The PSORS1 locus gene CCHCR1 affects keratinocyte proliferation in transgenic mice

The PSORS1 locus gene CCHCR1 affectskeratinocyte proliferation in transgenic mice

Inkeri Tiala1, Janica Wakkinen1, Sari Suomela2, Pauli Puolakkainen3, Raija Tammi4,

Sofi Forsberg5, Ola Rollman5, Kati Kainu1,2, Bjorn Rozell6,7,8, Juha Kere1,6,8,�,

Ulpu Saarialho-Kere2,9 and Outi Elomaa1

1Department of Medical Genetics, 2Department of Dermatology and 3Department of Surgery, University of Helsinki

and Helsinki University Central Hospital, Helsinki, Finland, 4Department of Anatomy, University of Kuopio, Finland,5Department of Medical Sciences, Dermatology and Venereology, Uppsala University Hospital, Sweden, 6Division of

Clinical Research Center, 7Division of Pathology and 8Division of Biosciences and Nutrition, Department of Laboratory

Medicine, Huddinge University Hospital, Stockholm, Sweden and 9Department of Dermatology, Karolinska Institutet at

Stockholm Soder Hospital, Stockholm, Sweden

Received September 11, 2007; Revised and Accepted December 21, 2007

The CCHCR1 gene (Coiled-Coil a-Helical Rod protein 1) within the major psoriasis susceptibility locusPSORS1 is a plausible candidate gene for the risk effect. We have previously generated transgenic mice over-expressing either the psoriasis-associated risk allele CCHCR1�WWCC or the normal allele of CCHCR1. Alltransgenic CCHCR1 mice appeared phenotypically normal, but exhibited altered expression of genes relevantto 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 1and 4 was delayed, and TPA-induced epidermal hyperproliferation was less pronounced in mice with theCCHCR1�WWCC risk allele than in mice with the normal allele or in wild-type animals. Finally, we demon-strated that overexpression of CCHCR1 affects basal keratinocyte proliferation in mice; CCHCR1�WWCCmice had less proliferating keratinocytes than the non-risk allele mice. Similarly, keratinocytes isolatedfrom risk allele mice proliferated more slowly in culture than wild-type cells when measured by BrdU labelingand 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 featureof psoriatic epidermis.

INTRODUCTION

The most important region for psoriasis predisposition isPSORS1 on chromosome 6p near the HLA-C gene (1,2).Three psoriasis-associated susceptibility alleles HLACw6,CCHCR1�WWCC and CDSN�5 (corneodesmosin) have beenidentified within it, but strong linkage disequilibrium hasmade it difficult to distinguish their individual genetic effect(3,4). In our previous studies, we have demonstrated that theCCHCR1 gene (Coiled-Coil a-Helical Rod protein 1, earlier

known as HCR) may be an essential gene for the effect ofPSORS1 in psoriasis. CCHCR1 protein is differentiallyexpressed in psoriatic lesions compared with healthy skin orother hyperproliferative skin disorders, such as eczema (3,5).Furthermore, we have demonstrated that the expression ofCCHCR1 is downregulated in cultured non-lesional psoriatickeratinocytes when compared with non-psoriatic cells (6).We have recently generated transgenic mice expressingeither the psoriasis-associated risk allele or the normal alleleof CCHCR1 under control of cytokeratin 14 (K14) promoter.

�To whom correspondence should be addressed at: Department of Biosciences and Nutrition, Karolinska Institutet, 14157 Huddinge, Sweden. Tel: þ46734213550; Fax: þ46 87745538; Email: [email protected]

# The Author 2008. Published by Oxford University Press. All rights reserved.For Permissions, please email: [email protected]

Human Molecular Genetics, 2008, Vol. 17, No. 7 1043–1051doi:10.1093/hmg/ddm377Advance Access published on January 3, 2008

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Although transgenic mice were phenotypically and histologi-cally normal, overexpression of CCHCR1 in mouse skinresulted in altered expression of several genes relevant inthe pathogenesis of psoriasis (7).

