Hyaluronan synthases (HAS1-3) in UV-treated epidermis. Specimens from mouse back skin were stained with antibodies against HAS1 (a–d), HAS2 (e–h), and HAS3 (i–l). In d, h, and l, the antibodies were preincubated with the peptides used in the immunization. All HAS antibodies gave a low-level signal in control, unirradiated epidermis, whereas dermal cells showed a stronger immunostaining (a, e, i). In UV-treated skin, hyperplastic areas (c, g, k) and SCCs (b, f, j) keratinocytes and fibroblasts showed increased staining intensity for all HASes compared with control skin. Magnification bar 20 μm (in a for a, e, i and in b for b–d, f–h, and j–l). 

Contexts in source publication

Context 1

... carcinomas. The area of HA and CD44 stainings in the epidermis was estimated with a four-level scoring from 0 to 3. Score 0 was given when no staining or only minimal staining around 1–2 hair follicles was detected. Score 1 was given when less than 33% of the interfollicular area was stained, score 2 when 33–66% of the interfollicular area was stained, and score 3 when more than 66% of the interfollicular area was stained. The intensity of epidermal staining was estimated with a four-level scoring from 0 to 3. The homogeneity of the staining was recorded as well as the intensity of dermal staining and the staining of skin areas showing abnormal morphology. The statistical calculations were performed using the SPSS program for MacIntosh (version 11.0, SPSS, Chicago, IL). The Kruskal-Wallis and Mann-Whitney U-tests were used to compare the extent and intensity of the stainings between treatments. The amount of epidermal hyperplasia was correlated to the HA and CD44 parameters using Kendall’s test. Probability values less than 0.05 were considered significant. In the present work, mouse back skin was exposed to 1 MED of UVR three times a week for 10.5 months. Histological evaluation indicated that in most of the treated animals the epidermal tissue showed marked, approximately 3–4-fold thickening (Fig. 1, e and f) compared with control skin (Fig. 1, a and b, Table 1). One fifth of the UVR-exposed animals developed squamous cell carcinomas (SCC, Fig. 2, d–i, and Table 1), and one third showed dysplastic changes (Fig. 2, a–c). Only one of the control animals showed mild hyperplasia, whereas dysplasia or SCC was not detected in any of the control animals (Table 1). In normal, untreated skin, dermal connective tissue was intensely stained for hyaluronan, whereas most of the epidermis either was negative (Fig. 1a) or showed a weak positive signal around the orifices of hair follicles (Table 1), as previously described in mouse ear and tail (Tammi et al. 2005). Although CD44 immunostaining in control skin was generally weak, some CD44-positive cells were found throughout the whole interfollicular epidermis, in both the basal and suprabasal layers (Fig. 1b, Table 2). In UVR-treated animals, hyaluronan-positive cells cov- ered more than two thirds of the epidermal area in most of the specimens (Table 2), and the intensity of epidermal hyaluronan staining was significantly increased compared with untreated epidermis (Table 2, Fig. 1 and 2). CD44 immunostaining also showed significantly increased epidermal staining intensity and coverage in UVR-treated animals (Figs. 1 and 2, Table 2). Epidermal areas showing benign hyperplasia showed moderate or strong hyaluronan and CD44 stainings, even in the case of moderate hyperplasia (Fig. 1, c and d); however, the number of positive layers was increased when the degree of hyperplasia increased (Fig. 1, e and f), with all vital cell layers except granular cells being positive for hyaluronan and CD44. Epidermal hyperplasia showed a significant positive correlation with both intensity and coverage of hyaluronan staining (Kendall’s τ-b test, p <0.01). Similar correlation was also observed between epidermal hyperplasia and CD44 staining intensity and coverage (Kendall’s τ-b test, p <0.01 and 0.05, respectively). Epidermal areas containing dysplastic cells were generally moderately or intensely positive for hyaluronan and CD44; however, they often contained