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Interferon α-Inducible Protein 27 (IFI27) is Upregulated in Psoriatic Skin and Certain Epithelial Cancers

      IFI27 is an interferon α-inducible protein found to be upregulated in lesional and non-lesional psoriatic skin in a gene array study. To further characterize its function, we studied by in situ hybridization whether IFI27 is expressed in psoriasis, other inflammatory skin diseases, and wound repair in vivo. We also examined its regulation by different growth factors and anti-psoriatic agents using quantitative RT-PCR (TaqMan). IFI27 mRNA expression was highly upregulated in lesional psoriatic epidermis and also detected in non-lesional keratinocytes. It was also expressed in lichen planus, chronic eczema, cutaneous squamous cell cancers, and during normal wound repair when IFI27 was found in the proliferating subpopulation of keratinocytes. A 3–17-fold upregulation of IFI27 mRNA expression was observed when keratinocytes were stimulated with IFN-γ, TNF-α, or TGF-β1 while retinoids and vitamin D downregulated its expression. Our results suggest that IFI27 is a novel marker of epithelial proliferation and cancer.

      Keywords

      Abbreviations

      SCC
      squamous cell cancer
      TNF-α
      tumor necrosis factor alpha
      Interferons exert their biological function mainly through the activation of interferon-stimulated genes (ISG). IFI27 (also known as ISG12 or p27) belongs to a family of small, interferon-alpha (IFN-α)-inducible genes of unknown function. The cDNA for IFI27 was originally cloned as an estrogen-inducible gene in the human epithelial cell line MCF-7 and designated as p27 (
      • Rasmussen U.B.
      • Wolf C.
      • Mattei M.-G.
      • et al.
      Identification of a new interferon a-inducible gene (p27) on human chromosome 14q32 and its expression in breast carcinoma.
      ). In several types of cell lines (B-lymphoid, cervical and breast epithelial, SV40-transformed keratinocytes, and fibroblasts) IFI27 is strongly induced by IFN-α and to a lower extent by IFN-gamma (IFN-γ) as assessed by RT-PCR (
      • Gjermandsen I.M.
      • Justesen J.
      • Martensen P.M.
      The interferon-induced gene ISG12 is regulated by various cytokines as the gene 6–16 in human cell lines.
      ). Immunohistochemical analyses reveal that upon treatment of HeLa cells with IFN-α, the IFI27 gene product resides in the nuclear envelope. IFI27 is thus the first interferon-induced protein that localizes to this site (
      • Martensen P.M.
      • Sogaard T.M.
      • Gjermandsen I.M.
      • et al.
      The interferon alpha induced protein ISG12 is localized to the nuclear membrane.
      ).
      Psoriatic keratinocytes elaborate IFN-γ (
      • Ortonne J.P.
      Recent developments in the understanding of the pathogenesis of psoriasis.
      ) and injection of this cytokine into healthy skin results in a psoriasis-like skin reaction (
      • Barker J.N.
      • Goodlad J.R.
      • Ross E.L.
      • Yu C.C.
      • Groves R.W.
      • MacDonald D.M.
      Increased epidermal cell proliferation in normal human skin in vivo following local administration of interferon-gamma.
      ). This is likely to be mediated in part by the expression of the type I cytokines such as tumor necrosis factor alpha (TNF-α) (
      • Austin L.M.
      • Ozawa M.
      • Kikuchi T.
      • Walters I.B.
      • Krueger J.G.
      The majority of epidermal T cells in psoriasis vulgaris lesions can produce type I cytokines, interferon-gamma, IL-2, and TNF-alpha, defining TC1 (cytotoxic T lymphocyte) and TH1 effector populations, a type 1 differentiation bias is also measured in circulating blood cells of psoriasis patients.
      ). IFI27 mRNA was recently found to be upregulated 13-fold in lesional and 10-fold in uninvolved skin compared to normal skin in an extensive gene array study (
      • Bowcock A.M.
      • Shannon W.
      • Du F.
      • et al.
      Insights into psoriasis and other inflammatory diseases from large-scale gene expression studies.
      ). IFI27 maps to chromosome 14q32, a region where a putative psoriasis susceptibility locus has been found in several studies (
      • Bowcock A.M.
      • Shannon W.
      • Du F.
      • et al.
      Insights into psoriasis and other inflammatory diseases from large-scale gene expression studies.
      ;
      • Veal C.D.
      • Clough R.L.
      • Barber R.C.
      • et al.
      Identification of a novel psoriasis susceptibility locus at 1p and evidence of epistasis between PSORS1 and candidate loci.
      ). The aim of this study was to examine the expression pattern and regulation of IFI27 in skin with special emphasis on psoriasis, other inflammatory skin diseases, and wound repair. In the course of this study, we also investigated a variety of epithelial cancers and demonstrated upregulation of IFI27 in a subset of them. We observed that IFI27 is a marker of epithelial proliferation and cancer.

