Low Dose Ultraviolet B Irradiation Increases Hyaluronan Synthesis in Epidermal Keratinocytes via Sequential Induction of Hyaluronan Synthases Has1–3 Mediated by p38 and Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Signaling*

Background: Accumulation of epidermal hyaluronan is an early event in UVB-induced squamous cell carcinomas. Results: UVB increases keratinocyte hyaluronan synthesis by up-regulating hyaluronan synthases Has1 and -2 via p38 and Has3 via Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling. Conclusion: UVB triggers deposition of epidermal hyaluronan through at least two signaling pathways. Significance: Increased hyaluronan synthesis is an inherent feature of keratinocyte adaptation to radiation injury. Hyaluronan, a major epidermal extracellular matrix component, responds strongly to different kinds of injuries. This also occurs by UV radiation, but the mechanisms involved are poorly understood. The effects of a single ultraviolet B (UVB) exposure on hyaluronan content and molecular mass, and expression of genes involved in hyaluronan metabolism were defined in monolayer and differentiated, organotypic three-dimensional cultures of rat epidermal keratinocytes. The signals regulating the response were characterized using specific inhibitors and Western blotting. In monolayer cultures, UVB increased hyaluronan synthase Has1 mRNA already 4 h postexposure, with a return to control level by 24 h. In contrast, Has2 and Has3 were persistently elevated from 8 h onward. Silencing of Has2 and especially Has3 decreased the UVB-induced accumulation of hyaluronan. p38 and Ca2+/calmodulin-dependent protein kinase II pathways were found to be involved in the UVB-induced up-regulation of Has2 and Has3 expression, respectively, and their inhibition reduced hyaluronan deposition. However, the expressions of the hyaluronan-degrading enzymes Hyal1 and Hyal2 and the hyaluronan receptor Cd44 were also up-regulated by UVB. In organotypic cultures, UVB treatment also resulted in increased expression of both Has and Hyal genes and shifted hyaluronan toward a smaller size range. Histochemical stainings indicated localized losses of hyaluronan in the epidermis. The data show that exposure of keratinocytes to acute, low dose UVB increases hyaluronan synthesis via up-regulation of Has2 and Has3. The simultaneously enhanced catabolism of hyaluronan demonstrates the complexity of the UVB-induced changes. Nevertheless, enhanced hyaluronan metabolism is an important part of the adaptation of keratinocytes to radiation injury.

Proper responsiveness to ultraviolet (UV) radiation and other external stimuli is crucial for the structural integrity and function of the skin, which protects us from physical, biological, and chemical hazards. UVB 2 (280 -315 nm) is an especially potent inducer of biochemically and genetically significant changes in the epidermis and its main cell type, keratinocyte. Elucidating the effects and mechanisms of action of UVB is increasingly significant as the incidence of skin carcinomas and melanomas continues to rise (1). However, UVB may also have beneficial effects on barrier regulation and natural immunity (2).
Like all epithelia, epidermis is characterized by a scanty extracellular matrix. However, in the extracellular matrix of dividing and differentiating basal and spinous cell layers, hyaluronan reaches high concentrations (3). Hyaluronan is clearly one of the main extracellular matrix molecules of epidermal keratinocytes as well as of the skin as whole (4,5). Hyaluronan is a high molecular weight, non-sulfated, free glycosaminoglycan consisting of a linear polymer of repeating disaccharide units of N-acetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcUA). In mammals, it is synthesized by three hyaluronan synthases (HAS1-3) on the inner surface of the plasma membrane and extruded directly into the extracellular space (6,7). In the pericellular matrix, it is organized into diffuse coats often supported by distinct membrane protrusions (8 -10) or longer, cable-like structures that are induced by inflammation or cell stress (11,12). The most prominent hyaluronan receptor on the keratinocyte surface is the transmembrane protein CD44 (13), which is also crucial for mediating hyaluronan signaling (14).
Hyaluronan is very hygroscopic and a good space filler. Moreover, it is needed by various cell types for attachment, migration, proliferation, and signaling, and its roles in embryonic development (15,16), inflammation (17,18), and cancer initiation and progression are widely recognized (19,20). In the adult skin, hyaluronan participates in hydration (5), wound healing (21,22), and maintenance of the balance between cell division and differentiation in the epidermis (23).
Hyaluronan content and HAS transcript levels change in response to UV irradiation in cultured keratinocytes and fibroblasts as well as in intact skin. Both up-regulation (24 -28) and down-regulation (29 -31) have been reported. The response undoubtedly depends on the cellular background, radiation type (narrowband versus broadband) and wavelength, dosage, exposure scheme (single, repeated, or chronic), and the time point of observation (32)(33)(34). Release of KGF and IL-1␤ has been implicated in the hyaluronan response seen in UV-treated keratinocyte cultures (25). In fibroblasts, collagen fragments produced by UVB-activated matrix metalloproteinases downregulate HAS2 expression by altering pERK-pELK1 signaling (31). It has also been shown that HAS3 is up-regulated via p38 signaling in human skin treated with simulated solar radiation (26). Despite these advances, our understanding of the specific signaling pathways leading to the observed changes in hyaluronan synthesis is essentially lacking.
In general, alterations in cellular metabolism in response to UVB involve the interplay of several factors. The effects of high energy radiation range from direct DNA damage to generating reactive oxygen species (ROS) that can modify other biomolecules and trigger signaling cascades. Activated plasma membrane receptors, their phosphorylated effector kinases, and transcription factors downstream are often central to the responses (35)(36)(37)(38). For example, a key role has been demonstrated for epidermal growth factor receptor (EGFR), a prominent receptor tyrosine kinase in keratinocytes (39), in relaying UVB-induced responses (40). It is also important to distinguish between immediate and delayed effects (41). This versatility in responses and the multitude of pathways potentially activated could account for the dynamic temporal patterns previously observed in hyaluronan secretion and HAS expression.