patches with reduced staining or even no staining at all (Fig. 2, b and c). Similarly, invasive squamous cell carcinomas showed generally moderate or strong hyaluronan and CD44 stainings (Fig. 2, e and f), but patches of lower staining intensity made the appearance inhomogeneous in some of the tumors (Fig. 2, h and i). Dermal connective tissue stained for hyaluronan in all samples. In semiquantitative scoring of dermal hyaluronan staining intensity, all samples in control group were graded as moderately stained, whereas in the UVR group 1 of 21 was graded as weakly stained, 2 were graded as strongly stained, and the remaining were graded as moderate (NS). However, in UVR-treated animals showing epidermal hyperplasia, there was a loss of subepidermal hyaluronan staining in 69% of the hyperplastic samples (Fig. 1e). We performed immunostainings with antibodies raised against hyaluronan synthase enzymes 1, 2, and 3 (Fig. 3). In the control skin that was not exposed to UVB (Fig. 3, a, e and i) just a few epidermal cells showed HAS- immunoreactivity above the level of diffuse, low-intensity background signal. The dermal cells stained weakly or with moderate intensity for HAS1, HAS2, and HAS3 (Fig. 3, a, e and i). The staining intensity in specimens exposed to the UVR was clearly higher with all HAS antibodies, in both epidermis and dermis (Fig. 3, b, c, f, g, j and k). Strongest induction of staining intensity was found with the HAS2 antibody (Fig. 3, f and g). The increased HAS immunostainings were seen both in hyperplastic areas (Fig. 3, c, g and k) and in the squamous cell carcinomas (Fig. 3, b, f and j). Fig. 3, d, h and l shows specimens stained with HAS- antibodies preincubated with peptides used for immunization. Most of the staining was lost, indicating the specificity of the signal. The retrieval at high temperature required for the exposure of the epitopes (Rilla et al, unpublished) tended to tear the fragile mouse skin sections and precluded HAS immunostainings of sufficient quality for comprehensive scoring of all specimens. The present study shows for the first time that chronic UV irradiation of skin leads to the accumulation of hyaluronan and its receptor CD44 in the epidermis and that this accumulation correlates with the development of epidermal hyperplasia. Although the effect of chronic UVR on GAG and hyaluronan metabolism in the dermal compartment has been studied previously (Schwartz 1988; Margelin et al. 1996; Koshiishi et al. 1999; Dai et al. 2007), none of the previous publications has reported changes in the epidermal compartment. The reports concerning the response of epidermal cells to acute UVR have been contradictory (Calikoglu et al. 2006; Averbeck et al. 2007; Kakizaki et al. 2008), probably because of differences in the dose and UV source. The single dose used in the present work (20 mJ/cm corresponding one minimum erythema dose) is comparable to the doses that have been reported to stimulate hyaluronan synthesis in in vitro experiments (Averbeck et al. 2007; Kakizaki et al. 2008). However, the doses cannot be directly compared, because under in vitro conditions epidermal cells lack the natural protective mechanisms, e.g., melanosomes and stratum corneum, and are therefore more sensitive to the UVR. The UV source used in the present work simulated solar irradiation, containing a broad spectrum of wavelengths from 310 to 400 nm (Kumlin et al. 1998). The main part (98%) of the physical dose is comprised of UVA; however, about 70% of the biologically effective (erythema-inducing) irradiation is expected to come from the UVB wavelength. UVA may either be ineffective in stimulating hyaluronan synthesis (Kakizaki et al. 2008) or even inhibit it when used at high doses (Calikoglu et al. 2006). Although in the present experiment we cannot differentiate between the effects of UVB and UVA on hyaluronan metabolism, our findings indicate that in a chronic setting, UVR resembling solar irradiation at intensities causing skin erythema (1 MED) causes a dramatic change in epidermal hyaluronan metabolism. In the present material, the increase in