      Results

      To investigate the expression pattern and regulation of IFI27 in skin with special emphasis on psoriasis and other inflammatory skin diseases, we first performed northern analyses of keratinocyte and fibroblast cultures. IFI27 mRNA could not be detected in fibroblasts (data not shown), whereas it was detected in many keratinocyte cell lines from psoriasis patients and carriers Figure 1a. Interestingly, IFI27 expression levels varied considerably in different keratinocyte lines Figure 1a and treatment of keratinocytes with TNF-α increased IFI27 levels by approximately 2-fold Figure 1a. Primary human keratinocytes from several normal individuals, cultured as previously described (
      • Suomela S.
      • Kariniemi A.-L.
      • Impola U.
      • et al.
      MMP-19 is expressed by keratinocytes in psoriasis.
      ), were then treated with various cytokines and IFI27 expression was investigated with quantitative Taqman RT-PCR (
      • Rechardt O.
      • Elomaa O.
      • Vaalamo M.
      • et al.
      Stromelysin-2 is upregulated during normal wound repair and is induced by cytokines.
      ). Its expression was not modified by LPS, PMA, IL-1β, EGF, TGF-α, KGF, bFGF, VEGF, IL-10, or IGF-1; however, IFN-γ, TNF-α, and TGF-β1 all upregulated IFI27 expression 3–17-fold Figure 1b, in agreement with the northern analyses. Upregulation of IFI27 by TNF-α was more pronounced when the cells were stimulated for 48 h (16-fold) compared to 24 h (8-fold). Of the agents used for the therapy of psoriasis, retinoic acid and vitamin D downregulated IFI27 expression (expression level 26%–44% of the untreated control) Figure 1c, whereas dexamethasone showed no effect.
      Figure thumbnail gr1
      Figure 1IFI27 is variably expressed in keratinocyte lines by northern analysis and upregulated by IFN-γ, TNF-α and TGF–β1 and downregulated by retinoic acid (RA) and vitamin D as assessed by quantitative real-time PCR. (a) Northern analysis of IFI27 in keratinocyte lines reveals variable levels of expression and upregulation following TNF-α treatment. Keratinocyte lines from psoriasis patients K1-1, K25-6, K5-14, K21-2 and psoriasis carriers from multiply affected families (K21-1 and K22-2). Cultures were split and TNF-α was added to one half of the culture at 10 ng per mL: - (control lines), + (treated lines). The size of the major IFI27 transcript is indicated. Beneath the autoradiograph of the northern is an agarose gel showing the quantity and integrity of the RNA used to prepare the northern blot. (b) Relative expression of IFI27 in normal and stimulated keratinocytes assessed by quantitative, real-time PCR. Keratinocytes were cultured in KGM. Total RNA was isolated and reverse transcribed to cDNA. TaqMan was performed as described in Materials and Methods using 18S RNA as an endogenous control. Results are shown relative to mRNA levels from nonstimulated normal keratinocytes, assigned the value 1. IFI27 was upregulated by IFN-γ, TNF-α (after 24 h and 48 h stimulation), and TGF-β1. (c) IFI27 expression was downregulated by RA and vitamin D in primary keratinocytes cultured and assessed by Taqman as described above.
      IFI27 was abundantly expressed in psoriatic skin Figure 2a, b, e, in agreement with previous gene array data (
      • Bowcock A.M.
      • Shannon W.
      • Du F.
      • et al.
      Insights into psoriasis and other inflammatory diseases from large-scale gene expression studies.
