role for CCHCR1 in keratinocyte proliferation. In psoriatic skin, regions positive for the cell proliferation marker Ki67 were almost negative for CCHCR1 (Fig. 4) (3). Furthermore, expression of keratins 6, 16 and 17, all hyperproliferation markers of psoriasis, were altered in risk allele mice when compared with the non-risk mice or wild-type animals (7). Also our gene regulation studies suggested a role for the CCHCR1 gene in keratinocyte proliferation; agents, such as {gamma}-interferon, insulin and EGF involved in the regulation of proliferation were shown to affect CCHCR1 expression in keratinocytes (5,6). In the present study, we show that CCHCR1 indeed affects keratinocyte proliferation in transgenic skin. Immunostaining for Ki67 demonstrated that keratinocyte proliferation is reduced in risk allele animals when compared with non-risk animals. Similarly, cultured keratinocytes isolated from the skin of CCHCR1 mice proliferated more slowly than wild-type cells. In addition, the wound and TPA-induced keratinocyte proliferation was less prominent in risk allele mice than in other mouse groups. These results demonstrated that overexpression of CCHCR1*WWCC results in reduced keratinocyte proliferation in mice. In several mouse models, TPA treatment and wounding have been used to trigger psoriasis-like phenotypes (9,12,13). In transgenic CCHCR1 mice, these treatments did not lead to an apparent phenotype, in accordance with the hypothesis of CCHCR1 as a negative regulator of proliferation.


Figure 4
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  		Figure 4. CCHCR1 and the proliferation marker Ki67 are expressed in different areas in human psoriatic skin. Two different sections are shown (A and B). Regions positive for Ki67 are almost negative for CCHCR1. CCHCR1 is mainly expressed in basal and suprabasal cells overlying the dermal papillae, whereas Ki67 is expressed particularly where the rete ridges project into the dermis. Arrows indicate the most proliferating regions. Arrowheads show sites where CCHCR1 is highly expressed. Scale bars 50 µm.

 
In vivo wounding experiments suggested that re-epithelialization is faster in the skin of CCHCR1 non-risk allele mice than in the risk allele mice, but there was no difference in cell migration when studied with cultured cells using the Transwell chamber or in vitro scratch systems. The experiment with skin explants from transgenic mice did not reveal any abnormalities in epidermal outgrowth either. Differences in the composition of basement membrane and matrix, cell–matrix or cell–cell contacts or cytokine composition may explain why in vitro models did not reveal any aberrations although delayed early wound repair was noted in vivo.

We have recently proposed that CCHCR1 may influence the pathogenesis of psoriasis through vitamin D/steroid metabolism by interacting with the steroidogenic acute regulatory protein (StAR) and thus enhancing the synthesis of steroids (6). The function in steroid metabolism may partially explain the mechanism of CCHCR1 action in proliferation as steroids such as estrogen and progesterone are known to affect keratinocyte proliferation (35,36). Steroids can also modulate wound healing in human and mouse skin (37–39). Estrogen and testosterone affect the rate and quality of cutaneous wound healing (37,39) and the delay in wound healing observed in elderly patients can be improved by topical estrogen application (38). Interestingly, estradiol induces the expression of c-fos and c-jun genes that are relevant in the pathogenesis of psoriasis, as discussed in what follows. Furthermore, estrogen activates cyclin-D1, a protein important for the progression of cells through the G1 phase of the cell cycle (36).

Another hypothesis for the action of CCHCR1 in proliferation is based on the interaction of CCHCR1 with the RNA polymerase II subunit 3 (RPB3) (40). Recently CCHCR1 was shown to regulate migration of this nuclear protein from cytoplasm to nucleus. Interestingly, RPB3 interacts and activates the activating transcription factor 4 (ATF4 or CREB2) (41) that is able to form heterodimers with other members of the AP-1 family, including Jun and JunD. ATF4 expression is associated with growth arrest (42–44) and Jun and JunB are known to regulate keratinocyte proliferation (20). Furthermore, Jun proteins have been implicated in psoriasis in previous studies; JunB has an altered expression pattern in psoriatic skin when compared with normal healthy skin and double knock-out mice Jun/JunB develop spontaneously a psoriasis-like phenotype (11,13). Interestingly, both the cutaneous wounding and TPA treatment are known to activate AP-1-mediated signaling (15,18,45,46), which in turn regulates a large number of genes associated with cell proliferation including epidermal growth factor receptor (20). As the most evident antiproliferative effect was observed after wounding and TPA treatment of skin in transgenic mice, CCHCR1 might affect AP-1 signaling pathway through regulating RPB3 protein. However, there are several other possible signaling pathways that are relevant in cell proliferation and pathogenesis of psoriasis. Interleukin-23 was recently suggested to induce epidermal thickening and inflammation in mouse skin via STAT3 (Signal transducers and activators of transcription) signaling (47). We have now re-examined our previous Affymetrix data (7), but there were no significant differences in the expression of Stat3 and Il-23 related genes, including Il-12b (Interleukin-12 beta gene encoding subunit of IL-23) and Il-12rb1 (Interleukin-12 receptor beta 1 encoding subunit of IL-23 receptor), when transgenic CCHCR1 and wild-type mice were compared (t-test threshold P < 0.01). The Affymetrix result of Stat3 was confirmed by quantitative RT–PCR (Supplementary Material, Fig. S1). In addition to IL-23 several other cytokines, such as, IFN-{gamma}, TGF-β, IL-1-{alpha}, TNF-{alpha} and KGF can induce epidermal thickening and hyperproliferation in mouse skin, and a number of pathways relevant for the pathogenesis of psoriasis, including STAT1, STAT3, AP-1, NF-{kappa}B signaling, are activated by these cytokines (48,49). Further studies are indicated to understand the signaling pathways of CCHCR1 protein.