HAS- immunostaining intensities in the UVR exposed epidermis suggests that the increased hyaluronan content in UVR- treated epidermis is at least partly explained by increased HAS activity. All HAS isoforms showed increased immunostaining in UVR exposed skin. In line with the previous cell culture experiments (Averbeck et al. 2007), HAS2 showed highest increase due to UVR. However, possible differences in the sensitivity of the antibodies preclude direct conclusions concerning HAS2 as the major target. UVR is known to activate several signaling routes, but there are no comprehensive data showing which of the signals are involved in the UV-induced upregulation of hyaluronan synthesis and HAS expression (Averbeck et al. 2007; Kakizaki et al. 2008). The acute phase upregulation of HAS2 and HAS3 was inhibited by blocking IL-1β, suggesting that an inflammatory pathway is involved (Kakizaki et al. 2008). Chronic UVB has been reported to cause enhanced activation of pathways associated with EGFR and ErbB2, PI3K and AKT, JNK, NFkB, and Stat3 (Sano et al. 2005; Katiyar and Meeran. 2007; Wunderlich et al. 2008), all known to influence HAS2 or HAS3 expression (Pienimäki et al. 2001; Saavalainen et al. 2005; Madson and Hansen. 2007; Han et al. 2008). The strongly elevated level of CD44 is likely to contribute to the increased epidermal content of hyaluronan by binding and immobilizing hyaluronan on the plasma membranes of keratinocytes. The close correlation between CD44 and hyaluronan staining patterns observed in the present work and in previous publications (Pirinen et al. 1998; Hirvikoski et al. 1999; Karvinen et al. 2003; Kosunen et al. 2004) supports this conclusion. However, acute high- dose UVB and UVA irradiation was reported to cause depletion of CD44 (Calikoglu et al. 2006), and our own data in cultured keratinocytes support this finding (Rauhala et al. unpublished), indicating that the CD44 response to UVR is biphasic. Mutations of p53 are typically found in epidermal keratinocytes exposed to solar radiation very early, before the actual tumor (de Gruijl and Rebel. 2008; Klein et al. 2010), and cause upregulation of CD44 expression (Godar et al. 2008), suggesting a plausible mechanism for the increased CD44 levels in our model. The p53-induced effect of CD44 on cell growth and survival was mediated by EGFR (Godar et al. 2008). In our material the epidermal hyperplasia ...

Context 2

... with a four-level scoring from 0 to 3. Score 0 was given when no staining or only minimal staining around 1–2 hair follicles was detected. Score 1 was given when less than 33% of the interfollicular area was stained, score 2 when 33–66% of the interfollicular area was stained, and score 3 when more than 66% of the interfollicular area was stained. The intensity of epidermal staining was estimated with a four-level scoring from 0 to 3. The homogeneity of the staining was recorded as well as the intensity of dermal staining and the staining of skin areas showing abnormal morphology. The statistical calculations were performed using the SPSS program for MacIntosh (version 11.0, SPSS, Chicago, IL). The Kruskal-Wallis and Mann-Whitney U-tests were used to compare the extent and intensity of the stainings between treatments. The amount of epidermal hyperplasia was correlated to the HA and CD44 parameters using Kendall’s test. Probability values less than 0.05 were considered significant. In the present work, mouse back skin was exposed to 1 MED of UVR three times a week for 10.5 months. Histological evaluation indicated that in most of the treated animals the epidermal tissue showed marked, approximately 3–4-fold thickening (Fig. 1, e and f) compared with control skin (Fig. 1, a and b, Table 1). One fifth of the UVR-exposed animals developed squamous cell carcinomas (SCC, Fig. 2, d–i, and Table 1), and one third showed dysplastic changes (Fig. 2, a–c). Only one of the control animals showed mild hyperplasia, whereas dysplasia or SCC was not detected in any of the control animals (Table 1). In normal, untreated skin, dermal connective tissue was intensely stained for hyaluronan, whereas most