      ). In situ hybridization also revealed expression of IFI27 in non-lesional skin, although the signal was lower Figure 2e. Normal skin Figure 2d did not express IFI27 in accordance with previous RT-PCR findings (
      • Rasmussen U.B.
      • Wolf C.
      • Mattei M.-G.
      • et al.
      Identification of a new interferon a-inducible gene (p27) on human chromosome 14q32 and its expression in breast carcinoma.
      ). Despite the different clinical treatments used prior to biopsy, the level of signal was roughly equal in all specimens of lesional skin. In situ hybridization, however, is a qualitative and not a quantitative technique, and there could have been differences that were not detected.
      Figure thumbnail gr2
      Figure 2In situ hybridization reveals that IFI27 is expressed in the epithelia of patients with psoriasis, lichen planus, and squamous cell carcinoma. (a) Dark-field image for IFI27 mRNA in an untreated psoriatic lesion displaying abundant signal in keratinocytes. (b) The corresponding bright-field image. (c) A parallel section hybridized with a sense cRNA probe for IFI27 is devoid of signal. (d) Normal skin expresses no signal for IFI27. (e) Another psoriasis sample expressing the border area between lesional and non-lesional skin. The positive signal in non-lesional skin is marked but not as strong as in lesional skin. The arrow depicts the border between the non-lesional and lesional skin. (f) A strong signal is also seen in a sample of hypertrophic lichen planus. (g) No signal for IFI27 mRNA in a 1-d acute wound where laminin-5 immunostaining reveals numerous migrating cells in an adjacent section (j). (h) In a 6-d wound, hyperproliferative keratinocytes are positive in an area, where laminin-5 staining is diminishing (k). (i) In a 9-d acute wound, IFI27 mRNA is seen in hyperproliferative keratinocytes, while laminin-5 staining is still weaker and concentrates along the basement membrane (l). Arrows depict corresponding areas. (m) Signal for IFI27 mRNA in an SCC. (n) Corresponding bright-field image. Scale bars: (e, f) 125 μm, (a, c, g–n) 50 μm, (b, d) 25 μm.
      IFI27 mRNA was also detected in the epidermis of patients with chronic eczema (three of three), lichen planus (three of three), and lichenoid dermatitis (three of four) Figure 2f. Two diseases, however, representing differential diagnoses of psoriasis, palmoplantar pustulosis and pityriasis rubra pilaris, were negative (data not shown). Chronic venous ulcers were devoid of IFI27 mRNA, whereas normally healing wounds from pinch graft sites at the upper thigh (
      • Vaalamo M.
      • Weckroth M.
      • Puolakkainen P.
      • Saarinen P.
      • Kere J.
      • Lauharanta J.
      • Saarialho-Kere U.
      Patterns of matrix metalloproteinase and TIMP-1 expression in chronic and normally healing human cutaneous wounds.
      ) expressed IFI27. The signal was present at the areas of proliferating keratinocytes since migrating cells, showing strong cytoplasmic laminin-5 staining (
      • Rechardt O.
      • Elomaa O.
      • Vaalamo M.
      • et al.
      Stromelysin-2 is upregulated during normal wound repair and is induced by cytokines.
      ), were not positive Figure 2g, h, i, j, k and l. The number of IFI27 positive cells clearly increased from 1- to 9-d wounds Figure 2g, h, i, j, k and l. Basal cell cancers lacked IFI27 mRNA (data not shown), although 9 of 13 SCC, representing grades I–III, were strongly positive Figure 2m. Thus, the degree of differentiation of the tumor did not seem to influence its expression. No signal was detected with the sense probe.

