Proliferating Keratinocytes Are Putative Sources of the Psoriasis Susceptibility-Related EDA+(Extra Domain A of Fibronectin) Oncofetal Fibronectin

Proliferating Keratinocytes Are Putative Sources of the Psoriasis Susceptibility-Related EDA þ (Extra Domain A of Fibronectin) Oncofetal Fibronectin Ma´rta Sze´ll,1 Zsuzsanna Bata-Cso¨rgo´´,w1 Andrea Koreck,w Andor Pivarcsi,w Hilda Polya´nka,w Csilla Szeg,w Magdolna Gaa´l,w Attila Dobozy,w and Lajos Keme´nyw

Dermatological Research Group of the Hungarian Academy of Sciences and the University of Szeged, Szeged, Hungary; wDepartment of Dermatology and Allergology, University of Szeged, Szeged, Hungary

The extra domain A of fibronectin (EDA) þ oncofetal isoform of fibronectin was recently reported to be overexpressed in psoriatic uninvolved epidermis. It has been proposed that the abnormal presence of EDA þ oncofetal protein at the dermal–epidermal junction in the uninvolved skin may provide the ‘‘psoriatic’’ environment in which keratinocytes are in a preactivated state with regard to mitogenic signals (e.g., T cell lymphokines). To determine the possible sources of cellular fibronectin in the non-lesional psoriatic skin, we aimed to investigate whether keratinocytes could produce the EDA þ oncofetal form of fibronectin. RT-PCR studies revealed that both cultured normal keratinocytes and HaCaT cells express the EDA þ splice variant of fibronectin mRNA, and in HaCaT cells the EDA þ /EDA
transcript ratio was elevated compared with normal keratinocytes. Cultured keratinocytes and HaCaT cells showed intracytoplasmic staining with an EDA þ fibronectin-specific antibody and among the positively stained cells many showed mitosis. Using RT-PCR, western blot analysis, and flow cytometry, we showed that in synchronized HaCaT cells the amount of both total fibronectin and its EDA þ isoform change with the proliferation/ differentiation state of HaCaT cells and peak in highly proliferating cells. We show that in short-term ex vivo cultures, a small population of EDA þ fibronectin containing cell population appear among psoriatic uninvolved, but not normal epidermal cells. We also demonstrate that cell attachment has a strong influence on the expression of both total and EDA þ fibronectin. Our results suggest that proliferating keratinocytes could be the sources of the psoriasis susceptibility-related EDA þ oncofetal fibronectin in the epidermis.

Key words: fibronectin alternative splicing/HaCaT cells/non-involved psoriatic epidermis/psoriatic extracellular matrix environment J Invest Dermatol 123:537 – 546, 2004 Psoriasis is a multifactorial hyperproliferative inflammatory skin disease that affects approximately 2%–3% of the European population. Both genetic and environmental factors contribute to the precipitation of psoriatic lesions. It is generally accepted that the genetic background for psoriasis susceptibility is pivotal for the appearance of the symptoms. Intensive family studies since the early 1950s and linkage analysis studies pointed out several genetic loci that play a role in psoriasis (Bhalerao and Bowcock, 1998). In the last decade, a molecular biology approach emerged to identify abnormally expressed genes and proteins contributing to psoriasis (Jackson et al, 1999; Chen et al, 2000). Previously, we have shown (Bata-Csorgo et al, 1995) that the clonogenic basal stem cell population in psoriatic uninvolved skin is more sensitive to lymphokines than basal cells derived from normal epidermis. The T cell lymphokine, interferon-g, has a pronounced growth-stimulatory effect on uninvolved psoriatic keratinocytes and its effect is synergis-

tically elevated by fibronectin. The keratinocyte receptor for fibronectin, a5b1 integrin, is upregulated both in uninvolved and involved skin of psoriatic patients (Pellegrini et al, 1992). Moreover, we demonstrated that keratinocytes isolated from uninvolved skin of psoriatic patients overexpress the a5, but not a2 and a3 integrins, on their membranes (BataCsorgo et al, 1998). In addition to the elevated a5 integrin protein, its ligand, fibronectin, is overexpressed in the uninvolved skin of psoriatic patients in an alternative spliced variant (extra domain A of fibronectin (EDA) þ or oncofetal fibronectin) form. RT-PCR and immunohistochemistry showed that the EDA þ form of fibronectin is overexpressed at the dermal–epidermal junction (DEJ) of uninvolved skin, but not in control skin (Ting et al, 2000). Rothaupt et al have demonstrated that the EDA þ isoform of fibronectin is colocalized with CD11c þ cells at the DEJ of lesional psoriatic skin and hypothesized that the macrophage-derived EDA þ fibronectin might contribute to the initiation of keratinocyte hyperproliferation. Although in the non-lesional skin bone marrow-derived (CD45RO þ ) cells also showed EDA þ staining in the dermis, given their very low number it is likely that there could be other sources of cellular fibronectin in the non-lesional tissue (Rothaupt et al, 2000).

Abbreviations: EDA, extra domain A of fibronectin; PCNA, proliferating cell nuclear antigen 1 These two authors contributed equally to this work.

Copyright r 2004 by The Society for Investigative Dermatology, Inc.