We have previously proposed that in addition to the allele specific effects, the amount of CCHCR1 protein may be critical for the pathogenesis of psoriasis (6). This was based on the observation that expression of CCHCR1 was altered in psoriasis patients; it was downregulated in cultured non-lesional keratinocytes of psoriatics when compared with normal healthy keratinocytes. The present study demonstrated that overexpression of both the risk and the non-risk allele form of CCHCR1 affects cell proliferation, but the expression of the risk allele has more obvious effects on cell proliferation. Taken together, we suggest that the downregulation of CCHCR1 in basal keratinocytes may result in enhanced cell proliferation in epidermis, which is relevant for the pathogenesis of psoriasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 
Transgenic mice
Generation of transgenic mice expressing either the normal allele or the psoriasis-associated risk allele of CCHCR1 has been described previously (7). Briefly, transgenic lines were established in FVB/N background using the K14-expression vector (PG3Z-K14 cassette). The K14 promoter predominantly targets transgene expression to basal keratinocytes and to outer root sheath keratinocytes of hair follicles. Multiple lines of transgenic mice were generated overexpressing CCHCR1 gene and two normal allele and three risk allele lines were studied in detail. For the present study, heterozygote mice from non-risk lines 12 and 34 or risk lines 106 and 132 (7) were crossed to produce homozygote mice for experiments. Experimental procedures performed in animals were approved by the Provincial State Office of Southern Finland (licence number STU402A).

Wounding experiments
Age-matched adult mice were anesthetized with ketamine hydrochloride (50 mg/kg s.c.) and xylazine hydrochloride (10 mg/kg s.c.). The backs of the mice were shaved and two-to-three 5 mm diameter circular full-thickness wounds were made using punch biopsy device on both sides of the back. At 1, 4, 11 and 30 days post wounding, mice were sacrificed and wound areas were measured. Four mice from each mouse group were used at each time point analyzed. Wound tissues were harvested, fixed in neutral formalin, dehydrated and embedded in paraffin. Hematoxylin and eosin (H&E)-stained sections were photographed and analyzed with Olympus BX41 microscope and computer using the Olympus DP-soft version 3.2 image program. Eight wounds per genotype were analyzed. Re-epithelialization was estimated on skin sections by measuring the distance between the original wound site (the edge of the muscular layer) and the leading edge of the epithelium.

TPA treatment
Mice were shaved on the backs and treated with 10 µg 12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma) in 100 µl acetone. The solution was applied over an area of ~2 x 2 cm. Four mice were used for each time point in each group. Animals were sacrificed at defined time points (24, 48 or 72 h) and the skin was processed for histological analysis. Two pieces of skin from each mouse were analyzed. Epidermal thickness (from the bottom of the stratum corneum to the basement membrane of the interfollicular epidermis) was measured in cross-sections using Olympus BX41 microscope and computer using the Olympus DP-soft version 3.2 image program. Four measurements were made on four-to-seven fields per mouse.