      Discussion

      Our present results confirm that whereas IFI27 mRNA cannot be detected in normal skin, it is highly upregulated in involved skin and is also detected in uninvolved psoriatic skin (
      • Bowcock A.M.
      • Shannon W.
      • Du F.
      • et al.
      Insights into psoriasis and other inflammatory diseases from large-scale gene expression studies.
      ). IFI27 mRNA was also detected in many benign skin disorders characterized by keratinocyte hyperproliferation such as hypertrophic lichen planus and chronic eczema. This overexpression may well be mediated through IFN-γ, which is elaborated by keratinocytes of both lichen planus and psoriasis (
      • Fayyazi A.
      • Schweyer S.
      • Soruri A.
      • et al.
      T lymphocytes and altered keratinocytes express interferon-gamma and interleukin 6 in lichen planus.
      ;
      • Ortonne J.P.
      Recent developments in the understanding of the pathogenesis of psoriasis.
      ). Furthermore, elevated IFN-γ levels have been linked to eczema both in vitro and in vivo (
      • Carroll J.M.
      • Crompton T.
      • Seery J.P.
      • Watt F.M.
      Transgenic mice expressing IFN-gamma in the epidermis have eczema, hair hypopigmentation, and hair loss.
      ).
      There have been previous reports of IFI27 expression in other tissues such as colon, stomach, and lung (
      • Rasmussen U.B.
      • Wolf C.
      • Mattei M.-G.
      • et al.
      Identification of a new interferon a-inducible gene (p27) on human chromosome 14q32 and its expression in breast carcinoma.
      ). We observed that IFI27 is upregulated during normal skin repair, but that IFI27 mRNA is not detected in chronic wounds that have existed more than 3 mo. This may reflect the fact that the cytokine profile (e.g., levels of active TNF-α and IFN-γ) and the composition of the inflammatory cell infiltrates of the chronic wound environment are very different from re-epithelializing wounds (
      • Hübner G.
      • Brauchle M.
      • Smola H.
      • Madlener M.
      • Fassler R.
      • Werner S.
      Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-treated mice.
      ;
      • Loots M.A.
      • Lamme E.N.
      • Zeegelaar J.
      • Mekkes J.R.
      • Bos J.D.
      • Middelkoop E.
      Differences in cellular infiltrate and extracellular matrix of chronic diabetic and venous ulcers versus acute wounds.
      ). Based on our serial specimens, keratinocytes expressing IFI27 did not seem to be migratory but rather located in the zone of proliferating keratinocytes. Our results suggest that IFI27 is temporarily and spatially regulated during wound repair as is the case with IP-10 (IFN-γ-inducible protein 10) and Mig (monokine induced by IFN-γ), two other proteins reported to be upregulated in psoriasis (
      • Engelhardt E.
      • Toksoy A.
      • Goebeler M.
      • Debus S.
      • Brocker E.B.
      • Gillitzer R.
      Chemokines IL-8, GROalpha, MCP-1, IP-10, and Mig are sequentially and differentially expressed during phase-specific infiltration of leukocyte subsets in human wound healing.
      ;
      • Gillitzer R.
      • Goebeler M.
      Chemokines in cutaneous wound healing.
      ). This reflects the features of proliferating keratinocytes in psoriasis that are reminiscent of the second phase of normal re-epithelialization. At this stage, one finds proliferation of the subadjacent keratinocytes after migration has taken place. IFI27 was also strongly expressed in a subpopulation of SCC, unlike in BCC, which may reflect their histopathological differences and the higher proliferation index of SCC (
      • al-Sader M.H.
      • Doyle E.
      • Kay E.W.
      • et al.
      Proliferation indexes — A comparison between cutaneous basal and squamous cell cancers.
      ). IFI27 may therefore be a marker of certain epithelial cancers.
      In keratinocyte cultures, IFI27 was upregulated by IFN-γ, TNF-α, and TGF-β1. These are all cytokines known to inhibit keratinocyte proliferation in vitro (
      • Symington F.W.
      Lymphotoxin, tumor necrosis factor, and gamma interferon are cytostatic for normal human keratinocytes.
      ). This discrepancy may be explained by the fact that many cytokines have different effects on keratinocytes in vivo and in culture (
      • Turksen K.
      • Kupper T.
      • Degenstein L.
      • Williams I.
      • Fuchs E.
      Interleukin 6: Insights to its function in skin by overexpression in transgenic mice.
      ). In a previous study, TNF-α, GM-CSF, and EGF did not upregulate IFI27 in SV40-transformed human keratinocytes (
      • Gjermandsen I.M.
      • Justesen J.
      • Martensen P.M.
      The interferon-induced gene ISG12 is regulated by various cytokines as the gene 6–16 in human cell lines.
      ). Furthermore, IFN-γ-induced IFI27 upregulation was much lower than in this study (3-fold vs 17-fold). We however observed upregulation of IFI27 in keratinocytes immortalized with E6/E7 viral proteins. These discrepant results may reflect the fact that primary keratinocytes are in many aspects different from immortalized SV40-transformed cells (
      • Celis J.E.
      • Olsen E.
      A qualitative and quantitative protein database approach identifies individual and groups of functionally related proteins that are differentially regulated in simian virus 40 (SV40) transformed human keratinocytes: An overview of the functional changes associated with the transformed phenotype.
      ).
      TNF-α is a multifunctional cytokine that is induced by inflammation and is found in psoriatic lesions (
      • Ortonne J.P.
      Recent developments in the understanding of the pathogenesis of psoriasis.
      ). Our results suggest that together with IFN-γ, it may be upregulating IFI27 expression in psoriatic skin. Retinoids and vitamin D downregulated IFI27 expression in keratinocytes, suggesting that as psoriasis heals during oral or topical therapy, IFI27 expression ceases in keratinocytes. It is possible that novel TNF-α antagonist drugs may exert their beneficial effects partly via pathways, in which IFI27 is involved. In fact, their use might also slow down the growth of SCC through inhibition of IFI27. In summary, IFI27 appears to be a novel marker of epithelial proliferation and cancer.