In this work, we wanted to characterize the mechanisms of hyaluronan response to acute, low dose UVB. We used both monolayer and organotypic cultures of rat epidermal keratinocytes, which provide a useful platform for modeling epidermal biology and hyaluronan metabolism (42)(43)(44)(45)(46). We found a rapid increase of hyaluronan production induced by UVB exposure via up-regulation of hyaluronan synthases (Has1-3) through the p38 and Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII) pathways. Displaying the complexity of the UVB response, the expression of enzymes involved in hyaluronan catabolism was also up-regulated. Experiments using organotypic three-dimensional cultures suggested UVB-induced fragmentation of hyaluronan, in addition to enhanced synthesis.
For the organotypic cultures, REKs were cultured on semipermeable inserts coated with type I collagen as described by Pasonen-Seppänen et al. (48). The three-dimensional cultures were maintained for 2 weeks in DMEM (Invitrogen) supplemented with 10% FBS (HyClone), 4 mM L-glutamine (Euroclone), and penicillin/streptomycin (50 g/ml streptomycin, 50 units/ml penicillin; Euroclone). Fresh medium was changed every 1-2 days. L-Ascorbic acid (40 g/ml; Sigma-Aldrich) was added from 1 day after lifting the cultures onto the air-liquid interface (culture day 4) to facilitate normal differentiation but left out 1 day before the UVB exposures.
UVB Irradiation-REKs were seeded on 6-well plates at 150,000 cells/well in 2 ml of growth medium and grown for 24 h prior to UVB exposure. The light source was a portable UV lamp (UVM-57; UVP, Upland, CA) that emits midrange UV at a nominal wavelength of 302 nm and emission maximum at 312 nm (supplemental Fig. 1). The characteristics of the lamp were verified by spectroradiometry (Macam SR9910; Macam Photometrics Ltd., Livingston, Scotland, UK), and the exposure distance and time were set to obtain effective doses of 2.5, 5, 10, 15, 20, 30, and 40 mJ/cm 2 .
For monolayer exposure, the culture medium was removed, and 0.5 ml of Dulbecco's PBS (Euroclone) per well was added. The UVB and sham treatments (UV light off) were performed with the plate lid removed. After the UVB or sham treatment, Dulbecco's PBS was removed, and 1 ml of fresh medium was added. For the experiments on dose dependence, the plates were further incubated for 24 h, and the culture medium was quantified for hyaluronan. To measure temporal changes after a 10-mJ/cm 2 dose, hyaluronan was assayed 12, 24, 36, and 48 h after exposure.
UVB exposure of the organotypic cultures was done 13-15 days after starting the cultures. For this, the medium beneath each insert was briefly replaced with 0.8 -1.5 ml of Dulbecco's PBS to wash the culture. Subsequently, 800 l of Dulbecco's PBS was added for the duration of the UVB or sham treatment. Afterward, 1.0 -1.5 ml of fresh medium was added, and the cultures were incubated for 24 h and collected for histology and/or biochemical analyses. In a subset of experiments, the organotypic cultures were maintained in a base medium (DMEM; Invitrogen) without phenol red and irradiated directly in 1.5 ml of the medium with lower serum (5% FBS). The cultures were exposed to 10 -40 mJ/cm 2 for the histological examinations and 20 -30 mJ/cm 2 for the metabolic labeling of newly synthesized glycosaminoglycans.
Treatments with Inhibitors of Signaling Pathways-To investigate the intracellular signaling pathways involved, REKs were seeded on 6-well plates at 150,000 -175,000 cells/well in 2 ml of monolayer growth medium, grown for 24 h, and treated for 2 h with inhibitors targeting EGFR, MEK1/2, PI3K, Akt1/2, p38, JNK, STAT3, and CaMKII before exposure to 10 mJ/cm 2 of UVB. Following irradiation, the cells were grown for either 8 and 36 h (for gene expression analyses) or 24 h (for hyaluronan assay) in fresh medium containing the respective inhibitors (supplemental Table 1).
Quantification of Hyaluronan-Hyaluronan concentration of the media was quantified using a sandwich-type enzymelinked sorbent assay as described previously (49). Briefly, 96-well plates were coated with hyaluronan-binding complex (HABC; 0.5 or 1.0 g/ml, prepared in-house as described previously (50)), blocked with 1% BSA in PBS, and incubated with 0 -50 ng/ml hyaluronan standards (Provisc; Algon Laboratories, Fort Worth, TX) or samples diluted with 1% BSA in PBS. Biotinylated HABC (prepared in-house) was added to detect the bound hyaluronan. Visualization was performed by sequential additions of horseradish peroxidase, 3,3Ј,5,5Ј-tetramethylbenzidine, H 2 O 2 , and 50 l of 2 M H 2 SO 4 for absorbance at 450 nm. Standards and samples were run as duplicates or triplicates.
Metabolic Labeling-In the organotypic cultures, hyaluronan synthesis was quantified using metabolic labeling as described previously (51). The organotypic cultures were irradiated as described above and incubated for 24 h in 1 ml of medium containing 1% FBS and 20 Ci/ml [ 3 H]glucosamine and 100 Ci/ml [ 35 S]Na 2 SO 4 (both from PerkinElmer Life Sciences). The media were collected, and the culture inserts were rinsed with 300 l of HBSS that was combined with the medium. The epidermis and the collagenous support layers were separated, and the three compartments (medium, epidermis, and collagen) were analyzed individually. The epidermal samples were incubated in pure acetone at 4°C with three changes and then air-dried and weighed.
The samples were subsequently purified and quantified for hyaluronan and chondroitin sulfate as described previously (42,51). The samples were digested overnight at 60°C with papain (Sigma-Aldrich), boiled, and centrifuged. Standard hyaluronan (6 g; Healon, Advanced Medical Optics Uppsala Ab, Uppsala, Sweden) was added to the supernatant as a carrier and internal standard. The samples were precipitated with 1% cetylpyridinium chloride, and the pellets were washed with distilled H 2 O, dissolved in 4 M guanidium chloride, and reprecipitated with ethanol. The pellets were digested with Streptococcus hyaluronidase (1 milliunit; Seikagaku Kogyo Co., Tokyo, Japan) and chondroitinase ABC (25 milliunits; Seikagaku). Hyaluronan-and chondroitin sulfate-derived disaccharides were separated on a Superdex Peptide column (GE Healthcare) and counted for [ 3 H]hexosamines and [ 35 S]sulfate to quantify the newly synthesized hyaluronan and chondroitin sulfates (52). The hyaluronan disaccharide absorbance at 232 nm was used to correct for possible losses in the purification.