of the epidermis either was negative (Fig. 1a) or showed a weak positive signal around the orifices of hair follicles (Table 1), as previously described in mouse ear and tail (Tammi et al. 2005). Although CD44 immunostaining in control skin was generally weak, some CD44-positive cells were found throughout the whole interfollicular epidermis, in both the basal and suprabasal layers (Fig. 1b, Table 2). In UVR-treated animals, hyaluronan-positive cells cov- ered more than two thirds of the epidermal area in most of the specimens (Table 2), and the intensity of epidermal hyaluronan staining was significantly increased compared with untreated epidermis (Table 2, Fig. 1 and 2). CD44 immunostaining also showed significantly increased epidermal staining intensity and coverage in UVR-treated animals (Figs. 1 and 2, Table 2). Epidermal areas showing benign hyperplasia showed moderate or strong hyaluronan and CD44 stainings, even in the case of moderate hyperplasia (Fig. 1, c and d); however, the number of positive layers was increased when the degree of hyperplasia increased (Fig. 1, e and f), with all vital cell layers except granular cells being positive for hyaluronan and CD44. Epidermal hyperplasia showed a significant positive correlation with both intensity and coverage of hyaluronan staining (Kendall’s τ-b test, p <0.01). Similar correlation was also observed between epidermal hyperplasia and CD44 staining intensity and coverage (Kendall’s τ-b test, p <0.01 and 0.05, respectively). Epidermal areas containing dysplastic cells were generally moderately or intensely positive for hyaluronan and CD44; however, they often contained patches with reduced staining or even no staining at all (Fig. 2, b and c). Similarly, invasive squamous cell carcinomas showed generally moderate or strong hyaluronan and CD44 stainings (Fig. 2, e and f), but patches of lower staining intensity made the appearance inhomogeneous in some of the tumors (Fig. 2, h and i). Dermal connective tissue stained for hyaluronan in all samples. In semiquantitative scoring of dermal hyaluronan staining intensity, all samples in control group were graded as moderately stained, whereas in the UVR group 1 of 21 was graded as weakly stained, 2 were graded as strongly stained, and the remaining were graded as moderate (NS). However, in UVR-treated animals showing epidermal hyperplasia, there was a loss of subepidermal hyaluronan staining in 69% of the hyperplastic samples (Fig. 1e). We performed immunostainings with antibodies raised against hyaluronan synthase enzymes 1, 2, and 3 (Fig. 3). In the control skin that was not exposed to UVB (Fig. 3, a, e and i) just a few epidermal cells showed HAS- immunoreactivity above the level of diffuse, low-intensity background signal. The dermal cells stained weakly or with moderate intensity for HAS1, HAS2, and HAS3 (Fig. 3, a, e and i). The staining intensity in specimens exposed to the UVR was clearly higher with all HAS antibodies, in both epidermis and dermis (Fig. 3, b, c, f, g, j and k). Strongest induction of staining intensity was found with the HAS2 antibody (Fig. 3, f and g). The increased HAS immunostainings were seen both in hyperplastic areas (Fig. 3, c, g and k) and in the squamous cell carcinomas (Fig. 3, b, f and j). Fig. 3, d, h and l shows specimens stained with HAS- antibodies preincubated with peptides used for immunization. Most of the staining was lost, indicating the specificity of the signal. The retrieval at high temperature required for the exposure of the epitopes (Rilla et al, unpublished) tended to tear the fragile mouse skin sections and precluded HAS immunostainings of sufficient quality for comprehensive scoring of all specimens. The present study shows for the first time that chronic UV irradiation of skin leads to the accumulation of hyaluronan and its receptor CD44 in the epidermis and that this accumulation correlates with the development of epidermal hyperplasia. Although the effect of chronic UVR on GAG and hyaluronan metabolism in the dermal compartment has been studied previously (Schwartz 1988; Margelin et al. 1996; Koshiishi et al. 