Many mouse models exhibit a psoriasis-like phenotype, butnone has shown all of the typical histological and immunophe-notypical abnormalities of psoriatic lesions (8). The majorityof engineered mice are transgenic, overexpressing genes thatexhibit altered expression in psoriatic lesions. The most com-plete animal models to date are the K5-Stat3 (Signal transdu-cers and activators of transcription) (9) and the Tie2 (Tyrosinekinase receptor) (10) transgenic mice and the JunB/c-Jundouble knock out mouse (11) which all develop a spontaneousphenotype, but none of these genes map in human to thePSORS 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-likephenotypes in mouse models (9,12,13). Both manipulationsare able to induce biological reactions persistently occurringin psoriatic lesions. These include processes such as prolifer-ation, migration and re-stratification of keratinocytes, in-flammation and neovascularization (14,15). Furthermore,wounding of healthy skin in psoriatic patients can trigger acomplete psoriatic response, in a classic reaction termed theKoebner phenomenon (16). The mechanisms of TPA actionleading to epidermal hypertrophy and hyperplasia are lesswell understood. One of the possible mechanisms involvesactivation of protein kinase C (17,18), which in turn regulatestranscription factors, including Jun proteins that play a criticalrole in cell cycle control (19,20).

In the present study, we investigated the effects of TPA andwounding of skin on CCHCR1 transgenic mice. These treat-ments revealed that CCHCR1 affects keratinocyte prolifer-ation in transgenic mice. Wound healing was delayed inmice with the psoriasis associated risk allele of CCHCR1and TPA-induced epidermal hyperproliferation was less pro-nounced in transgenic mice than in wild-type animals. Simul-taneously, the number of proliferating Ki67 positive cells wasreduced in CCHCR1 mice. Finally, we demonstrated that theoverexpression of CCHCR1 affects also normal keratinocyteproliferation in mice. Immunostaining for Ki67 in untreatedmice revealed that CCHCR1�WWCC allele mice had less pro-liferating basal keratinocytes than the non-risk allele mice.Similarly keratinocytes isolated from risk allele mice prolifer-ated more slowly than wild-type cells in culture. We proposethat CCHCR1 participates in regulating keratinocyte prolifer-ation and that disturbance of this function may contribute tothe pathophysiology of psoriasis.

RESULTS

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-typeand transgenic mice expressing either the risk or the normalallele of the CCHCR1 transgene (7). On days 1, 4, 11 and30 post-wounding the mice were sacrificed, wound areaswere measured and skin sections processed for histologicalstudies. Eight wounds from each group were studied at each

time point. Wound size measurements on day 1 and 4suggested that early wound closure was delayed in micewith the CCHCR1�WWCC allele when compared withnon-risk allele mice (day 1, P , 0.01 and day 4, P , 0.05;Fig. 1A). Also, histological evaluation and measurement ofnewly formed epithelium supported the macroscopic results(Fig. 1B–D). On day 4, wounds in risk allele mice were stilllargely devoid of the epithelial layer, whereas in non-riskmice neoepidermis was visible already on day 1 (data notshown) and complete re-epithelialization was seen in 50% ofthe wounds on day 4. On day 11, wounds were closed andregeneration of the epidermis was complete in all mousegroups. During the observation period of 30 days, no clinicalor histological features of psoriasis developed in woundedskin.

Histological evaluation of dermal components inHE-stained wounds based on the modified Greenhalg criteria(21,22) did not reveal any abnormalities in inflammation, for-mation of granulation tissues or neovascularization (data notshown). This suggested that the primary cause of retardedwound healing might be impaired regeneration of theepidermis which involves proliferation and migration of kera-tinocytes. Cytoplasmic laminin-5 staining for migrating kera-tinocytes (23) or TUNEL staining for apoptosis did notreveal any differences between mice groups. Immunostainingfor Ki67 on day 1 demonstrated only a few proliferative ker-atinocytes next to the wound edges and there was no differ-ence between the groups. On day 4, however risk allelemice had significantly fewer proliferating keratinocytes innewly formed epidermis than wild-type or non-risk allelemice (P , 0.001) (Fig. 1E), suggesting that this could be thereason for delayed wound closure in risk allele mice. Onday 11, the number of Ki67-positive cells was lower in riskmice than non-risk mice (P ¼ 0.01) and still on day 30 asimilar trend was observed (data not shown). Staining formast cells (toluidine blue) and immunohistochemical analyseswith antibodies against cytokeratin 6 (hyperproliferation),a-smooth muscle actin (myofibroblasts) and von Willebrandfactor (neoangiogenesis) did not reveal any abnormalities intransgenic mice.