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In a recent report, it was demonstrated that basal keratinocytes from uninvolved psoriatic skin exhibit a significantly higher level staining for focal adhesion kinase (FAK) in response to fibronectin. Moreover, FAK tyrosine phosphorylation also had a greater degree in uninvolved psoriatic keratinocytes than in normal keratinocytes. Based on these observations it has been proposed that keratinocytes in the non-lesional psoriatic skin are in a ‘‘pre-activated’’ state to signalling through integrin interactions such as a5b1 integrin signalling (Chen et al, 2001). Ting et al (2000) have demonstrated that the source of the EDA þ fibronectin at the DEJ of uninvolved skin was not the dermal fibroblast. Rothaupt et al (2000) attributed the origin of EDA þ fibronectin to macrophages at the basement membrane zone. Since the presence of EDA þ fibronectin could be responsible for the hyper-responsiveness of uninvolved psoriatic keratinocytes, it is very important to know whether keratinocytes themselves could be the sources of EDA þ fibronectin. We aimed to demonstrate the presence of EDA þ fibronectin in cultured keratinocytes and compare the ratio of EDA þ and EDA
fibronectin mRNA in cultured dermal fibroblasts, in cultured keratinocytes, and in HaCaT keratinocytes. We have also investigated the correlation between the proliferation/differentiation state of keratinocytes and the ratio of EDA þ /EDA
fibronectin mRNA and protein expression using synchronized HaCaT keratinocytes that resemble the characteristics of epidermal keratinocytes in different stages of proliferation and differentiation (Pivarcsi et al, 2001).

Results HaCaT cells express a higher-level fibronectin mRNA with a shifted EDA þ /EDA
splice variant ratio compared with normal keratinocytes In order to study the fibronectin mRNA profile of epidermal cells, cultured keratinocytes, HaCaT cells, and fibroblasts, a primer pair was designed that border the EDA motif of fibronectin (Fig 1). After RT-PCR analysis, the resulting bands were easily distinguishable: the 847 bp band corresponds to the EDA
form of fibronectin, whereas the 1221 bp indicates the EDA þ oncofetal form of fibronectin. After densitometry, both total fibronectin mRNA accumulation and EDA þ /EDA
splice variant ratio (Fig 2) were compared in the three different cell types. All three cell types were studied at the same proliferation stage: the culture flasks were approximately 70%–80% covered with the cells at the time of sample collection. G3PDH-specific RT-PCR analysis proved the equal quality of total RNA preparations from three different cell types (data not shown). Normal human keratin-

Figure 1 The primers were designed around the EDA motive of fibronectin. With the EDA motive, the PCR product was 1221 bp long, without it was 847 bp long.

Figure 2 Fibroblasts, normal cultured keratinocytes, and HaCaT cells express different amounts of total fibronectin mRNA and EDA þ : EDA
isoforms. Since fibroblasts, normal human keratinocytes, and HaCaT cells express considerably different levels of fibronectin, different amounts of cDNAs were used for fibronectin-specific PCR reactions in order to obtain good-quality presentation for the comparison of isoform ratios. The resulting PCR products were run on 1% agarose gel (A) and after densitometry the amount of total fibronectin as well as the ratio of EDA þ and EDA
isoforms were calculated (B). The gel photo (A) shows a representative experiment; data (B) (mean (  SE)) were obtained from two independent fibroblast, three independent normal keratinocyte, and four independent HaCaT cell cultures.

ocytes express a dramatically lower level (0.35  0.02%, n ¼ 3) of fibronectin mRNA compared with cultured fibroblasts taken as 100% (n ¼ 2). The immortalized HaCaT keratinocytes exhibit a significantly higher level of total fibronectin mRNA when compared with normal keratinocytes, that is 1.69%  0.41% (n ¼ 4) of the fibronectin mRNA accumulation of fibroblasts. In addition to differences in the total fibronectin mRNA levels in the studied cell types, the ratio of the EDA þ /EDA
splice variants is also different in each cell type. Both fibroblasts and cultured normal human keratinocytes express the two splice variants at about the same level at the studied state of proliferation: the ratio of EDA þ /EDA
splice variants is 0.91  0.24:1 in fibroblasts and 1.21  0.12:1 in keratinocytes. In contrast, in HaCaT keratinocytes, the EDA þ oncofetal form of fibronectin clearly dominates; the EDA þ /EDA
ratio is 2.55  0.20:1. Proliferating normal keratinocytes and HaCaT cells express the EDA þ oncofetal isoform of fibronectin Immunocytochemical staining with a mouse monoclonal antibody specific for the EDA motif demonstrates that actively proliferating, subconfluent cultures of fibroblasts, normal keratinocytes, and HaCaT cells express the oncofetal isoform of fibronectin (Fig 3). The level of staining is apparently different in the three examined cell types: all fibroblasts showed uniform intracytoplasmatic staining, but

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et al (2001) have demonstrated that discrete stages of a synchronized HaCaT culture after release from cell quiescence resemble different populations of epidermal keratinocytes. The serum-starved, contact-inhibited HaCaT cells resemble suprabasal non-proliferating differentiated (K1/ K10 þ ) keratinocytes of normal epidermis, whereas the highly proliferative HaCaT cells (a5 integrin þ , K1/K10 þ ) after release from cell quiescence resemble the activated, differentiated, transiently amplifying keratinocytes. Here, we demonstrate that the serum-starved, contact-inhibited HaCaT cells express a very low-level fibronectin mRNA that increases dramatically with time after passaging and serum re-addition (Fig 4A and B). The highest levels of total fibronectin mRNA (4.64  1.14-fold and 4.12  0.71-fold, compared with 0 h, n ¼ 2) coincide with the highest proliferation rates of HaCaT culture (48 and 72 h), demonstrated by PI staining and the highest levels of a5 integrin mRNA as well as protein expression (Pivarcsi et al, 2001). As the proliferation of the cells slows, the amount of fibronectin