Molecular Mass Distribution of Hyaluronan-To analyze the size distribution of hyaluronan secreted in the medium, gel filtration on a 1 ϫ 30-cm Sephacryl S-1000 column (HR 10/30; GE Healthcare) was done as described previously (51). 150 mM sodium acetate buffer (pH 6.8) containing 0.1% CHAPS, at a flow rate of 24 ml/h, was used for elution. For samples collected from monolayer cultures, 1-ml fractions were lyophilized and redissolved in 250 l of 1% BSA-PBS for the determination of hyaluronan content with an enzyme-linked sorbent assay as described above. For the organotypic cultures, epidermal sheets were separated and digested with 250 g of proteinase K in 100 mM ammonium acetate for 2 h at 60°C. After inactivating the enzyme, supernatants were collected and analyzed with S-1000 as described above. For the organotypic specimens, no concentration step was necessary. The column was calibrated using hyaluronan standards with known molecular mass (150, 500, and 2500 kDa), purchased from Hyalose (Oklahoma City, OK).
RNA Extraction and Quantitative Real-time PCR-The transcript levels of Has1-3, Hyal1 and -2 (hyaluronidase 1 and 2), and Cd44 were measured using quantitative real-time PCR (qRT-PCR). For the monolayer cultures, cells were plated and cultured as above, exposed to 10 mJ/cm 2 of UVB, and incubated for an additional 2-36 h. Cells were lysed by adding 1 ml of RNA extraction reagent/well (EuroGOLD RNAPure TM or EUROzol from Euroclone, TRI Reagent solution from Applied Biosystems/Invitrogen, or TRIzol reagent from Invitrogen). Total RNA was extracted with chloroform-isopropyl alcohol according to standard procedures, washed once with 75% ethanol, dissolved in nuclease-free water, and stored at Ϫ70°C.
For the organotypic cultures, the epidermal layer was separated with fine tweezers and frozen in liquid nitrogen 24 h after the UVB exposure (30 mJ/cm 2 ). The samples were then homogenized in Lysing Matrix D tubes (MP Biomedicals, Santa Ana, CA) with a FastPrep homogenizer (Savant, Thermo Fisher Scientific), and total RNA was extracted with the High Pure RNA Tissue Kit (Roche Applied Science) according to the kit protocol.
For cDNA synthesis and qRT-PCR, 1 g of total RNA was amplified with the Verso TM SYBR Green two-step qRT-PCR kit (Abgene Ltd., Thermo Fisher Scientific) using anchored oligo(dT) primers. The cDNA was diluted 1:5, and 2 l was added to each PCR (FastStart Universal SYBR Green Master (Rox)). The samples were amplified with a Stratagene Mx3000P realtime PCR machine (Agilent, La Jolla, CA) using 45 cycles of 20 s at 95°C, 20 s at 60°C, and 20 s at 72°C, preceded by activation of the enzyme for 15 min at 95°C. Ribosomal protein LP0 (Rplp0) was used as the reference gene. The sequences of the primers and the sizes of the amplicons are presented in supplemental Table 2.
siRNA Transfections-The specific siRNAs against Has1 and Has3 were ordered from Eurogentec (Liège, Belgium), and siRNAs against Has2 were from Ambion (Austin, TX). The control siRNA for all was from Origene (Rockville, MD). For Has1 and Has3, three different siRNAs were used in combination at a final concentration of 50 nM, whereas for Has2, a single prescreened oligonucleotide was chosen (supplemental Table  3). The transfections were performed in antibiotic-free medium (MEM with 5% FBS; Invitrogen), mixing either the control or the specific siRNAs with RNAiMAX (Invitrogen). All of the reagents were diluted in ␣-MEM (HyClone). RNAiMAX mixed with ␣-MEM served as the transfection reagent control.
The cells were transfected for 4 -6 h, after which they were grown overnight in fresh, antibiotic-free medium. Thereafter, the cells were split on 6-well plates at 175,000 cells/well, grown overnight, and irradiated with 10 mJ/cm 2 of UVB as described above. Total RNA was collected 8 h after and medium for hyaluronan assays 24 h after the exposure.
Histochemistry and Microscopy-2-Week-old REK three-dimensional cultures were fixed in 2% paraformaldehyde, 0.5% glutaraldehyde or in Histochoice MB (Amresco, Solon, OH) overnight at 4°C and embedded in paraffin. Hematoxylin and eosin (H&E) stainings were performed according to standard procedures to confirm the general morphology of the cultures. Hyaluronan stainings were performed with biotinylated HABC with Mayer's hematoxylin counterstaining and mounted in DePex (BDH Laboratory Supplies, Poole, UK) as described previously (54).
For CD44 immunostaining, the sections were first incubated in target retrieval solution (Dako, Glostrup, Denmark) for 30 min at 95°C, followed by blocking of endogenous peroxidases and nonspecific binding as described above. After an overnight incubation at 4°C with the anti-CD44 antibody (OX-50; Chemicon/Millipore, Billerica, MA) (diluted 1:200 with 1% BSA in 0.1 M sodium phosphate buffer, pH 7.4), the sections were incubated with a biotinylated anti-mouse antibody (Vector Laboratories, Inc., Burlingame, CA) (diluted 1:200 with 1% BSA in 0.1 M sodium phosphate buffer, pH 7.4) for 1 h, followed by sequential incubations with the ABC reagent and 3,3Јdiaminobenzidine, counterstaining, and mounting as described above (54).
The slides were analyzed and photographed on a Nikon Microphot-FXA microscope (Nikon Corp., Tokyo, Japan) using a CoolSNAP digitizing system (Photometrics, Tucson, AZ) or on a Zeiss AxioImager M2 microscope (Carl Zeiss AG, Oberkochen, Germany) with a digital color camera (AxioCam MRc, Carl Zeiss AG).