      Materials and Methods

      Northern analyses

      Keratinocytes were cultured in keratinocyte growth medium (KGM) supplemented with epidermal growth factor (EGF) and bovine pituitary extract (BPE) until confluency. The cells were then washed twice with PBS and transferred to keratinocyte basic medium without supplementary nutrients. Cultures were split and TNF-α was added to one half of the culture at 10 ng per mL. After 24 h, total cellular RNA was extracted with TRIzol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, California). Primary fibroblast cultures from psoriatic and normal skin were established with routine methods, cells were harvested, and RNA was prepared. Total RNA was denatured and separated on 1% agarose gel containing 2.2% formaldehyde, and transferred to nylon-supported nitrocellulose membrane (Osmonics, Massachusetts). A 482 bp fragment of human IFI27 cDNA was labeled with [α-32P]dCTP. Hybridization was performed with PerfectHyb Plus buffer according to the procedure provided by the manufacturer (Sigma, St Louis, Missouri). Hybridization signal was detected by autoradiography.

      Cell cultures, cytokines and growth factors

      Keratinocytes were cultured in KGM supplemented with EGF and BPE, equal numbers were plated on 24-well tissue culture plates, and the next day the cells were incubated in KGM without supplements for 18–24 h. The cells were subsequently treated with phorbol 12-myristate 13-acetate (PMA, 10 ng per mL, Sigma), interleukin-1 beta (IL-1β, 5 U per mL, Roche Molecular Biochemicals, Mannheim, Germany), IFN-γ (100 U per mL, Roche), transforming growth factor alpha (TGF-α, 30 ng per mL, Sigma), keratinocyte growth factor (KGF, 10 ng per mL, Sigma), EGF (10 ng per mL, Sigma), insulin-like growth factor-1 (IGF-1, 100 ng per mL, R & D Systems, Minneapolis, Minneapolis), basic fibroblast growth factor (bFGF, 10 ng per mL, Sigma), TNF-α (10 ng per mL, Sigma), transforming growth factor beta-1 (TGF-β1, 10 ng per mL, Sigma), vascular endothelial growth factor (VEGF, 10 ng per mL, R & D Systems), lipopolysaccharide (LPS, 2,5 μg per mL, Sigma), retinoic acid (RA, 10-6 M, Sigma), interleukin-10 (IL-10, 10 ng per mL, R & D Systems), dexamethasone (10-5 M, Sigma), or 1,25-(OH)2 D3 (calcitriol, 10-7 M, Sigma). Untreated cells were used as controls. After 24 and 48 h, total RNA was extracted from the cells using an RNeasy miniprep-kit (Qiagen, Chatsworth, California), and reverse transcribed to complementary DNA with Taqman Reverse Transcription reagents (Applied Biosystems, Foster City, California) with random hexamers and used as a template for TaqMan quantitative, real-time PCR.

      PCR primers and probes and quantitative real-time PCR (TaqMan RT-PCR)

      PCR primers and probes (Applied Biosystems) for IFI27 were designed with the computer program Primer Express 1.5 (Applied Biosystems). Primers used for the amplification were TGC CTC GGG CAG CCT (forward nucleotides) and TTG GTC AAT CCG GAG AGT CC (reverse nucleotides). Human 18S RNA labeled with VIC™ reporter dye (pre-developed TaqMan assay reagents for endogenous control human 18S RNA, Applied Biosystems) was used as an endogenous control. Reactions were performed with the ABI PRISM 7700 Sequence Detector System (Applied Biosystems) in 20 μL volumes, IFI27 and 18S RNA in separate reactions. For IFI27 the mixture contained 5 μL 1:5 diluted cDNA, 100 nM of each primer, and 200 nM of the FAM-labeled probe. For 18S RNA the mix contained 1 μL 18S RNA control reagents, 5 μL of the diluted cDNA, and 1× Taqman Universal Master Mix. The PCR was started with 2 min at 50°C and an initial 10 min denaturation at 94°C, followed by a total of 40 cycles of 15 s denaturation at 94°C, and 1 min of annealing and elongation at 60°C.