Single-dose TPA application

To induce epidermal hyperplasia, dorsal skin was treated withTPA. Skin samples were harvested 24, 48 or 72 h after treat-ment and analyzed histologically. A single topical applicationof TPA resulted in increased thickening of epidermis in allgroups already 24 h after application. However, the TPA-treatment of transgenic CCHCR1 mice showed a less well-developed epidermal hyperplasia when compared withwild-type controls. The difference was most evident betweenmouse groups 48 h after treatment (Fig. 2A and B) when therisk allele mice exhibited an epidermis of almost normal thick-ness. Immunostaining with Ki67 revealed that at that timepoint the number of proliferating cells was 20–30% lowerin the epidermis of transgenic CCHCR1 mice than in wild-type animals. In transgenic mice, Ki67-positive cells wereconfined mainly to the basal layer of epidermis, whereas inwild-type mice proliferation was observed also suprabasally(Fig. 2C). Comparison between transgenic mice demonstrated

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that the number of Ki67 positive cells was 14% lower in riskallele mice than in non-risk mice (P , 0.05) (Fig. 2D) 48 hafter treatment. TPA is known to induce an inflammatoryresponse in the skin (17,18), but there was no difference inthe number of infiltrating T-cells between mice groups (datanot shown). Immunostaining for the keratinocyte differen-tiation marker cytokeratin 10 did not reveal any differencesbetween mouse groups (data not shown).

Normal cell proliferation in untreated skinand cultured keratinocytes

To study the effects of CCHCR1 on normal keratinocyte pro-liferation, untreated mice were injected intraperitoneally withbromodeoxyuridine (BrdU) and labeled cells were detected byimmunohistochemistry. After 6 h labeling, the proportion ofBrdU positive cells was 63% in the risk allele mice and 77and 73% in the non-risk allele and wild-type mice, respect-ively, suggesting that cell proliferation was impaired in riskmice when compared with the non-risk mice (n ¼ 4, P ,0.05) (Fig. 3A). Immunostaining for Ki67 showed a similartrend (n ¼ 3) in cell proliferation of untreated skin; thenumber of proliferating cells was decreased in CCHCR1 riskallele mice when compared with non-risk mice (data notshown).

Cultured keratinocytes derived from newborn transgenicCCHCR1 mice or from wild-type animals were used toexamine proliferation. Cells were labeled at different time

points with BrdU in the presence or absence of EGF afterwhich the amount of incorporated BrdU was determinedwith ELISA. Absorbance values were plotted as a functionof time and relative proliferation rates were estimated fromthe slope of a line (Fig. 3B and C). Both in the presence orabsence of EGF relative proliferation rate was slower in kera-tinocytes expressing the risk allele of CCHCR1 when com-pared with wild-type keratinocytes. There was no obviousdifference in proliferation rate between cells expressing therisk allele and non-risk allele of CCHCR1.

CCHCR1 does not affect cell migrationor skin explant outgrowths

We studied migration in keratinocytes isolated from thenewborn transgenic and wild-type mice using the Transwellchambers (Costar) or in vitro scratch system (24,25). Radialoutgrowth of mouse epidermis was also measured using therecently described skin explant model (26,27) in whichre-epithelialization of acellular dermis is demonstrated bysequential fluorescence imaging. According to these exper-iments, the different alleles of CCHCR1 did not affect cellmigration (data not shown).

DISCUSSION

There is unequivocal evidence that PSORS1 is by far the stron-gest genetic determinant of psoriasis, and it is the only one

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 theCCHCR1�WWCC risk allele, suggesting that early wound closure is delayed in risk allele mice. The largest difference was seen between the transgenicCCHCR1 animals (day 1, P , 0.01 and day 4, P , 0.05). Histological evaluation (B–D) and measurement of migrating epithelium on sections (B) supportedthe macroscopic results. On day 4, the distance (mm) between the original wound site (arrow head showing the edge of the muscular layer) and the leading edgeof the epithelium (arrows) was shorter in CCHCR1 risk allele mice (D) than in non-risk (C) mice (P , 0.001, scale bars 200 mm). 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 day4. Eight wounds from each mouse group were analyzed at each time point. Error bars represent mean+SEM. Statistical comparisons between data sets weremade with Student’s t-test (��P , 0.01, �P , 0.05).