Statistical Analyses-Statistical analyses were performed with the PASW Statistics version 18.0 software package for Mac OS X and IBM SPSS Statistics, version 19 (SPSS Inc., Chicago, IL), R for Windows version 2.7.2 (55), and GraphPad Prism version 5.03 for Windows (GraphPad Software, Inc., La Jolla, CA). Clear outliers were removed based on Grubbs' test with a significance level of p Ͻ 0.01. Mathematical transformation of variables with unequal error variances or lacking normal distribution was performed using log 10 or ln transformations to correct the situation.
The data were subjected to analyses of variance using either univariate, mixed, or two-way models depending on the experimental set-up, followed by analyses of pairwise comparisons using Tukey's honestly significant difference and Bonferroni's tests or estimated marginal means (LSD), as indicated in the figure legends. In the inhibitor experiments, differences between the individual treatment groups were analyzed using the estimated marginal means, and each inhibitor-treated group was compared with control by calculating the cumulative distribution function in R for Windows (pnorm), in both cases correcting the p value (LSD) for the number of comparisons.

Acute UVB Exposure Stimulates Hyaluronan Synthesis in
Monolayer REK Cultures-Monolayer REK cultures were subjected to a single exposure of broadband UVB, and hyaluronan secreted in the culture medium during a 24-h follow-up was analyzed. In the first experimental set-up, we used irradiation doses ranging from 2.5 to 10 mJ/cm 2 (Fig. 1A). Whereas 5 mJ/cm 2 of UVB caused a slight increase in the amount of extracellular hyaluronan, 10 mJ/cm 2 caused a statistically significant stimulation when compared with controls.
To study whether higher UVB doses would further increase the hyaluronan response, additional experiments were conducted, including doses up to 20 mJ/cm 2 (Fig. 1B). 15 mJ/cm 2 caused an effect comparable with that of 10 mJ/cm 2 , whereas 20 mJ/cm 2 reversed the trend. Because 15 and 20 mJ/cm 2 elicited up to 35% dead cells (data not shown), compared with a maximum of ϳ10% seen in cultures treated with the lower doses, 10 mJ/cm 2 was chosen for further experiments. With this level of irradiation, a steady, 2-3 times higher hyaluronan secretion was evident in all subsequent data sets (Figs. 1C, 3, and 4). 10 mJ/cm 2 caused a transient inhibition in cell proliferation, as indicated by significantly lower cell counts at 12 h after the irradiation (supplemental Fig. 2). Thereafter, the cell numbers increased at apparently equal rates.
Temporal changes in hyaluronan secretion after a single dose (10 mJ/cm 2 ) were examined by collecting culture media at different time points after the exposure. The content of hyaluronan in the UVB-treated cultures was 142% higher compared with the non-irradiated controls already 12 h postexposure, indicating that the response was rapid. At later time points, the difference between the UVB-and sham-treated groups was increased even further (Fig. 1C).
Because UV irradiation can break hyaluronan chains both in vivo and in vitro (56,57), we analyzed the size distribution of the hyaluronan released into the growth medium during a 24-h period after a single UVB exposure. As reported previously (58), the majority of hyaluronan secreted by control REKs represented high molecular mass molecules (Ͼ2000 kDa; Fig. 1D). UVB treatment did not cause significant changes in this distribution.

UVB Treatment Causes Up-regulation of All Has Isoforms-
To establish the cause for the increased hyaluronan secretion, transcript levels of Has1-3 were evaluated by qRT-PCR. The mRNA levels of Has1 and Has3 were relatively low in REK monolayers as compared with Has2, with relative C t values of about 30, 32, and 27 for Has1, Has3, and Has2, respectively (data not shown). There were clear, dynamic, and statistically significant changes in the mRNA levels of the Has enzymes after a single exposure to 10 mJ/cm 2 (two-way analysis of variance; overall effect of UVB, p Ͻ 0.05 for Has1, p Ͻ 0.001 for Has2 and Has3; Fig. 2, A-C). Has1 showed the earliest induction (2-fold) after 4 h, declining thereafter back to the basal level ( Fig. 2A). Has2 increased about 2-fold at 8 -12 h after an initial decline (Fig. 2B). It remained up-regulated until 36 h, where the highest induction (about 3-fold) was seen. Has3 showed the highest overall induction of all Has isoforms. The up-regulation of Has3 expression started 8 h postirradiation and lasted up to 36 h (Fig. 2C).
To see whether UVB exposure influences the expression of proteins involved in the degradation of hyaluronan, we analyzed its effects on the expression of Hyal1 and Hyal2, and the hyaluronan receptor Cd44. The mRNA levels of Hyal1 appeared lower than those of Hyal2 and Cd44 in untreated cultures (relative C t values of about 31, 26, and 24, respectively; data not shown). Hyal1 and Hyal2 transcript levels were significantly up-regulated by UVB. The two Hyal transcripts showed very similar temporal patterns after the UVB exposure, with a significant increase from 4 to 12 h. However, the -fold changes in Hyal1 transcript levels were higher than those in Hyal2 (Fig.  2, D and E). About 50% up-regulation of Cd44 was seen at the transcript level at 24 -36 h after an initial 50% drop at 2-8 h post-UVB (Fig. 2F). Western blot analyses (supplemental Fig. 3) showed that REK cells express both standard (90 kDa) and several higher molecular mass isoforms similar to human keratinocytes (59). No differences between the controls and the UVBtreated cultures were detected, however.
Taken together, the data indicate that the expression of the three Has isoforms, the hyaluronan receptor Cd44, and the hyaluronan degrading enzymes Hyal1 and Hyal2 are all significantly modulated after an acute exposure to UVB. Although Has2, Has3, and CD44 transcripts follow a broadly similar pattern with a delayed and sustained up-regulation, Has1 and Hyal transcripts show a more rapid and transient increase.
Knockdown of Has3 and Has2 with siRNA Reduces the UVBinduced Accumulation of Hyaluronan-To get an idea about the contribution of the different HAS isoenzymes for the accumulation of hyaluronan in REK cultures, we used siRNAs that reduced both Has2 and Has3 mRNAs in control and UVBirradiated samples by about 60 -65% (Fig. 3A). Silencing of Has2 resulted in a modest reduction in accumulated hyaluronan both in the non-UVB-treated cultures (on average 38%) and in UVB-treated cultures (on average 26%; Fig. 3B). Silencing of Has3 expression resulted in a more substantial reduction in both the basal hyaluronan production (on average 49%) and UVB-stimulated hyaluronan production (on average 57%; Fig.  3B). Unfortunately, we were unable to effectively silence Has1 expression (data not shown).