Immunohistochemistry
Paraffin sections of skin were processed according to standard procedures. Immunostainings of tissue sections were performed using the avidin–biotin–peroxidase complex method (Vectastain ABC Kit, Vector Laboratories or Histomouse-SP kit, Zymed) and following primary antibodies: mouse monoclonals for cytokeratin 6 (Labvision co, Neomarkers), bromodeoxyuridine (Dako) and {alpha}-smooth muscle actin (Sigma), and rabbit polyclonals for cytokeratin 10 (Covance, PRB159P), Ki67 (Novocastra, NCL-Ki67p), laminin 5 (Pyke et al. 1994), von Willebrand factor (DakoCytomation) and CCHCR1 (Asumalahti 2002). Sections were pretreated with trypsin (10 mg/ml; HCR) or by microwaving in citrate buffer (Ki67 and BrdU). Aminoethylcarbazole or diaminobenzidine were used as chromogenic substrates and hematoxylin for counterstaining. Controls with preimmune sera or normal rabbit immunoglobulin were used as negative controls. Cell apoptosis was visualized using the TUNEL labeling system ApoTag Peroxidase In Situ Apoptosis Detection Kit (Chemicon). The number of Ki67 positive cells was determined on two-to-three sections per animal and on five-to-seven fields of each section. Immunohistochemistry of human psoriatic skin samples with antibodies against CCHCR1 and Ki67 has been described previously (3).

In vivo cell proliferation assay with untreated mice
Epithelial cell proliferation was measured by intraperitoneal injection of BrdU (Roche) 50 mg/kg body weight. Four mice were used for each time point in each group. Mice were sacrificed 2 or 6 h after injection and BrdU incorporation was detected by immunohistochemical staining with mouse anti-BrdU monoclonal antibody (Dako). The number of BrdU positive cells relative to total number of epidermal keratinocytes on field were determined on two-to-three sections per animal and on five-to-seven fields of each section.

Isolation and proliferation assay of primary mouse keratinocytes
Keratinocytes were isolated from 0- to 3-day-old transgenic mice or from wild-type animals as described elsewhere (50), with modifications. Mice were sacrificed, the skin was removed and incubated in 0.25% dispase in Earle's balanced salt solution (EBSS) overnight at 4°C. The following day, epidermis was separated from dermis, washed and incubated in 0.25% trypsin solution 15 min 37°C. Epidermal pieces were suspended by pipetting in keratinocyte growth medium (KGM-2 media supplemented with growth factors, Clonetics, Cambrex Bio Science Walkersville, Inc., MD, USA) supplemented with 8% decalcified FCS and penicillin–streptomycin and centrifuged (200g, 10 min). Pelleted keratinocytes were resuspended in KGM-2 medium supplemented with 2% decalcified FCS and penicillin–streptomycin, counted and seeded (5000 or 10 000 cells) in 96-well plates for cell proliferation assays. For proliferation assay, cells were cultured in the presence or absence of EGF (15 ng/ml, Sigma) for 2–3 days after which cells were labeled 2, 4, 8 or 24 h with 10 µM BrdU (Roche). The amount of incorporated BrdU was determined with colorimetric ELISA system (Roche Diagnostics, Germany) according to the manufacturer's instructions. Absorbance (450 nm) values were plotted as a function of time and proliferation rates ({Delta}A450 nm/{Delta}h) were estimated from the slope of a line.

In vitro cell migration assays
Keratinocytes isolated from skin of newborn mice were counted and plated in 6-well plates or in Transwell inserts (8 µm membrane, Costar) that were coated with 10 µg/cm2 rat collagen type I (Sigma). Cells in 6-well plates were cultured in the presence of absence of 15 ng/ml EGF (Sigma) 2–3 days until they were 70–90% confluent on the day of experiment. For examining cell motility, a cell free area was introduced by scraping the cell layer with a pipette tip (24,25). After 24 and 48 h of migration, the cell-free area was evaluated. Photographs were taken using a phase contrast microscope. Cells in Transwell chambers were let to migrate 24 or 48 h after which migrated cells were fixed with methanol and stained with hematoxylin. Inserts were mounted and migrated cells were counted with microcope. The rate of re-epithelialization was studied from mouse skin explants using fluorescence imaging technique (26,27).

Statistical analysis
All statistical analyses were made using Microsoft Office Excel. Statistical comparisons between data sets were made with Student's t-test and P < 0.05 was considered significant.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 REFERENCES
 
Supplementary material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
REFERENCES
 
This study was supported by the Academy of Finland, the Sigrid Juselius Foundation, Finska Läkaresällskapet, the Finnish Konkordia Fund (IT), the Maud Kuistila Memorial Foundation (IT), the Biomedicum Helsinki Foundation (IT) and the Helsinki University Hospital Research Fund (TYH 4226 and TYH 7113), Finland and the Swedish Research Council, the Swedish Psoriasis Association and the Finsen-Welander Foundation, Sweden.


   ACKNOWLEDGEMENTS
 
We thank Hong Jiao for her help with Affymetrix data and Ranja Eklund and Alli Tallqvist for their excellent technical assistance.

Conflict of Interest statement: none declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 REFERENCES
 

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