Figure 3 Immunocytochemistry on cultured human fibroblasts, keratinocytes, and HaCaT cells. Subconfluent cell cultures were stained with a mouse monoclonal antibody specific for human extra domain (EDA sequence) of cellular fibronectin (A, C, E ) and mouse IgG1 as isotypic control (B, D, F ). Fibroblasts showed a uniform intracytoplasmic staining (A), whereas among keratinocytes (C) and HaCaT cells (E ), mostly mitotic cells revealed positive intracytoplasmic staining.

among keratinocytes and HaCaT cells, mostly mitotic cells revealed positive intracytoplasmic staining. The ratio of EDA þ /EDA
splice variants depends on the proliferation/differentiation stage of HaCaT cells After detecting a shifted EDA þ /EDA
splice variant ratio in subconfluent HaCaT cells, compared with cultured subconfluent keratinocytes, we aimed to study whether the proliferation/differentiation stages of HaCaT cells affect the fibronectin mRNA profile. Using propidium iodide DNA staining, and a5 integrin, keratin 1, and keratin 10 expression as proliferation and differentiation markers, Pivarcsi

Figure 4 The amount of total fibronectin mRNA as well as the ratio of EDA þ /EDA
mRNA splice variants depend on the proliferation/ differentiation stage of HaCaT cells. HaCaT cells were synchronized by contact inhibition and serum-starvation (0 h), and then released from cell quiescence by serum re-addition and passage. Samples for RTPCR analysis were taken at the indicated time points. After running the resulted PCR products on 1% agarose gel (A), both the EDA þ and EDA
bands were measured by densitometry. Changes of total EDA þ and EDA
mRNAs at different times, relative to the amounts measured in the 0 h samples, are shown (B). The calculated ratios of EDA þ and EDA
mRNA splice variants are also shown at different times (C). Values represent the mean of two independent experiments (  SE).

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mRNA decreases and in the one-week-old culture it almost reaches the level of the serum-starved HaCaT cells (1.22  0.39-fold, compared with 0 h level, n ¼ 2). In addition to changes in total fibronectin mRNA, the ratio of EDA þ /EDA
splice variants also varies in the synchronized HaCaT cell culture. The serum-starved HaCaT cells exhibit approximately the same amount of EDA þ and EDA
forms of fibronectin mRNA (Fig 4A and C). Twenty-four hours after release from cell quiescence, when the HaCaT cells have already attached to the surface of the culture flask and start to proliferate, there is three times more EDA þ fibronectin mRNA splice variant than EDA
. The most pronounced difference between the two splice variants (5.70  1.42:1) appears at 24 h that precedes the highest overall fibronectin mRNA level and the highest proliferation rate of HaCaT culture at 48 and 72 h. In parallel with the dramatic decrease of total fibronectin mRNA, the ratio between the EDA þ /EDA
splice variants reduces from the 72nd h in the culture, and in the 1-wk culture, it reaches a level similar to the serum-starved cells at 0 h. The amount of EDA þ fibronectin protein peaks in highly proliferating HaCaT cells In order to follow changes in total fibronectin at the protein levels in synchronized HaCaT cells after release from cell quiescence, western blot analysis was carried out. Goat polyclonal antibody was used to detect the amount of total fibronectin in cell lysates. Total fibronectin (Fig 5) was undetectable in the 0 and 12 h samples; its expression appeared in the 24 h sample (1.28  0.58 normalized band intensity in relative unit, (n ¼ 2)) and was highest in the 36–48 h samples (1.77  0.45 and 1.48  0.33 normalized band intensities in relative units, respectively (n ¼ 2)), and then gradually decreased to a still detectable level (0.46  0.20 normalized band intensity in relative unit (n ¼ 2)) at 168 h. This pattern of total fibronectin expression was identical in four independent experiments, with the exception of the 0 h samples, in which a small amount of fibronectin was detectable if the protein was harvested directly from the plates (n ¼ 2), but no detectable fibronectin was present when cells were lysed after removal from the tissue culture plates (n ¼ 2). This observed difference indicates that HaCaT cells discharge some amount of fibronectin during the 2-wk period of synchronization. To follow the changes of EDA þ oncofetal fibronectin protein in HaCaT cells after release from cell quiescence, western blot and flow cytometry experiments were carried out. Although the monoclonal antibody specific for EDA þ fibronectin showed very faint bands at the appropriate molecular weight in the 48, 72, and 96 h samples on western blot in four independent experiments, its sensitivity was not sufficient in HaCaT cells (data not shown). Therefore, we used flow cytometric analysis to follow the EDA þ fibronectin protein expression. In repeated experiments (n ¼ 3), the level of EDA þ fibronectin was indistinguishable from isotype staining in serumstarved, contact-inhibited HaCaT cells (0 h) (Fig 6A). As cells started to proliferate in the culture, FITC fluorescence above the isotype level appeared. The maximal EDA þ fibronectin expression was seen at 72 h (12.56%, 6.54%, and 6.66% of cells were found above the isotype level in the three different experiments) (Fig 6B). In one experiment,