FIGURE 1. UVB irradiation induces hyaluronan secretion in REK monolayers in a dose-dependent manner without affecting the molecular mass distribution.
A and B, REK monolayer cultures were exposed to the indicated doses of UVB, and the culture media were collected after a 24-h incubation and analyzed for hyaluronan content. The data in A and B represent means and S.E. (error bars) of three independent experiments. Statistical significance was analyzed using univariate analysis of variance. Individual means between groups were compared using Tukey's honestly significant difference (*, p Ͻ 0.05; ***, p Ͻ 0.001). C, hyaluronan secretion in the culture medium during various time intervals following a single UVB dose of 10 mJ/cm 2 . The overall effect of the UVB treatment was significant (p Ͻ 0.001; two-way analysis of variance). The data represent means and S.E. from three independent experiments, each containing two replicates for each time point and treatment. The results of Bonferroni's tests for individual time points are shown (**, p Ͻ 0.01; ***, p Ͻ 0.001). D, the molecular mass distribution of hyaluronan released in the culture medium during a 24-h incubation after a single UVB exposure (10 mJ/cm 2 ) was analyzed using Sephacryl S-1000 gel chromatography. The data represent means and half-range from two independent experiments.
p38 Inhibition Suppresses the UVB-induced Hyaluronan Synthesis-To find the signaling pathways involved in the UVBinduced hyaluronan response, we treated REK monolayers with inhibitors specific to several pathways previously reported to be UVB-responsive and also demonstrated to regulate hyaluronan secretion, like EGFR and its downstream effectors ERK1/2, Akt, STAT3, and the stress-responsive p38 and JNK (supplemental Table 1). The experiments showed that under the basal experimental conditions, EGFR and the MAPK p38 regulated hyaluronan production (Fig. 4A). The EGFR inhibitor AG1478 and the p38 inhibitor BIRB796 caused an ϳ25% reduction in the basal level of hyaluronan secretion in REK cells, whereas the other inhibitors did not exert a significant influence (supplemental Fig. 4). Of these inhibitors, AG1478 and BIRB796 also reduced the UVB-induced increase of hyaluronan secretion (Fig. 4A). The decrease caused by the EGFR inhibitor was ϳ28%, whereas the p38 inhibitor BIRB796 cut the induction by 46% (Fig. 4A).
However, inhibitors of the common downstream pathways of EGFR, UO126, and PD 98059 for MEK1/2 and wortmannin and Akt inhibitor VIII for PI3K and Akt, respectively, failed to counteract the UVB-induced hyaluronan production (supplemental Fig. 4). Similarly, the inhibitor of STAT3 (Cpd188), a downstream target of the EGFR and JAK pathways, exerted no effect on hyaluronan release (supplemental Fig. 4), not even with a higher dose (10 M; data not shown). Furthermore, inhibition of stress-related signaling pathways by targeting JNK with SP600125 had no influence on hyaluronan secretion (supplemental Fig. 4). In conclusion, of the signals previously known to stimulate hyaluronan synthesis, p38 was clearly associated with the UVB-induced response, whereas the contribution of EGFR was more modest.
Because UV radiation has been shown to activate CaMKII by regulating intracellular calcium levels in immune cells and fibroblasts (60,61), and CaMKII can control various signaling pathways, including MAPKs, we tested the effect of a specific CaMKII inhibitor, KN93, on hyaluronan synthesis in REK cells (Fig. 4A). This inhibitor exerted a strong inhibitory activity both on the basal hyaluronan level (Ϫ51%) and the UVB-induced increase in hyaluronan accumulation (Ϫ71%; Fig. 4A). However, KN92, the generally used inactive control compound for KN93, also decreased the UVB-induced response by 39%, whereas its effect on the basal secretion was small (Ϫ11%; Fig.  4A).

Regulation of UVB-induced Has Expression Is Associated with p38 and CaMKII-In order to see if the observed changes in hyaluronan synthesis and
Has expression correlated with activation of the corresponding signaling pathways, we performed Western blots with phosphospecific antibodies (Fig. 4, B and C, and supplemental Fig. 5). Phosphorylated MAPK p38 showed a constant 2-3-fold increase compared with the control specimens collected at the same time point (Fig. 4, B and C), whereas JNK had a lower level of activation (supplemental Fig. 5), and ERK1/2 showed no activation at all (supplemental Fig. 5). STAT3 phosphorylation was down-regulated (although not statistically significantly) at 4 h postexposure and returned back to the control level at 6 h post-UVB (supplemental Fig. 5), a pattern similar to the one previously suggested by Kim et al. (62).
Because the effects of inhibitors on hyaluronan synthesis suggested involvement of p38, EGFR, and CaMKII in the UVBinduced hyaluronan response, we wanted to see if inhibitors of these signaling routes influence Has expression. The 8 h time point was checked first, because all Has isoenzymes were upregulated then (Fig. 2, A-C). The p38 inhibitor BIRB796 effectively cut the UVB-induced Has1 and Has2 expression down to the level of the control cultures (Fig. 5A), although it also affected the basal expression of both Has isoforms. In contrast, the EGFR inhibitor AG1478 had no effect on the mRNA levels of Has2 and Has3, whereas there was actually an increase in Has1 (supplemental Fig. 6). At 8 h postirradiation, the CaMKII inhibitor KN93 had no significant effect on Has1 and Has3 expression, whereas it slightly increased the basal expression of Has2 (Fig. 5B).
Because there appeared to be a later activation of Has2 and Has3 expression at the 36 h time point (Fig. 2, A-C), we also tested whether p38 and CaMKII were involved in those up-regulations. The p38 inhibitor BIRB796 tended to reduce the UVBinduced Has2 response also at this time point but had no effect on the induction of Has3 (Fig. 5A). At 36 h, the CaMKII inhibitor KN93 up-regulated the basal expression of Has1 and Has2, whereas the UVB-induced Has2 response was not affected. In contrast, the UVB response of Has3 mRNA was strongly down-regulated by KN93 at this time point, whereas the ineffective analog KN92 had no influence (Fig. 5B).