1999; Dai et al. 2007), none of the previous publications has reported changes in the epidermal compartment. The reports concerning the response of epidermal cells to acute UVR have been contradictory (Calikoglu et al. 2006; Averbeck et al. 2007; Kakizaki et al. 2008), probably because of differences in the dose and UV source. The single dose used in the present work (20 mJ/cm corresponding one minimum erythema dose) is comparable to the doses that have been reported to stimulate hyaluronan synthesis in in vitro experiments (Averbeck et al. 2007; Kakizaki et al. 2008). However, the doses cannot be directly compared, because under in vitro conditions epidermal cells lack the natural protective mechanisms, e.g., melanosomes and stratum corneum, and are therefore more sensitive to the UVR. The UV source used in the present work simulated solar irradiation, containing a broad spectrum of wavelengths from 310 to 400 nm (Kumlin et al. 1998). The main part (98%) of the physical dose is comprised of UVA; however, about 70% of the biologically effective (erythema-inducing) irradiation is expected to come from the UVB wavelength. UVA may either be ineffective in stimulating hyaluronan synthesis (Kakizaki et al. 2008) or even inhibit it when used at high doses (Calikoglu et al. 2006). Although in the present experiment we cannot differentiate between the effects of UVB and UVA on hyaluronan metabolism, our findings indicate that in a chronic setting, UVR resembling solar irradiation at intensities causing skin erythema (1 MED) causes a dramatic change in epidermal hyaluronan metabolism. In the present material, the increase in HAS- immunostaining intensities in the UVR exposed epidermis suggests that the increased hyaluronan content in UVR- treated epidermis is at least partly explained by increased HAS activity. All HAS isoforms showed increased immunostaining in UVR exposed skin. In line with the previous cell culture experiments (Averbeck et al. 2007), HAS2 showed highest increase due to UVR. However, possible differences in the sensitivity of the antibodies preclude direct conclusions concerning HAS2 as the major target. UVR is known to activate several signaling routes, but there are no comprehensive data showing which of the signals are involved in the UV-induced upregulation of hyaluronan synthesis and HAS expression (Averbeck et al. 2007; Kakizaki et al. 2008). The acute phase upregulation of HAS2 and HAS3 was inhibited by blocking IL-1β, suggesting that an inflammatory pathway is involved (Kakizaki et al. 2008). Chronic UVB has been reported to cause enhanced activation of pathways associated with EGFR and ErbB2, PI3K and AKT, JNK, NFkB, and Stat3 (Sano et al. 2005; Katiyar and Meeran. 2007; Wunderlich et al. 2008), all known to influence HAS2 or HAS3 expression (Pienimäki et al. 2001; Saavalainen et al. 2005; Madson and Hansen. 2007; Han et al. 2008). The strongly elevated level of CD44 is likely to contribute to the increased epidermal content of hyaluronan by binding and immobilizing hyaluronan on the plasma membranes of keratinocytes. The close correlation between CD44 and hyaluronan staining patterns observed in the present work and in previous publications (Pirinen et al. 1998; Hirvikoski et al. 1999; Karvinen et al. 2003; Kosunen et al. 2004) supports this conclusion. However, acute high- dose UVB and UVA irradiation was reported to cause depletion of CD44 (Calikoglu et al. 2006), and our own data in cultured keratinocytes support this finding (Rauhala et al. unpublished), indicating that the CD44 response to UVR is biphasic. Mutations of p53 are typically found in epidermal keratinocytes exposed to solar radiation very early, before the actual tumor (de Gruijl and Rebel. 2008; Klein et al. 2010), and cause upregulation of CD44 expression (Godar et al. 2008), suggesting a plausible mechanism for the increased CD44 levels in our model. The p53-induced effect of CD44 on cell growth and survival was mediated by EGFR (Godar et al. 2008). In our material the epidermal hyperplasia strongly correlated with the levels of epidermal CD44 and hyaluronan. Likewise, ...