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detected in all genetic mapping and association studies.However, identifying the effector gene within PSORS1 hasturned out remarkably difficult. While the association ofHLA-Cw6 to psoriasis has been known for over two decades,more recent association mapping results first excludedHLA-C and implicated a region including the CDSN andCCHCR1 genes (1,28–31). Most recently, however, the stron-gest association has again been attributed to markers near butoutside the coding region of HLA-C (4,32–34). Geneticmapping alone may not answer the question about the effectorgene, because long-range regulatory effects are documentedfor a number of loci. Such mechanisms, however, have notbeen studied for PSORS1. Studies on SNP or haplotypeeffects on gene expression are technically challenging,because only some cell types in skin express the main func-tional candidates HLA-C, CCHCR1 and CDSN, andcell-type-specific regulatory effects may get diluted if wholeskin samples are used. As another line of investigation, it iswell motivated to test the hypothesis of the impact of each

of these genes through functional studies, an approach thatwe have chosen to understand better the functions ofCCHCR1.

The first and so far only putative risk gene mapping to aconfirmed locus for psoriasis susceptibility examined in amouse model is the CCHCR1 (7). The CCHCR1 transgenicmice were phenotypically normal, but detailed analyses ofgene expression in skin by Affymetrix array revealedgenotype-dependent, allele-specific differences between thetransgenic mice, such as upregulation of cytokeratins 6, 16and 17, SPRRs (small proline-rich proteins) and certainmatrix metalloproteinases in risk allele mice (7). These obser-vations are compatible with a model that a susceptibility genefor psoriasis induces changes that are contributory but not suf-ficient alone to produce the clinical phenotype. This suggestedthat additional genes and/or external stimuli might be neededto trigger a phenotype in CCHCR1 mice. In the present study,wounding and TPA treatment were used to challenge the skinof transgenic CCHCR1 mice. These treatments did not lead to

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 thickeningof epidermis in all mice groups already 24 h after application (A). However, after TPA treatment the epidermis of transgenic mice was significantly thinner thanin wild-type mice. The difference was most evident between mouse groups 48 h after treatment when mice with the CCHCR1 risk allele (B) exhibited thinnerepidermis 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 waslower 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 animalsrevealed 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 proliferativeKi67-positive cells were confined mainly to the basal layer of epidermis, whereas in wild-type mice proliferation was also observed suprabasally (C). Eight skinsamples from each mouse group were analyzed at each time point. Error bars represent mean+SEM. Statistical comparisons between data sets were made withStudent’s t-test (��P , 0.01, �P , 0.05). Scale bars 50 mm.




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psoriasis-like skin phenotype. However, our experimentssuggested that CCHCR1 may function as a negative regulatorof keratinocyte proliferation. This function supports our pre-vious hypothesis that downregulation of CCHCR1 maypromote the pathogenesis of psoriasis (6).

In our earlier studies, we have highlighted a role forCCHCR1 in keratinocyte proliferation. In psoriatic skin,regions positive for the cell proliferation marker Ki67 werealmost negative for CCHCR1 (Fig. 4) (3). Furthermore,expression of keratins 6, 16 and 17, all hyperproliferationmarkers of psoriasis, were altered in risk allele mice whencompared with the non-risk mice or wild-type animals (7).Also our gene regulation studies suggested a role for theCCHCR1 gene in keratinocyte proliferation; agents, such asg-interferon, insulin and EGF involved in the regulation ofproliferation were shown to affect CCHCR1 expression in ker-atinocytes (5,6). In the present study, we show that CCHCR1indeed affects keratinocyte proliferation in transgenic skin.Immunostaining for Ki67 demonstrated that keratinocyte pro-liferation is reduced in risk allele animals when compared withnon-risk animals. Similarly, cultured keratinocytes isolatedfrom the skin of CCHCR1 mice proliferated more slowlythan wild-type cells. In addition, the wound and TPA-inducedkeratinocyte proliferation was less prominent in risk allelemice than in other mouse groups. These results demonstratedthat overexpression of CCHCR1�WWCC results in reducedkeratinocyte proliferation in mice. In several mouse models,TPA treatment and wounding have been used to triggerpsoriasis-like phenotypes (9,12,13). In transgenic CCHCR1mice, these treatments did not lead to an apparent phenotype,in accordance with the hypothesis of CCHCR1 as a negativeregulator of proliferation.

In vivo wounding experiments suggested that re-epithelialization is faster in the skin of CCHCR1 non-riskallele mice than in the risk allele mice, but there was no differ-ence in cell migration when studied with cultured cells usingthe Transwell chamber or in vitro scratch systems. The exper-iment with skin explants from transgenic mice did not revealany abnormalities in epidermal outgrowth either. Differencesin the composition of basement membrane and matrix, cell–matrix or cell–cell contacts or cytokine composition mayexplain why in vitro models did not reveal any aberrationsalthough delayed early wound repair was noted in vivo.