Figure 5 The amount of total fibronectin changes with the proliferation/differentiation stage of HaCaT cells. HaCaT cells were synchronized by contact inhibition and serum-starvation (0 h), and then released from cell quiescence by serum re-addition and passage. Samples for analysis were taken at the indicated time points, run on SDS-PAGE, and stained with Coomassie Brilliant Blue (CBB). The staining indicates equal loading (A). The same amounts of proteins were blotted on nylon membrane, and western blot analysis was performed for the detection of total fibronectin by colorimetric method (B). Three relatively steady bands from the CBB gel were chosen, their average calculated, and used to normalize the total fibronectin bands of the western blot. Normalized band intensities (  SE) of two independent experiments are shown (C).

double staining with the proliferating cell nuclear antigen (PCNA) mAb showed that all the EDA þ fibronectin-containing cells were among the PCNA þ population (Fig 6B). We also stained freshly separated epidermal cells from normal epidermis, psoriatic non-lesional, and lesional epidermis with antibodies against EDA þ fibronectin (n ¼ 4) and PCNA (n ¼ 2). We could not clearly detect EDA þ fibronectin cells in these freshly separated epidermal samples. Although EDA þ fibronectin fluorescence always exceeded that of the normal in both non-lesional and lesional psoriatic samples, almost similar shifts were detectable in the isotype control samples (data not shown).

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Figure 6 The EDA þ oncofetal form of fibronectin is expressed in highly proliferating HaCaT cells. HaCaT cells were synchronized by contact inhibition and serum-starvation (0 h), and then released from cell quiescence by serum re-addition and passage. Flow cytometry demonstrated that in the 0 h, contact-inhibited and serum-starved HaCaT cells, EDA þ fibronectin (continuous line), were indistinguishable from isotype staining (dashed line) (A). 72 h after re-entering the cell cycle, the EDA þ fibronectin staining exceeded isotype staining in HaCaT cells (B, left panel). 12.56% of the cells fall above the background level in this representative experiment. Double staining with PCNA demonstrated that the EDA þ fibronectin-expressing cells (B, right panel) were all positive for PCNA (upper right quadrant).

Next, we compared differences in EDA þ fibronectin production between normal and non-lesional psoriatic epidermal cells in short-term (72 h) ex vivo cultures. In the psoriatic non-lesional cultures, a small cell population (3.06% of all plated cells) (Fig 7C), appearing clearly above the isotype level (Fig 7D), was detected, indicating the presence of EDA þ fibronectin in these cells. This cell population was not seen in normal epidermal cell cultures (Fig 7A and B). These data indicate that in the non-lesional psoriatic epidermis, there are cells that are more capable of producing EDA þ fibronectin than cells in normal epidermis under certain conditions such as cell–cell contact disruption followed by in vitro culturing. Cell attachment substantially affects the expression of fibronectin mRNA and the ratio of EDA þ /EDA
splice variants To test how cell attachment affects the expression of fibronectin mRNA and the EDA þ /EDA
fibronectin mRNA splice variant ratio, different cell culture surfaces were used. Pre-confluent, proliferating HaCaT cells were seeded on tissue culture plates, fibronectin-coated tissue culture plates, and Teflon and bacterial plates and then cultured for 72 h. (In a preliminary experiment, cells were harvested either at 24 or at 72 h after plating and the 24 and 72 h harvests gave similar results.) When semiconfluent HaCaT cells were seeded on tissue culture dish (TC) and on fibronectin-coated tissue culture plates (F), there was no drastic change in the total amount of fibronectin mRNA (84.53%  0.70% and 95.78%  13.90%, compared with the 0 h sample, n ¼ 2, 24 h harvests), and we detected a slight increase in the EDA þ fibronectin mRNA splice variant ratio (1.26  0.05:1 and 1.25  0.13:1, respectively, n ¼ 2, 24 h harvests). In contrast to this, on Teflon (TF) and bacterial plates (B), both total fibronectin mRNA (37.20%  7.86% and 25.54%  3.77%, compared with the 0 h sample, n ¼ 2, 24 h harvests) and the EDA þ isoform production were downregulated. The ratio of EDA þ /EDA


Figure 7 A small population of uninvolved psoriatic, but not normal epidermal cells, express EDA þ fibronectin after a short-term culture. Normal (A and B) and psoriatic non-lesional (C and D) epidermal cells were cultured for 72 h, and then stained with an mAb for EDA þ fibronectin and with mouse IgG1 as isotype control. Although in the normal cultured epidermal cells the EDA þ FITC fluorescence level did not exceed the level of isotype fluorescence, among the uninvolved epidermal cells a small population (3.06%) of cells fall above the isotype level in the EDA þ FITC samples.

fibronectin mRNA splice variants was higher on Teflon (1.40  0.09:1) than on the bacterial plate (0.64  0.01:1) (Fig 8A and B). These data demonstrate that without

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Figure 8 Culture conditions substantially affect the expression of fibronectin mRNA and the EDA þ /EDA
spice variant ratio. The effects of different culture surfaces were tested on HaCaT cells. A pre-confluent culture of HaCaT cells (0 h) was passaged to tissue-culture dish (TC), fibronectin-coated tissue culture dish (F), Teflon (TF), and bacterial plate (B). Cells were harvested, and the expression of fibronectin was studied by RT-PCR. Cells grown on tissue culture dish and fibronectin-coated tissue culture dish did not show drastic changes in the total fibronectin mRNA expression, but showed a slight shift in the EDA þ /EDA
mRNA splice variants. HaCaT cells that were seeded onto teflon and bacterial plates expressed substantially lower levels of fibronectin mRNA (A). The most pronounced decrease in EDA þ /EDA
mRNA splice variant ratio was detected in HaCaT cells cultured on bacterial plates (B). The gel photo (A) shows a representative experiment; data (B) (mean (  SE)) were obtained from two independent experiments.

sufficient attachment, the cells are not able to express fibronectin mRNA and its EDA þ splice variant.