Altogether, the experiments with specific inhibitors suggest that the ERK, Akt, and STAT3 pathways have no significant role in the UVB-induced hyaluronan synthesis in rat keratinocytes. Instead, p38 increases the expression of Has2, and CaM-KII increases the expression of Has3, apparently accounting for the hyaluronan response.
Acute UVB Exposure Activates Hyaluronan Metabolism in an Epidermal Equivalent-To study whether UVB can also regulate epidermal hyaluronan synthesis under conditions that resemble the situation in vivo, we employed organotypic threedimensional cultures of the same REK cells. When raised to the air-liquid interface, they form a fully differentiated epidermis in 2 weeks. We exposed the differentiated cultures to slightly higher doses of UVB (20 -40 mJ/cm 2 ) than the monolayer cultures, because the radiation is filtered through an intact stratum corneum. These were biologically relevant doses, as indicated by the increased number of apoptotic cells. With 20 -30  . p38 activation is involved in the accumulation of hyaluronan after UVB exposure. REKs pretreated with specific inhibitors for 2 h were exposed to 10 mJ/cm 2 UVB, followed by a 24-h incubation in the presence of the corresponding inhibitor, as explained in supplemental Table 1. The control cultures contained an equal concentration of DMSO, which was used to dissolve the inhibitors. A, hyaluronan accumulation with an inhibitor for p38 MAPK (BIRB796), EGFR (AG1478), and Ca 2ϩ/ calmodulin-dependent protein kinase II (KN93) and its supposedly inactive control compound (KN92). The data represent means and S.E. (error bars) from three independent experiments. Statistical significance was analyzed using univariate analysis of variance, and the differences between treatments were tested using estimated marginal means (LSD) and between controls and treatments using pnorm as described under "Experimental Procedures" (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001). B, Western blot of UVB-exposed and control specimens at different time points after a single exposure to UVB, detected with a phosphospecific antibody against active p38 (p-p38), total actin, and total p38␣. Normalization against actin and total p38␣, respectively, was done from the same samples, on separate blots. C, actin-normalized (gray bars) and p38␣-normalized (black bars) band intensities of active p-p38 (means and S.E. from three independent experiments). Two-way analysis of variance was used to analyze the data, and the results of Bonferroni's tests for individual comparisons are shown (**, p Ͻ 0.01; ***, p Ͻ 0.001, as compared with the control at the same time point).

UVB-induced Up-regulation of Has1-3 via p38 and CaMKII
mJ/cm 2 the general tissue morphology was normal, whereas 40 mJ/cm 2 caused tissue necrosis. 3 Hyaluronan synthesis was analyzed using metabolic labeling, which gives additional information on the synthesis of sulfated glycosaminoglycans. The total amount of hyaluronan synthesized during the 24-h labeling period, including that in the epidermis, the supporting collagen gel, and the culture medium, was increased after UVB (p ϭ 0.05; Fig. 6A). A similar trend was seen in the epidermal layer alone (Fig. 6B); however, the effect was not statistically significant. This assay also indicated that the synthesis of chondroitin sulfates was not significantly stimulated by UVB irradiation (Fig. 6C).
The transcript levels of Has1-3, Cd44, and Hyal1 and -2 were evaluated by qRT-PCR in the organotypic cultures 8 and 24 h after irradiation with 30 mJ/cm 2 . In the unirradiated organotypic cultures, the transcripts of Cd44 and Hyal2 appeared most abundant (C t values around 24 and 25, respectively; data not shown), whereas Has1, Has2, Has3, and Hyal1 showed relatively lower transcript levels (C t values ϳ32, 28, 30, and 28, respectively; data not shown).
No change in Has1 mRNA level was detected in the organotypic model at either 8 or 24 h post-UVB (Fig. 6D). The mRNA level of Has2 was up-regulated by 1.6-fold at 24 h, and that of Has3 was up-regulated by 1.6-and 1.8-fold at the 8 and 24 h time points, respectively (Fig. 6D). The rise in Hyal2 and Hyal1 mRNA levels was modest although statistically significant (1.4fold for Hyal1 at 24 h and 1.2-and 1.4-fold for Hyal2 at 8 and 24 h; Fig. 6D). Similarly, a modest, 1.4-fold increase was found in the mRNA level of Cd44 at 8 h.
Hyaluronan staining intensity was higher in the UVB-treated epidermis than in the control cultures (Fig. 7, A-D). Although the distribution of hyaluronan staining was not affected by the lower UVB dose, increasing the dose tended to make the staining pattern irregular, with regions of increased staining interspersed with areas showing loss of hyaluronan (Fig. 7, C and D). Like hyaluronan, its receptor CD44 was localized in the basal and spinous layers (Fig. 7, E-H). With the lower UVB doses, no substantial changes in the intensity or localization of CD44 staining were detectable. However, at the highest dose (40 mJ/cm 2 ), CD44 staining became irregular with locally reduced signals, similar to the pattern of hyaluronan (Fig. 7H).
Because the histological analyses suggested local acceleration of hyaluronan degradation in the UVB-treated samples, we assayed the molecular mass distribution of epidermal hyaluronan. The analyses showed that the UVB-treated epidermal cultures contained more hyaluronan in the lower, ϳ50 -500 kDa size range (Fig. 7, I and J). At 24 h, the increase of the low molecular mass hyaluronan (fractions 25-33) was 61%, as compared with 25% in the high molecular mass material (fractions 17-23; Fig. 7J). A similar trend was also seen in the size distribution of hyaluronan associated with the collagen matrix (data not shown). Because the newly synthesized hyaluronan in these cells is of high molecular mass (58) (Fig. 1D), the data suggest accelerated fragmentation of hyaluronan in the UVB-treated specimens. Thus, the organotypic epidermis, containing keratinocytes at all differentiation stages and with UVB filtered through an intact stratum corneum, also elicits a clear hyaluronan response to acute irradiation, with  both the synthetic and degradative mechanisms activated after the exposure.