We have recently proposed that CCHCR1 may influence thepathogenesis of psoriasis through vitamin D/steroid metab-olism by interacting with the steroidogenic acute regulatoryprotein (StAR) and thus enhancing the synthesis of steroids(6). The function in steroid metabolism may partiallyexplain the mechanism of CCHCR1 action in proliferationas steroids such as estrogen and progesterone are known toaffect keratinocyte proliferation (35,36). Steroids can alsomodulate wound healing in human and mouse skin (37–39).Estrogen and testosterone affect the rate and quality ofcutaneous wound healing (37,39) and the delay in woundhealing observed in elderly patients can be improved bytopical estrogen application (38). Interestingly, estradiolinduces the expression of c-fos and c-jun genes that are rel-evant in the pathogenesis of psoriasis, as discussed in whatfollows. Furthermore, estrogen activates cyclin-D1, a proteinimportant for the progression of cells through the G1 phaseof the cell cycle (36).

Another hypothesis for the action of CCHCR1 in prolifer-ation is based on the interaction of CCHCR1 with the RNApolymerase II subunit 3 (RPB3) (40). Recently CCHCR1was shown to regulate migration of this nuclear protein from

Figure 3. Cell proliferation in untreated skin of transgenic CCHCR1 and wild-type mice. In in vivo proliferation assay, four mice from each mouse groupwere injected intraperitoneally with BrdU and labeled cells were detectedby immunohistochemistry (A). The proportion of proliferating cells waslower in mice with the CCHCR�WWCC risk allele than mice with non-riskallele (P , 0.05) mice after 6 h labeling. Proliferation of cultured keratino-cytes derived from transgenic CCHCR1 mice or from wild-type animals (Band C). Cells were labeled for 2, 4, 8 or 24 h with BrdU in the presence orabsence of EGF after which the amount of incorporated BrdU was determinedwith ELISA. Absorbance (450 nm) values were plotted as a function of timeand relative proliferation rates (DA450nm/Dh) were estimated from the slopeof a line. Both in the presence (B) and absence of EGF (C), the relative pro-liferation rate was slower in keratinocytes expressing the risk allele ofCCHCR1 when compared with wild-type keratinocytes. There was noobvious difference in proliferation rate between cells expressing the riskallele and non-risk allele of CCHCR1. Error bars represent mean+SEM.




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cytoplasm to nucleus. Interestingly, RPB3 interacts and acti-vates the activating transcription factor 4 (ATF4 or CREB2)(41) that is able to form heterodimers with other members ofthe AP-1 family, including Jun and JunD. ATF4 expressionis associated with growth arrest (42–44) and Jun and JunBare known to regulate keratinocyte proliferation (20). Further-more, Jun proteins have been implicated in psoriasis in pre-vious studies; JunB has an altered expression pattern inpsoriatic skin when compared with normal healthy skin anddouble knock-out mice Jun/JunB develop spontaneously apsoriasis-like phenotype (11,13). Interestingly, both thecutaneous wounding and TPA treatment are known to activateAP-1-mediated signaling (15,18,45,46), which in turn regu-lates a large number of genes associated with cell proliferationincluding epidermal growth factor receptor (20). As the mostevident antiproliferative effect was observed after woundingand TPA treatment of skin in transgenic mice, CCHCR1might affect AP-1 signaling pathway through regulatingRPB3 protein. However, there are several other possible sig-naling pathways that are relevant in cell proliferation andpathogenesis of psoriasis. Interleukin-23 was recentlysuggested to induce epidermal thickening and inflammationin mouse skin via STAT3 (Signal transducers and activatorsof transcription) signaling (47). We have now re-examinedour previous Affymetrix data (7), but there were no significantdifferences in the expression of Stat3 and Il-23 related genes,including Il-12b (Interleukin-12 beta gene encoding subunit ofIL-23) and Il-12rb1 (Interleukin-12 receptor beta 1 encodingsubunit of IL-23 receptor), when transgenic CCHCR1 andwild-type mice were compared (t-test threshold P , 0.01).The Affymetrix result of Stat3 was confirmed by quantitativeRT–PCR (Supplementary Material, Fig. S1). In addition toIL-23 several other cytokines, such as, IFN-g, TGF-b,IL-1-a, TNF-a and KGF can induce epidermal thickening

and hyperproliferation in mouse skin, and a number of path-ways relevant for the pathogenesis of psoriasis, includingSTAT1, STAT3, AP-1, NF-kB signaling, are activated bythese cytokines (48,49). Further studies are indicated to under-stand the signaling pathways of CCHCR1 protein.