Discussion Psoriasis is thought to be a multigenic disorder, and the susceptibility genes have been intensely investigated. Given the complexity of the disease, the modified expression of well-characterized genes in the psoriatic uninvolved epidermis might lead to the understanding of the molecular pathogenesis of this skin disorder. It has been demonstrated that the oncofetal EDA þ isoform of fibronectin is present in the uninvolved psoriatic, but not in normal epidermis (Ting et al, 2000). The presence of the EDA þ fibronectin in the non-lesional epidermis may be the key in providing a ‘‘psoriatic’’ environment to the keratinocytes that renders them hypersensitive to different signals. In in vivo nonlesional skin, fibroblasts do not seem to be the source of cellular fibronectin; only CD45RO þ cells double stain with EDA þ fibronectin (Rothaupt et al, 2000).

Here, we demonstrate the presence of EDA þ mRNA and EDA þ isoform of fibronectin protein in cultured fibroblasts, keratinocytes (both in third passage), and in HaCaT keratinocytes with RT-PCR analysis as well as with immunohistochemical staining. In contrast to normal cultured keratinocytes that express the EDA þ /EDA
splice variants approximately at the same ratio, HaCaT cells exhibit a dominating level of EDA þ fibronectin mRNA. HaCaT cells, although immortalized and genetically abnormal, are considered to be good models for studying the proliferation/differentiation of human keratinocytes (Boukamp et al, 1988; Ryle et al, 1989; Pivarcsi et al, 2001). HaCaT cells, however, have the ability to dedifferentiate and this unique feature may be related to their immortalized nature. Here, we provide yet another feature that may explain why HaCaT cells are immortalized. The EDA þ fibronectin produced and secreted by HaCaT cells could provide an elevated stability for the receptor of fibronectin, a5b1 integrin, and self-sustain a high-level proliferation rate that is characteristic of HaCaT cells. Our data indicate a correlation between proliferation and EDA þ fibronectin production in HaCaT cells. The fact that only a minor population of highly proliferating PCNA þ cells contain EDA þ fibronectin suggest that EDA þ fibronectin is synthetized during a well-defined, short period of the cell cycle and is released relatively quickly from the cells. Both the amount and the EDA þ /EDA
fibronectin mRNA splice variant ratio change with the proliferation/differentiation state of HaCaT keratinocytes: after release from cell quiescence, the synchronized cells show the highest level of total fibronectin expression and the highest EDA þ /EDA
ratio when the cells actively proliferate. The fact that certain genes such as keratin 1 and keratin 10 are downregulated during this period (Pivarcsi et al, 2001) excludes the possibility that the elevated level of fibronectin mRNA would reflect only a general increase in gene expression. We demonstrate that culture conditions that result in different cell attachment opportunities affect the total fibronectin mRNA expression as well as the EDA þ /EDA
transcript ratio. Our data also suggest that attachment signals are necessary for triggering upregulation of fibronectin mRNA and its EDA þ isoform. That the state of proliferation may direct fibronectin splicing has been indicated in cultured fibroblasts and MDCK epithelial cells (Inoue et al, 1999). Both in fibroblasts and MDCK epithelial cells, the EDA þ /EDA
fibronectin splice variant ratio was higher at low cell density than at high cell density. The same authors have also demonstrated that during migration, MDCK cells produce a lesser amount of fibronectin mRNA with an elevated EDA þ /EDA
ratio (Inoue et al, 2001). In contrast to the findings of elevated EDA þ /EDA
ratios occurring due to different stimuli, a decreased EDA þ /EDA
ratio has been demonstrated in interleukin-4-activated macrophages (Gratchev et al, 2001). Both cis- and trans-acting factors play important roles in the regulation of fibronectin mRNA splicing. Purine-rich sequence tracks have been described within exon B, which are important for proper 50 splice selection both in vivo and in vitro (Kuo and Norton, 1999). Moreover, an exonic splicing enhancer (ESE) that binds splicing regulatory (SR) proteins