DISCUSSION
The present data show that keratinocytes respond to acute, low dose UVB exposure by a rapid and long lasting synthesis and deposition of hyaluronan. The increase of hyaluronan was associated with sequential, partly overlapping inductions of the Has1, Has2, and Has3 genes, the latter two mediated by the p38 and CaMKII pathways, respectively. Increased expression of Hyal1 and Hyal2 as well as Cd44 suggests that not just the synthesis, but also degradation of hyaluronan was affected. Most of these responses were reproduced in a stratified, organotypic model, supporting the in vivo relevance of the results.
Dose Dependence of the UVB-induced Changes in Hyaluronan Metabolism-To avoid overt toxicity, we used relatively low UVB doses in most of the experiments. That these doses were biologically meaningful was shown by the fact that cell proliferation temporarily slowed down. Furthermore, the results of a genome-wide gene expression array using the organotypic model indicated highly significant activation of many biological processes relevant to irradiation damage, including DNA repair and cell cycle control. 3 Although an increase in hyaluronan content was first found with increasing doses of UVB, a trend toward reduction was observed at the highest dose used. A similar dose-dependent dichotomy in the acute effects of UVB irradiation on the synthesis of hyaluronan has been found in human keratinocytes (25), probably reflecting general toxicity of the high doses.
In the organotypic epidermis, UVB is filtered through stratum corneum and a part of the vital cell layers. This caused variation in the effective dose reaching the cells, and generally higher doses were required to reach effects similar to those seen in the monolayer cultures.
Time-dependent Expression of Has Isoenzymes-Whereas Has1 expression rapidly increased, Has2 was down-regulated 2-4 h after the UVB exposure, which correlates well with the previously reported decrease of Has2 transcripts at 3-6 h postirradiation in HaCaT cells (24,63). Regardless of the fluctuations of Has expressions during the first hours, hyaluronan synthesis rose rapidly, as indicated by the 2.4-fold elevation in its content already after 12 h. Interestingly, quite a persistent up-regulation of Has2 and Has3 took place in REKs at the later time points after a single UVB exposure, causing a robust increase in hyaluronan secretion. This is also in line with the recent data of Tobiishi et al. (28), who saw in mouse epidermis a significant up-regulation of Has2 and Has3 following 1 and 2 days of acute UVB irradiation, respectively.
The Role of Different Has Isoenzymes in the UVB-induced Synthesis of Hyaluronan-Has2 and Has3 gene up-regulation generally accompanies hyaluronan accumulation in epidermal keratinocytes subjected to various growth factors or injury (22, 46, 64 -66). The same was found in response to UVB in the present and previous reports (24,25,28). Due to their coexpression and similar reactions to many of the stimuli studied, the contribution of each individual Has to the deposition of hyaluronan in keratinocytes has been difficult. However, a drastic decline in the wounding-induced epidermal hyaluronan accumulation was observed in mice with deleted Has3 and Has1 genes, suggesting that one or both of these enzymes were responsible for the majority of hyaluronan accumulation (67). Our data using siRNA interference indicated that Has3 played a major role in UVB-induced hyaluronan synthesis in REK cells, whereas Has2 also contributed to hyaluronan accumulation, although its role was more modest. Because we were unable to silence Has1 expression, the role of Has1 remains unsettled at the moment. The less persistent up-regulation of Has1 by UVB and its dependence on high substrate levels for active hyaluronan synthesis, as compared with Has2 and Has3 (68,69), suggest that the role of Has1 in the UVB-induced synthesis of hyaluronan in keratinocytes is less prominent than that of Has3. UVB caused up-regulation of Has2 and Has3 expression also in the organotypic cultures. However, the magnitude of the UVB response in Has3 expression was more modest in the organotypic cultures, and no change at all was detected in Has1. Variation in the effective UVB dose received by different epidermal cell layers may contribute to the smaller response in the organotypic cultures. It is also likely that the signaling systems influencing Has expression vary between undifferentiated and differentiated cells. Likewise, release of bioactive molecules from the cornified and differentiating cells may contribute to the differential effects observed between the monolayer and organotypic models.
The Role of Hyaluronan Degradation in the UVB-induced Hyaluronan Response-While hyaluronan synthesis and content increased in the UVB-treated cultures, there was a simultaneous up-regulation of Hyal1 and Hyal2 expressions both in the monolayer and organotypic keratinocyte cultures, in line with a previous report (24). Increased hyaluronidase activity should lead to enhanced catabolism and decreased hyaluronan accumulation, counteracting the stimulated hyaluronan pro- FIGURE 7. Epidermal hyaluronan distribution in UVB-exposed organotypic cultures. Hyaluronan was stained with biotinylated HABC (bHABC; A-D) and CD44 antibody (OX50; E-H) 24 h after a single UVB or sham exposure, as indicated in the panels. The images are from one experiment, representative of four independent experiments, each experiment containing a minimum of two replicates for each condition. The arrows indicate areas of hyaluronan and CD44 depletion in cultures treated with higher doses. The magnification bar represents 50 m. I-J, molecular mass distribution of epidermal hyaluronan extracted 8 h (I) and 24 h (J) after a UVB (30 mJ/cm 2 ) or sham exposure and analyzed with S1000 gel filtration. The data represent means and half-range (error bars) from two cultures. duction by HAS activity. Although no hyaluronan fragmentation was visible by gel filtration in the REK monolayer cultures or previously in human keratinocytes (25), we observed a change in the molecular mass distribution toward smaller sizes in the organotypic model. The cause of this fragmentation, whether due to the Hyal enzymes or to non-enzymatic degradation via ROS (70) generated by the irradiation (71,72), remains unsettled at the moment. ROS may also have an indirect effect by activating Hyal enzymes (73,74), adding to the complex regulatory mechanisms.
However, the metabolism of epidermal hyaluronan is more efficient in organotypic cultures (51,75) than in monolayers. 4 Keratinocytes in the normal epidermis produce hyaluronan into the narrow intercellular space, where it is probably more accessible to the degradative machinery (receptors, enzymes, and ROS), compared with monolayers, where hyaluronan is mostly released into a large volume of culture medium.