We have previously proposed that in addition to the allelespecific effects, the amount of CCHCR1 protein may be criti-cal for the pathogenesis of psoriasis (6). This was based on theobservation that expression of CCHCR1 was altered in psoria-sis patients; it was downregulated in cultured non-lesional ker-atinocytes of psoriatics when compared with normal healthykeratinocytes. The present study demonstrated that overex-pression of both the risk and the non-risk allele form ofCCHCR1 affects cell proliferation, but the expression of therisk allele has more obvious effects on cell proliferation.Taken together, we suggest that the downregulation ofCCHCR1 in basal keratinocytes may result in enhanced cellproliferation in epidermis, which is relevant for the pathogen-esis of psoriasis.

MATERIALS AND METHODS

Transgenic mice

Generation of transgenic mice expressing either the normalallele or the psoriasis-associated risk allele of CCHCR1 hasbeen described previously (7). Briefly, transgenic lines wereestablished in FVB/N background using the K14-expressionvector (PG3Z-K14 cassette). The K14 promoter predomi-nantly targets transgene expression to basal keratinocytesand to outer root sheath keratinocytes of hair follicles. Mul-tiple lines of transgenic mice were generated overexpressingCCHCR1 gene and two normal allele and three riskallele lines were studied in detail. For the present study,

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 siteswhere CCHCR1 is highly expressed. Scale bars 50 mm.




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heterozygote mice from non-risk lines 12 and 34 or risk lines106 and 132 (7) were crossed to produce homozygote mice forexperiments. Experimental procedures performed in animalswere approved by the Provincial State Office of SouthernFinland (licence number STU402A).

Wounding experiments

Age-matched adult mice were anesthetized with ketaminehydrochloride (50 mg/kg s.c.) and xylazine hydrochloride(10 mg/kg s.c.). The backs of the mice were shaved andtwo-to-three 5 mm diameter circular full-thickness woundswere made using punch biopsy device on both sides of theback. At 1, 4, 11 and 30 days post wounding, mice were sacri-ficed and wound areas were measured. Four mice from eachmouse group were used at each time point analyzed. Woundtissues were harvested, fixed in neutral formalin, dehydratedand embedded in paraffin. Hematoxylin and eosin(H&E)-stained sections were photographed and analyzedwith Olympus BX41 microscope and computer using theOlympus DP-soft version 3.2 image program. Eight woundsper genotype were analyzed. Re-epithelialization was esti-mated on skin sections by measuring the distance betweenthe original wound site (the edge of the muscular layer) andthe leading edge of the epithelium.

TPA treatment

Mice were shaved on the backs and treated with 10 mg12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma) in100 ml acetone. The solution was applied over an area of�2 � 2 cm. Four mice were used for each time point ineach group. Animals were sacrificed at defined time points(24, 48 or 72 h) and the skin was processed for histologicalanalysis. Two pieces of skin from each mouse were analyzed.Epidermal thickness (from the bottom of the stratum corneumto the basement membrane of the interfollicular epidermis)was measured in cross-sections using Olympus BX41 micro-scope and computer using the Olympus DP-soft version 3.2image program. Four measurements were made onfour-to-seven fields per mouse.

Immunohistochemistry

Paraffin sections of skin were processed according to standardprocedures. Immunostainings of tissue sections were per-formed using the avidin–biotin–peroxidase complex method(Vectastain ABC Kit, Vector Laboratories or Histomouse-SPkit, Zymed) and following primary antibodies: mouse mono-clonals for cytokeratin 6 (Labvision co, Neomarkers), bromo-deoxyuridine (Dako) and a-smooth muscle actin (Sigma), andrabbit polyclonals for cytokeratin 10 (Covance, PRB159P),Ki67 (Novocastra, NCL-Ki67p), laminin 5 (Pyke et al.1994), von Willebrand factor (DakoCytomation) andCCHCR1 (Asumalahti 2002). Sections were pretreated withtrypsin (10 mg/ml; HCR) or by microwaving in citratebuffer (Ki67 and BrdU). Aminoethylcarbazole or diaminoben-zidine were used as chromogenic substrates and hematoxylinfor counterstaining. Controls with preimmune sera or normalrabbit immunoglobulin were used as negative controls. Cell

apoptosis was visualized using the TUNEL labeling systemApoTag Peroxidase In Situ Apoptosis Detection Kit (Chemi-con). The number of Ki67 positive cells was determined ontwo-to-three sections per animal and on five-to-seven fieldsof each section. Immunohistochemistry of human psoriaticskin samples with antibodies against CCHCR1 and Ki67 hasbeen described previously (3).