Context 3

... was used as control. The stainings were analyzed blind regarding the experimental group. General morphological features of the sections were analyzed and scored for epidermal hyperplasia (no hyperplasia, mild, moderate, strong hyperplasia). In addition, the sections were scored for the presence of tumors, dysplasia, and squamous cell carcinomas. The area of HA and CD44 stainings in the epidermis was estimated with a four-level scoring from 0 to 3. Score 0 was given when no staining or only minimal staining around 1–2 hair follicles was detected. Score 1 was given when less than 33% of the interfollicular area was stained, score 2 when 33–66% of the interfollicular area was stained, and score 3 when more than 66% of the interfollicular area was stained. The intensity of epidermal staining was estimated with a four-level scoring from 0 to 3. The homogeneity of the staining was recorded as well as the intensity of dermal staining and the staining of skin areas showing abnormal morphology. The statistical calculations were performed using the SPSS program for MacIntosh (version 11.0, SPSS, Chicago, IL). The Kruskal-Wallis and Mann-Whitney U-tests were used to compare the extent and intensity of the stainings between treatments. The amount of epidermal hyperplasia was correlated to the HA and CD44 parameters using Kendall’s test. Probability values less than 0.05 were considered significant. In the present work, mouse back skin was exposed to 1 MED of UVR three times a week for 10.5 months. Histological evaluation indicated that in most of the treated animals the epidermal tissue showed marked, approximately 3–4-fold thickening (Fig. 1, e and f) compared with control skin (Fig. 1, a and b, Table 1). One fifth of the UVR-exposed animals developed squamous cell carcinomas (SCC, Fig. 2, d–i, and Table 1), and one third showed dysplastic changes (Fig. 2, a–c). Only one of the control animals showed mild hyperplasia, whereas dysplasia or SCC was not detected in any of the control animals (Table 1). In normal, untreated skin, dermal connective tissue was intensely stained for hyaluronan, whereas most of the epidermis either was negative (Fig. 1a) or showed a weak positive signal around the orifices of hair follicles (Table 1), as previously described in mouse ear and tail (Tammi et al. 2005). Although CD44 immunostaining in control skin was generally weak, some CD44-positive cells were found throughout the whole interfollicular epidermis, in both the basal and suprabasal layers (Fig. 1b, Table 2). In UVR-treated animals, hyaluronan-positive cells cov- ered more than two thirds of the epidermal area in most of the specimens (Table 2), and the intensity of epidermal hyaluronan staining was significantly increased compared with untreated epidermis (Table 2, Fig. 1 and 2). CD44 immunostaining also showed significantly increased epidermal staining intensity and coverage in UVR-treated animals (Figs. 1 and 2, Table 2). Epidermal areas showing benign hyperplasia showed moderate or strong hyaluronan and CD44 stainings, even in the case of moderate hyperplasia (Fig. 1, c and d); however, the number of positive layers was increased when the degree of hyperplasia increased (Fig. 1, e and f), with all vital cell layers except granular cells being positive for hyaluronan and CD44. Epidermal hyperplasia showed a significant positive correlation with both intensity and coverage of hyaluronan staining (Kendall’s τ-b test, p <0.01). Similar correlation was also observed between epidermal hyperplasia and CD44 staining intensity and coverage (Kendall’s τ-b test, p <0.01 and 0.05, respectively). Epidermal areas containing dysplastic cells were generally moderately or intensely positive for hyaluronan and CD44; however, they often contained patches with reduced staining or even no staining at all (Fig. 2, b and c). Similarly, invasive squamous cell carcinomas showed generally moderate or strong hyaluronan and CD44 stainings (Fig. 2, e and f), but patches of lower staining intensity made the appearance inhomogeneous in some of the tumors (Fig. 2, h and i). Dermal connective tissue stained for hyaluronan in all samples. In semiquantitative scoring of dermal hyaluronan staining intensity, all samples in control group were graded as moderately stained, whereas in the UVR group 1 of 21 was graded as weakly stained, 2 were graded as strongly stained, and the remaining were graded as moderate (NS). However, in UVR-treated animals showing epidermal hyperplasia, there was a loss of subepidermal hyaluronan staining in 69% of the hyperplastic samples (Fig. 1e). We performed immunostainings with antibodies raised against hyaluronan synthase enzymes 1, 2, and 3 (Fig. 3). In the control skin that was not exposed to UVB (Fig. 3, a, e and i) just a few epidermal cells showed HAS- immunoreactivity above the level of diffuse, low-intensity background signal. The dermal cells stained weakly or with moderate intensity for HAS1, HAS2, and HAS3 (Fig. 3, a, e and i). The staining intensity in specimens exposed to the UVR was clearly higher with all HAS antibodies, in both epidermis and dermis (Fig. 3, b, c, f, g, j and k). Strongest induction of staining intensity was found with the HAS2 antibody (Fig. 3, f and g). The increased HAS immunostainings were seen both in hyperplastic areas (Fig. 3, c, g and k) and in the squamous cell carcinomas (Fig. 3, b, f and j). Fig. 3, d, h and l shows specimens stained with HAS- antibodies preincubated with peptides used for immunization. Most of the staining was lost, indicating the specificity of the signal. The retrieval at high temperature required for the exposure of the epitopes (Rilla et al, unpublished) tended to tear the fragile mouse skin sections and precluded HAS immunostainings of sufficient quality for comprehensive scoring of all specimens. The present study shows for the first time that chronic UV irradiation of skin leads to the accumulation of hyaluronan and its receptor CD44 in the epidermis and that this accumulation correlates with the development of epidermal hyperplasia. Although the effect of chronic UVR on GAG and hyaluronan metabolism in the dermal compartment has been studied previously (Schwartz 1988; Margelin et al. 1996; Koshiishi et al. 1999; Dai et al. 2007), none of the previous publications has reported changes in the epidermal compartment. The reports concerning the response of epidermal cells to acute UVR have been contradictory (Calikoglu et al. 2006; Averbeck et al. 2007; Kakizaki et al. 2008), probably because of differences in the dose and UV source. The single dose used in the present work (20 mJ/cm corresponding one minimum erythema dose) is comparable to the doses that have been reported to stimulate hyaluronan synthesis in in vitro experiments (Averbeck et al. 2007; Kakizaki et al. 2008). However, the doses cannot be directly compared, because under in vitro conditions epidermal cells lack the natural protective mechanisms, e.g., melanosomes and stratum corneum, and are therefore more sensitive to the UVR. The UV source used in the present work simulated solar irradiation, containing a broad spectrum of wavelengths from 310 to 400 nm (Kumlin et al. 1998). The main part (98%) of the physical dose is comprised of UVA; however, about 70% of the biologically effective (erythema-inducing) irradiation is expected to come from the UVB wavelength. UVA may either be ineffective in stimulating hyaluronan synthesis (Kakizaki et al. 2008) or even inhibit it when used at high doses (Calikoglu et al. 2006). Although in the present experiment we cannot differentiate between the effects of UVB and UVA on hyaluronan metabolism, our findings indicate that in a chronic setting, UVR resembling solar irradiation at intensities causing skin erythema (1 MED) causes a dramatic change in epidermal hyaluronan metabolism. In the present material, the increase in HAS- immunostaining intensities in the UVR exposed epidermis suggests that the increased hyaluronan content in UVR- treated epidermis is at least partly explained by increased HAS activity. All HAS isoforms showed increased immunostaining in UVR exposed skin. In line with the previous cell culture experiments (Averbeck et al. 2007), HAS2 showed highest increase due to UVR. However, possible differences in the sensitivity of the antibodies preclude direct conclusions concerning HAS2 as the major target. UVR is known to activate several signaling routes, but there are no comprehensive data showing which of the signals are involved in the UV-induced upregulation of hyaluronan synthesis and HAS expression (Averbeck et al. 2007; Kakizaki et al. 2008). The acute phase upregulation of HAS2 and HAS3 was inhibited by blocking IL-1β, suggesting that an inflammatory pathway is involved (Kakizaki et al. 2008). Chronic UVB has been reported to cause enhanced activation of pathways associated with EGFR and ErbB2, PI3K and AKT, JNK, NFkB, and Stat3 (Sano et al. 2005; Katiyar and Meeran. 2007; Wunderlich et al. 2008), all known to influence HAS2 or HAS3 expression (Pienimäki et al. 2001; Saavalainen et al. 2005; Madson and Hansen. 2007; Han et al. 2008). The strongly elevated level of CD44 is likely to contribute to the increased epidermal content of hyaluronan by binding and immobilizing hyaluronan on the plasma membranes of keratinocytes. The close correlation between CD44 and hyaluronan staining patterns observed in the present work and in previous publications (Pirinen et al. 1998; Hirvikoski et al. 1999; Karvinen et al. 2003; Kosunen et al. 2004) supports this conclusion. However, acute high- dose UVB and UVA irradiation was reported to cause depletion of CD44 (Calikoglu et al. 2006), and our own data in cultured keratinocytes support this finding (Rauhala et al. unpublished), indicating that the CD44 response to UVR is biphasic. Mutations of p53 are typically found in epidermal keratinocytes exposed to solar radiation very early, before the ...