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was also identified in the promoter region of the fibronectin gene (Cramer et al, 1999). The trans-acting factors that bind these cis-elements may act either as activators (T-Ag) or as inhibitors (VP16) of EDA inclusion (Kadener et al, 2001). Comparing the above cis-elements and trans-acting factors in different cell lines (normal cultured keratinocytes versus HaCaT cells) and under various pathological conditions (e.g., psoriasis), where the EDA þ /EDA
fibronectin mRNA splice variant ratios differ dramatically, may reveal variances in the fibronectin gene itself that affect splicing processes. So far, only two groups of growth factors are known that can affect the splicing of fibronectin: the hepatocyte growth factor/scatter factor (HGF/SF) (Inoue et al, 1999) and the transforming growth factor b (TGF-b) (Li et al, 2000). HGF/ SF increases the EDA þ /EDA
ratio in MDCK epithelial cells in a concentration-dependent manner up to 2.1-fold compared with untreated controls. The effects of two TGF-b isoforms (TGF-b1, TGF-b2) on fibronectin mRNA splicing have been compared in porcine trabecular cells. Both isoforms increased the EDA þ /EDA
ratio in the treated cells, although the effect of TGF-b2 was always more pronounced (Li et al, 2000). Among many other growth factors, the isoforms of the TGF-b family (Wataya-Kaneda et al, 1996), as well as their receptors (Leivo et al, 1998), show abnormalities in psoriasis. It is possible that the abnormal expression of TGF-b isoforms and their receptors contributes to the precipitation of psoriatic lesions via inducing alterations in the splicing of fibronectin pre-mRNA. Oyama et al have demonstrated that the anti-inflammatory effects of 1a,25-dihidroxyvitamin D3 on psoriatic lesional skin may be mediated by a complex TGF-b regulation in local fibroblasts. Through the regulation of TGF-b, 1a,25-dihidroxyvitamin D3 could affect fibronectin pre-mRNA splicing, which may be an important element in the antipsoriatic effect of this agent (Oyama et al, 2000). HaCaT cells would be good models to test this hypothesis due to the drastic changes in their EDA þ /EDA
fibronectin mRNA splice variant ratio during proliferation and differentiation. Our results demonstrate a clear correlation between the high proliferative state and total fibronectin expression both at the mRNA and protein levels in HaCaT cells. Our experiments suggest that the total fibronectin protein production is regulated at the transcriptional level. But we cannot exclude the possibility that under certain conditions besides transcriptional regulation, post-transcriptional mechanisms may also contribute to the final fibronectin protein profile. The protein stability and protease resistance of fibronectin synthesized and released at various stages of the cell culture may also be different. One possible mechanism by which fibronectin stability is regulated could be glycosylation. Using two-dimensional nuclear magnetic resonance and simulated annealing, Sticht et al (1998) showed that glycosylation modifies the three-dimensional structure of fibronectin, which provides a higher protein stability and protease resistance to the protein. That fibronectin synthesis could be regulated at different levels is already known. There is evidence that IFN-g, which elevates the level of fibronectin transcription in cultured normal human fibroblasts, decreases the stability of the fibronectin mRNA and results in a decreased protein level

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(Diaz and Jimenez, 1997). Dexamethasone, on the other hand, has no effect on the level of transcription, but elevates the level of protein synthesis via stabilizing fibronectin mRNA (Kucich et al, 2000). In contrast to this, TGF-beta1 produces a 4-fold increase in transcription of fibronectin gene, but only a small increase in mRNA stability and finally an increased fibronectin protein level (Raghow et al, 1986). Although we did not check whether HaCaT cells assembled the produced fibronectin, there is evidence that keratinocytes cultured in serum-containing media are capable of assembling fibronectin dimers (Altankov et al, 2001). On the other hand, it has also been shown that monomeric fibronectin can also bind purified a5b1 integrin receptor (Gailit and Rouslahti, 1987). We have recently demonstrated by a differential display experiment comparing the expression patterns of Dispaseseparated non-involved psoriatic and healthy epidermis that among many others, fibronectin mRNA is overexpressed in the non-lesional epidermis (manuscript in preparation). This result is in agreement with the findings of Ting et al (2000), who demonstrated EDA þ fibronectin mRNA expression in the epidermis and EDA þ fibronectin protein expression at the dermal–epidermal junction of psoriatic uninvolved and involved skin. Rothaupt and co-workers have reported (2000) that at the dermal–epidermal junction bone-marrowderived immunocytes showed positive staining for the EDA þ form of fibronectin. Although we could not show EDA þ fibronectin expression in freshly separated non-lesional and lesional psoriatic epidermal cells, in short-term cultures, a small difference between uninvolved psoriatic and normal epidermal cells in EDA þ fibronectin production response became detectable. Keratinocytes similar to HaCaT cells may produce EDA þ fibronectin during a short period when they cycle and release the protein quickly into the extracellular space. It is also possible that among keratinocytes with proliferative potential, only a subpopulation of the cells are capable of EDA þ fibronectin production. The observed minor difference in the readiness of non-lesional psoriatic keratinocytes to produce EDA þ fibronectin to signals of activation (ex vivo culturing) may explain the presence of EDA þ fibronectin in the uninvolved psoriatic, but not in normal skin, which was observed by Ting et al (2000). Wounding may be required for non-lesional psoriatic keratinocytes to produce EDA þ fibronectin. All indications are that psoriatic non-lesional keratinocytes exhibit a ‘‘wound’’ phenotype: increased a5 integrin expression, accelerated spreading, and focal adhesion kinase responsiveness to fibronectin (Pellegrini et al, 1992; Bata-Csorgo et al, 1998; Chen et al, 2001). The enhanced EDA þ fibronectin production that we observed in the psoriatic nonlesional ex vivo culture could reflect an inherent abnormality of psoriatic keratinocytes, or it could be the result of an altered regulatory extracellular milieu. The latter is supported by observations showing alterations in the basement membrane in non-lesional psoriatic skin and MMP-2 overexpression by non-lesional keratinocytes (Mondello et al, 1996; Fleischmajer et al, 2000; Vaccaro et al, 2002). Taken together, our results indicate that psoriatic uninvolved basal keratinocytes could be responsible for releasing EDA þ fibronectin into the extracellular matrix, and in