In vivo cell proliferation assay with untreated mice

Epithelial cell proliferation was measured by intraperitonealinjection of BrdU (Roche) 50 mg/kg body weight. Four micewere used for each time point in each group. Mice were sacri-ficed 2 or 6 h after injection and BrdU incorporation wasdetected by immunohistochemical staining with mouse anti-BrdU monoclonal antibody (Dako). The number of BrdU posi-tive cells relative to total number of epidermal keratinocyteson field were determined on two-to-three sections per animaland on five-to-seven fields of each section.

Isolation and proliferation assay of primarymouse keratinocytes

Keratinocytes were isolated from 0- to 3-day-old transgenicmice or from wild-type animals as described elsewhere (50),with modifications. Mice were sacrificed, the skin wasremoved and incubated in 0.25% dispase in Earle’s balancedsalt solution (EBSS) overnight at 48C. The following day, epi-dermis was separated from dermis, washed and incubated in0.25% trypsin solution 15 min 378C. Epidermal pieces weresuspended by pipetting in keratinocyte growth medium(KGM-2 media supplemented with growth factors, Clonetics,Cambrex Bio Science Walkersville, Inc., MD, USA) sup-plemented with 8% decalcified FCS and penicillin–streptomy-cin and centrifuged (200g, 10 min). Pelleted keratinocyteswere resuspended in KGM-2 medium supplemented with 2%decalcified FCS and penicillin–streptomycin, counted andseeded (5000 or 10 000 cells) in 96-well plates for cell pro-liferation assays. For proliferation assay, cells were culturedin 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 with10 mM BrdU (Roche). The amount of incorporated BrdU wasdetermined with colorimetric ELISA system (Roche Diagnos-tics, Germany) according to the manufacturer’s instructions.Absorbance (450 nm) values were plotted as a function oftime and proliferation rates (DA450nm/Dh) were estimatedfrom the slope of a line.

In vitro cell migration assays

Keratinocytes isolated from skin of newborn mice werecounted and plated in 6-well plates or in Transwell inserts(8 mm membrane, Costar) that were coated with 10 mg/cm2

rat collagen type I (Sigma). Cells in 6-well plates were cul-tured in the presence of absence of 15 ng/ml EGF (Sigma)2–3 days until they were 70–90% confluent on the day ofexperiment. For examining cell motility, a cell free area wasintroduced by scraping the cell layer with a pipette tip(24,25). After 24 and 48 h of migration, the cell-free areawas evaluated. Photographs were taken using a phase contrast




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microscope. Cells in Transwell chambers were let to migrate24 or 48 h after which migrated cells were fixed with methanoland stained with hematoxylin. Inserts were mounted andmigrated cells were counted with microcope. The rate ofre-epithelialization was studied from mouse skin explantsusing fluorescence imaging technique (26,27).

Statistical analysis

All statistical analyses were made using Microsoft OfficeExcel. Statistical comparisons between data sets were madewith Student’s t-test and P , 0.05 was considered significant.

SUPPLEMENTARY MATERIAL

Supplementary material is available at HMG Online.

ACKNOWLEDGEMENTS

We thank Hong Jiao for her help with Affymetrix data andRanja Eklund and Alli Tallqvist for their excellent technicalassistance.

Conflict of Interest statement: none declared.

FUNDING

This study was supported by the Academy of Finland, theSigrid Juselius Foundation, Finska Lakaresallskapet, theFinnish Konkordia Fund (IT), the Maud Kuistila MemorialFoundation (IT), the Biomedicum Helsinki Foundation (IT)and the Helsinki University Hospital Research Fund (TYH4226 and TYH 7113), Finland and the Swedish ResearchCouncil, the Swedish Psoriasis Association and the Finsen-Welander Foundation, Sweden.

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The PSORS1 locus gene CCHCR1 affects keratinocyte proliferation in transgenic mice

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