In any case, the change in the molecular mass distribution and the locally reduced staining pattern of hyaluronan after the highest UVB doses suggest that acute UVB causes activation of hyaluronan catabolism in the epidermis. This, together with the lower activation of Has expression, probably explains why the surge of hyaluronan remained more modest in the organotypic cultures than in the monolayers.
p38 Activation Is Involved in Increased Hyaluronan Synthesis and Has Expression-From the multitude of possible signaling routes activated by UVB (35, 40, 41, 62, 76 -78), we first checked EGFR and its downstream pathways, known to be of crucial importance in the regulation of epidermal hyaluronan synthesis (45,46,64,79). However, the inhibitor of EGFR exhibited a modest effect on the UVB-induced hyaluronan response, and it failed to prevent the UVB-induced up-regulation of Has expression. Lack of UVB-induced activation of ERK1/2, STAT3, and JNK and the inability of the inhibitors of these pathways to suppress hyaluronan production excluded these pathways as mediators of the UVB-induced hyaluronan response in REK cells.
Of the MAPKs tested, only p38 showed significant activation by UVB. Accordingly, a specific inhibitor of p38 significantly suppressed the UVB-induced increase of hyaluronan. The target of p38 signaling was clearly Has expression, because p38 inhibition efficiently blocked the UVB-mediated up-regulation of Has1 and particularly Has2.
Calcium Signals Involved in the UVB-induced Hyaluronan Synthesis-UVB has been reported to increase Ca 2ϩ flux into the cytosol, activating the production of ROS (80) and various signaling cascades (61). Our data, indicating that KN93, a specific inhibitor of CaMKII, reduced the UVB-induced hyaluronan synthesis clearly more than its inactive control compound, suggest the involvement of calcium-related pathways in the hyaluronan response. This would be in agreement with previous reports that perturbation of epidermal calcium homeostasis is associated with changes in hyaluronan synthesis (81) and that UV radiation can activate CaMKII (60,61). There are, however, no previous reports connecting CaMKII to the regulation of Has expression.
Of the three Has isoenzymes, Has3 appeared to be the one regulated by a CaMKII-related mechanism, explaining the strong effect of KN93 on hyaluronan synthesis. The effect on Has3 expression was especially obvious at the later time points, suggesting that the up-regulation of Has3 by CaMKII probably represents an adaptive response to more immediate changes triggered by UVB. Our preliminary data suggest that G-protein-mediated mechanisms (including the P2Y2 receptor) may be involved in the induction of Has3 by UVB. 5 However, further experimentation is needed to define the upstream factors evoking the CaMKII and p38 signals that lead to the up-regulation of the Has isoforms.
Importance of Hyaluronan Synthesis in UVB-induced Malignancies-The emerging link between epidermal hyaluronan and UVB is particularly noteworthy, considering the increasing incidence of basal and squamous cell carcinomas and melanomas. Squamous cell carcinomas induced by chronic UV irradiation show elevated levels of all HAS isoenzymes and accumulation of hyaluronan (27). Notably, chronic UV exposure causes hyaluronan accumulation in the epidermis already in benign lesions (27). This is in line with our present findings on acute hyaluronan response and supports the view that hyaluronan synthesis is activated early on during skin carcinogenesis. In the human skin tumors related to UV irradiation (i.e. SCC and melanoma), hyaluronan increases in early lesions, whereas it tends to be reduced in more advanced cancers 6 (82). In immunostainings of human melanomas, HAS1-3 are all present, and particularly HAS1 and HAS2 are elevated in dysplastic nevi and/or melanoma in situ but decreased in metastases. 6 The staining for HYAL2 is increased in both early and more advanced melanomas. 6 The present findings are thus largely parallel to those in early UV-induced tumors in vivo.
Interestingly, skin tumor development appears to be dependent on p38 function (83), the same signal found here in the acute hyaluronan response to UVB. CaMKII also appears to be a key mediator in carcinomas inducing keratinocyte differentiation with Wnt5a in regressing tumors (84). Moreover, the hyaluronan-CD44 axis has been indicated to convey signals via CaMKII in head and neck squamous carcinoma cells, causing cytoskeletal rearrangements and promoting migration (85). The activation of p38, CaMKII, and hyaluronan synthesis may thus represent changes in the epidermis that facilitate cell proliferation and later, after repeated exposure, tumorigenesis.
HAS Activation as a Part of Inflammation and Wounding-p38 is activated in inflammation, and the biological significance of the acutely increased hyaluronan synthesis after UVB-irradiation may be a part of this adaptive reaction to tissue trauma. This would be similar to the p38-dependent up-regulation of Has1 and Has2 noted in synovial fibroblasts and intestinal mesenchymal cells, respectively, when subjected to mediators of inflammation (86,87). Similarly, Has3 can be induced by proinflammatory cytokines in synovial membrane cells (88). This kind of acute increase in epidermal hyaluronan can be considered as an adaptive response to restore homeostasis, much like those seen after wounding (22) and epidermal barrier disruption (89).
Hyaluronan may also have a protective effect in itself by modulating apoptotic signals and cytokine production after UVB irradiation, like in corneal epithelial cell cultures pretreated with high molecular mass hyaluronan (90). Similarly, it was reported that exogenous hyaluronan increases the viability of UVB-exposed keratinocytes and decreases the secretion of inflammatory mediators (63). Moreover, the anti-inflammatory action of hyaluronan is supported by the finding that Has1/ Has3 knock-out mice show enhanced inflammatory response after wounding (67), and overexpressing Has1 reduces leukocyte infiltration and enhances wound healing in a mouse model (91).

CONCLUSION
The present work shows for the first time that the acute upregulation of epidermal hyaluronan synthesis by UVB radiation is mediated by p38 and CaMKII. Although Has1 and Has2 have previously been shown to be regulated through p38, our finding that Has3 is regulated by CaMKII reveals a new regulatory pathway. In addition to activating hyaluronan synthesis, UVB may also influence hyaluronan degradation, especially in the context of normal epidermal tissue architecture. The final outcome of the enhanced hyaluronan metabolism induced by UVB, whether accumulation or loss of hyaluronan, eventually depends on the balance of these opposite processes, as seen in UVB-induced skin tumors.