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this way they themselves could contribute to the cellular environment that predisposes them to hyperproliferate. Materials and Methods Cell cultures Human epidermal and dermal cells were obtained from healthy individuals undergoing plastic surgery. Psoriatic nonlesional and lesional keratome biopsies from the buttock area were taken from patients with psoriasis vulgaris. The skin-donating patients had a medication-free period of at least 1 mo systemic and 2 wk topical therapy. Informed consents approved by internal review board were obtained from all donors. After removal of the subcutaneous tissue, the tissue samples were cut into small strips and incubated in Dispase solution (Grade II, Roche Molecular Biochemicals, Mannheim, Germany) overnight at 41C. On the following day, the epidermis was peeled off the dermis. The epidermis was incubated in 0.25% trypsin solution (Sigma, Budapest, Hungary) at 371C for 30 min and aspirated using a Pasteur pipette to aid cell dissociation. A suspension of primary epidermal cells was prepared in Keratinocyte Serum Free Medium (Keratinocyte-SFM, GibcoBRL, Eggstein, Germany) supplemented with antibiotic/antimycotic solution (Sigma, Budapest, Hungary). Epidermal cells were seeded into 75 cm2 tissue culture flasks (Myriad Industries, San Diego, California) at a density of 4  104 cells cm2 in Keratinocyte-SFM. Human epidermal keratinocytes were cultured in Keratinocyte-SFM. The medium was changed every 2 d. In our experiments, third-passage keratinocytes were used at 70%
80% confluence. For short-term (72 h) cultures of epidermal cells from normal and psoriatic non-lesional biopsies, keratinocyte-SFM medium without EGF and bovine pituitary extract, supplemented with 1% FBS (Hyclone, Perbio Science, BE, Bonn, Germany), was used. The dermis was also cut into small pieces, and then the pieces were placed in 6-well plates. After 3 days, fibroblasts had migrated out from the dermis. The small pieces of dermis were removed and the fibroblasts were cultured in high glucose DMEM (GibcoBRL) containing 20% FBS (GibcoBRL) in 75 cm2 tissue culture flasks (Myriad Industries). Fibroblasts were passaged twice a week; thirdpassage fibroblasts at 70–80% confluence were used for the experiments. Human HaCaT cells, kindly provided by Dr N. E. Fusenig (Heidelberg, Germany), were cultured and synchronized as described in detail in a previous publication (Pivarcsi et al, 2001). To observe whether cell attachment was necessary for the HaCaT cells to produce fibronectin and its EDA þ isoform, subconfluent HaCaT cells cultured in high glucose and 10% FBS-containing DMEM were passaged onto 9 cm Ø bacterial plates (Ma´traplast, Budapest, Hungary), tissue-culture plates (Sarstedt, Budapest, Hungary), glass plates covered with Teflon sheets (PEMU¨ Rt, Budapest, Hungary), and fibronectin-coated tissue-culture plates. To prepare the fibronectin coat, tissue-culture plates were incubated with 3 mL of 1 mg per mL fibronectin (Sigma) for 1 h at 371C and then rinsed with PBS. HaCaT cells were grown for 72 h under different attachment conditions and then harvested at 24 and 72 h after plating with TRIzol reagent (Invitrogen, Carlsbad, California). The cell cultures were maintained in a humidified atmosphere containing 5% CO2. Reverse transcription polymerase chain reaction (RT-PCR) Total RNA was isolated from the epidermal samples derived from punch biopsies and from various cell cultures by the Trizol reagent of GibcoBRL, following the instructions of the manual. cDNA was generated from 1 mg of RNA using the First Strand cDNA Synthesis Kit of MBI Fermentas (Vilnius, Lithuania) in a final volume of 20 mL. After reverse transcription, amplification was carried out using Taq DNA polymerase and the dNTP set of MBI Fermentas. Specific primers used in the experiments were as follows: human fibronectin (50 -AAGCCAATTTCCATTAATTACCGAAC-30 and 50 -TCTCATACTTGATGATGTAGCCGGTAA-30 ) resulting in a 1221 bp (EDA þ ) and an 847 bp (EDA
) product (Fig 1) and human glyceraldehyde-3-