      Tissues

      Formalin-fixed, paraffin-embedded specimens were obtained from the Department of Dermatopathology, University of Helsinki. in compliance with Declaration of Helsinki guidelines. Informed consent was obtained from each patient prior to the biopsy of psoriatic skin. The study protocol was approved by the corresponding Ethical Committee. Twenty-three patients affected with psoriasis (duration of disease from newly diagnosed to several decades) were included in this study and biopsies were taken from the center of psoriatic lesions. Twelve of the patients were untreated, five used topical corticosteroids, two used topical calcipotriol, two used topical corticosteroids + calcipotriol, one used SUP and one was subjected to PUVA treatment. Biopsies from 16 of these patients were also taken from the normal-looking skin approximately 10 cm away from the psoriasis lesion or from the border between lesional and non-lesional skin. Specimens of lichen planus (n=3), lichenoid chronic dermatitis (=neurodermatitis) (n=3), palmoplantar pustulosis (n=3), pityriasis rubra pilaris (n=3), chronic eczema (n=3), squamous cell cancers (SCC) of different grades (n=13), basal cell cancers (n=4), chronic wounds (n=5), normally healing timed wounds (n=6) (
      • Vaalamo M.
      • Weckroth M.
      • Puolakkainen P.
      • Saarinen P.
      • Kere J.
      • Lauharanta J.
      • Saarialho-Kere U.
      Patterns of matrix metalloproteinase and TIMP-1 expression in chronic and normally healing human cutaneous wounds.
      ), and normal skin (n=6) were also examined by in situ hybridization.

      In situ hybridization

      A 482 bp cDNA fragment of IFI27 was amplified with the PCR and subcloned into pBluescript SK+ plasmid digested with EcoRI/BamHI. The insert was released with PvuII and its integrity was confirmed by DNA sequencing. The sense probe was transcribed as a T7 run-off. T3 was used for the antisense probe. Transcription of the sense and antisense probes has been described elsewhere (
      • Saarialho-Kere U.K.
      • Pentland A.P.
      • Birkedal-Hansen H.
      • Parks W.C.
      • Welgus H.G.
      Distinct populations of basal keratinocytes express stromelysin-1 and stromelysin-2 in chronic wounds.
      ). In situ hybridization was performed on 5 μm sections as described in detail (
      • Prosser I.W.
      • Stenmark K.R.
      • Suthar M.
      • Crouch E.C.
      • Mecham R.P.
      • Parks W.C.
      Regional heterogeneity of elastin and collagen gene expression in intralobular arteries in response to hypoxic pulmonary hypertension as demonstrated by in situ hybridization.
      ). Sections were hybridized with 35S-labeled probes (3.5×104 cpm per μL of hybridization buffer) at 55°C for at least 18 h in a humidified chamber. After 10–29 d of autoradiography, the photographic emulsion was developed, and the slides were stained with hematoxylin and eosin. No signal was detected with the sense probe. The slides were analyzed independently by two investigators (U.S.-K. and S.S.).

      Immunohistochemistry

      Migrating keratinocytes demonstrating strong cytoplasmic staining for laminin-5 were identified with polyclonal rabbit antibodies against the γ2 chain of laminin-5 (1:600, a gift from Professor Karl Tryggvason, Karolinska Institutet) as previously described (
      • Saarialho-Kere U.
      • Kerkela E.
      • Jahkola T.
      • Suomela S.
      • Keski-Oja J.
      • Lohi J.
      Epilysin (MMP-28) is associated with cell proliferation during epithelial repair.
      ). Diaminoethylcarbazole was used as chromogenic substrate and hematoxylin as counterstain.

      ACKNOWLEDGMENTS

      We thank Dr Arja-Leena Kariniemi for her pathology expertise and Ms Alli Tallqvist for her excellent technical assistance. This study was supported by the Paulo Foundation, Biomedicum Helsinki Foundation, Duodecim Research Foundation, Instrumentarium Foundation, Sigrid Juselius Foundation, Academy of Finland, National Psoriasis Foundation (A.B.), NIH grant AR049049 (A.B.), and Helsinki University Central Hospital Research Funds.

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