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY phosphate dehydrogenase (G3PDH) (50 -ACAGTCCATGCCATCACTGCC-30 and 50 -GCCTGCTTCACCACCTTCTTG-30 ) resulting in a 235 bp product. PCR was carried out using the above primers at a final concentration of 0.66 pmol per mL. The concentration of MgCl2 was 1.5 mM in the reactions. The amplification protocol contained one cycle of initial denaturation at 941C for 5 min, varying number of cycles of denaturation at 941C for 1 min, annealing at 551C for 1 min, extension at 721C for 2 min, and one cycle of terminal extension at 721C for 10 min. Since fibronectin is expressed at a wide range in different cell types, the cDNA amounts in the PCR reactions varied depending on the cell type studied: 0.1 mL cDNA (originated from 10 ng total RNA) was used for the detection of fibronectin mRNA in fibroblasts and 2.5 mL cDNA (originated from 250 ng RNA) was used in HaCaT cells and in keratinocytes. For detecting G3PDH in the cDNAs derived from the HaCaT cell synchronization experiments, 0.2 mL cDNA was used. The number of cycles was 35 and 25 for fibronectin and for G3PDH, respectively. Ten microliters of the PCR products were run on 1% agarose gel, stained with ethidium bromide, and photographed and evaluated using a Kodak Edas 290 densitometer and Kodak 1D Digital Science software (Scientific Imaging Systems, New Haven, Connecticut). Western blot analysis Total protein extracts were prepared from synchronized HaCaT cells at different times after the end of synchronization either by taking the protein directly from the plates (n ¼ 2) or harvesting the cells first from the plates with a brief trypsinization (n ¼ 2) before putting them into the lysis buffer of 1.5% sodium dodecyl sulfate (SDS), 62.5 mM Tris-HCL pH 6.8, 5 mM ethylenediamine tetraacetic acid (EDTA), 5% 2-mercaptoethanol (2-ME), 1 mg per mL antipain, 1 mg per mL chymostatin, and 1 mg per mL leupeptin (all chemicals were obtained from Sigma). Lysates were precleared by centrifugation, and supernatants were stored at
201C. The concentration of proteins were defined by UV280 absorption and on the basis of the calculation from the absorption data, 100 mg of HaCaT cell extracts were separated by sodium-dodecyl-sulfate-polyacrylamide-gel-electrophoresis (SDSPAGE) on 8% separating gel (SDS-PAGE) and stained by Coomassie Brilliant Blue (Sigma). The gel was dried, scanned, and all of the loaded lanes were analyzed by densitometry. Based on the measured density, the amounts of the loaded protein samples were further corrected and checked again on SDS-PAGE. For the western blot analysis, equal amounts of proteins were run on SDSPAGE and then transferred onto a nylon membrane (Amersham, Buckingamshire, England). Membranes were blocked by incubation in Tris-buffered saline (150 mM NaCl, 25 mM Tris pH 7.4) containing 0.05% Tween 20 (Sigma) and 3% non-fat dry milk (Fluka Chemie AG, Neu-Buchs, Switzerland) for 2 h at room temperature and subsequently incubated overnight at 41C with either goat antiserum to human fibronectin (ICN Biochemicals, Aurora, Ohio) in a dilution of 1:200 or with mouse monoclonal antibody specific for human extra domain (EDA sequence) of cellular fibronectin at 1:200 dilution (ICN Biochemicals). Alkaline phosphatase-conjugated rabbit anti-goat IgG (whole molecule) (Sigma) and alkaline phosphatase-conjugated goat anti-mouse IgG (Fc specific) (Sigma) were used as secondary antibodies at 1:2000 dilution in the blocking buffer for 2 h at room temperature. The blot for total fibronectin was developed using 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium as a substrate (BCIP/NBT, Sigma), and the blot for EDA þ fibronectin was developed by enhanced chemiluminescence detection (Amersham, Arlington Heights, Illinois). Immunocytochemistry on cultured human fibroblasts, keratinocytes, and HaCaT cells Fibroblasts, keratinocytes (both in third passage), and HaCaT cells were seeded into slide chambers (Nunc A/S, Roskilde, Denmark) at a density of 4  104 cells per cm2 in the appropriate media and were maintained in a humidified atmosphere containing 5% CO2 until the cultures reached the state of subconfluency. For immunocytochemical staining, the cells were

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fixed for 20 min at 41C in 2% paraformaldehyde (Sigma), and then incubated overnight at 41C in a humid chamber with the primary antibodies. Mouse monoclonal antibody (1 mg per mL) specific for human extra domain (EDA sequence) of cellular fibronectin (ICN Biochemicals) was used as the primary antibody. Mouse IgG1 (Sigma) at a concentration of 1 mg per mL served as an isotypic control. Immunostaining was performed using an avidin–biotin immunoperoxidase kit (Vectastain Elite kit, Vector Laboratories, Burlingame, California) with 3-amino-9-ethylcarbazol (AEC; Sigma) as the chromogen. The slides were counterstained with hematoxylin.

Flow cytometry The staining protocol for flow cytometric analysis with the monoclonal antibody against the EDA þ fibronectin was established on cultured fibroblasts as a positive control. Different fixation methods were tried and compared. EDA þ fibronectin detection by flow cytometry was most efficient when cells were fixed in 0.4% paraformaldehyde for 20 min at 41C and then placed in 70% cold ethanol, and maintained at
201C at least overnight. With this sequential paraformaldehyde and ethanol fixation, both membrane-bound and intracellular EDA þ fibronectin could be detected (Pollice et al, 1992). Cells were then stained with the primary monoclonal antibody specific for EDA þ fibronectin (10 mg per mL) (ICN Biochemicals, Inc, Aurora, OH, USA) for 30 min at room temperature. Cells were washed two times in staining buffer (PBS þ 0.5% BSA), and then an FITC-conjugated rat anti-mouse IgG1 secondary antibody (BD Pharmingen, San Diego, California) was added for 30 min. Cells were either analyzed by flow cytometry or further stained with a PE-conjugated mouse anti-human PCNA antibody (BD Pharmingen) for 30 min. After two washings, cells were resuspended in PBS and immediately analyzed on a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany). Mouse IgG1 (Sigma) for the EDA þ fibronectin mAb and a direct PE-conjugated mouse IgG2a for the PCNA mAb at concentrations identical to the mAbs were used to define the background stainings. In each case, a minimum of 50,000 and a maximum of 100,000 cells were acquired. Data were analyzed with Cell Quest software from Becton Dickinson. In cultured fibroblasts, a complete shift occurred in the EDA þ fibronectin samples relative to the isotype control (mean channel fluorescence for EDA þ samples: 116.58 and for isotype: 60.55), with 55.75% signal above the isotype level, indicating that all cells consisted of some level of EDA þ fibronectin (data not shown).

This work was supported by grants OTKA T030749, OTKA T032496, OTKA T032494, OTKA TS044826, ETT 142/2001, and NKFP1A/0012/ 2002. Ma´rta Sze´ll was supported by the Bolyai Foundation of the Hungarian Academy of Sciences. DOI: 10.1111/j.0022-202X.2004.23224.x Manuscript received July 22, 2002; revised March 24, 2004; accepted for publication March 29, 2004 Address correspondence to: Dr Ma´rta Sze´ll, Dermatological Research Group of the Hungarian Academy of Sciences and the University of Szeged, 6720 Szeged, Kora´nyi fasor 6, Hungary. Email: szell@derma. szote.u-szeged.hu

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