Context 4

... above. After washes, the sections were counterstained with Mayer’s hematoxylin for 1 min, washed, dehydrated, and mounted in DPX. Treatment of primary antibodies with corresponding peptides (sc-34021 P for HAS1, sc-34067 P for HAS2, and sc-34204 P for HAS3; Santa Cruz Biotechnology) was used as control. The stainings were analyzed blind regarding the experimental group. General morphological features of the sections were analyzed and scored for epidermal hyperplasia (no hyperplasia, mild, moderate, strong hyperplasia). In addition, the sections were scored for the presence of tumors, dysplasia, and squamous cell carcinomas. The area of HA and CD44 stainings in the epidermis was estimated with a four-level scoring from 0 to 3. Score 0 was given when no staining or only minimal staining around 1–2 hair follicles was detected. Score 1 was given when less than 33% of the interfollicular area was stained, score 2 when 33–66% of the interfollicular area was stained, and score 3 when more than 66% of the interfollicular area was stained. The intensity of epidermal staining was estimated with a four-level scoring from 0 to 3. The homogeneity of the staining was recorded as well as the intensity of dermal staining and the staining of skin areas showing abnormal morphology. The statistical calculations were performed using the SPSS program for MacIntosh (version 11.0, SPSS, Chicago, IL). The Kruskal-Wallis and Mann-Whitney U-tests were used to compare the extent and intensity of the stainings between treatments. The amount of epidermal hyperplasia was correlated to the HA and CD44 parameters using Kendall’s test. Probability values less than 0.05 were considered significant. In the present work, mouse back skin was exposed to 1 MED of UVR three times a week for 10.5 months. Histological evaluation indicated that in most of the treated animals the epidermal tissue showed marked, approximately 3–4-fold thickening (Fig. 1, e and f) compared with control skin (Fig. 1, a and b, Table 1). One fifth of the UVR-exposed animals developed squamous cell carcinomas (SCC, Fig. 2, d–i, and Table 1), and one third showed dysplastic changes (Fig. 2, a–c). Only one of the control animals showed mild hyperplasia, whereas dysplasia or SCC was not detected in any of the control animals (Table 1). In normal, untreated skin, dermal connective tissue was intensely stained for hyaluronan, whereas most of the epidermis either was negative (Fig. 1a) or showed a weak positive signal around the orifices of hair follicles (Table 1), as previously described in mouse ear and tail (Tammi et al. 2005). Although CD44 immunostaining in control skin was generally weak, some CD44-positive cells were found throughout the whole interfollicular epidermis, in both the basal and suprabasal layers (Fig. 1b, Table 2). In UVR-treated animals, hyaluronan-positive cells cov- ered more than two thirds of the epidermal area in most of the specimens (Table 2), and the intensity of epidermal hyaluronan staining was significantly increased compared with untreated epidermis (Table 2, Fig. 1 and 2). CD44 immunostaining also showed significantly increased epidermal staining intensity and coverage in UVR-treated animals (Figs. 1 and 2, Table 2). Epidermal areas showing benign hyperplasia showed moderate or strong hyaluronan and CD44 stainings, even in the case of moderate hyperplasia (Fig. 1, c and d); however, the number of positive layers was increased when the degree of hyperplasia increased (Fig. 1, e and f), with all vital cell layers except granular cells being positive for hyaluronan and CD44. Epidermal hyperplasia showed a significant positive correlation with both intensity and coverage of hyaluronan staining (Kendall’s τ-b test, p <0.01). Similar correlation was also observed between epidermal hyperplasia and CD44 staining intensity and coverage (Kendall’s τ-b test, p <0.01 and 0.05, respectively). Epidermal areas containing dysplastic cells were generally moderately or intensely positive for hyaluronan and CD44; however, they often contained patches with reduced staining or even no staining at all (Fig. 2, b and c). Similarly, invasive squamous cell carcinomas showed generally moderate or strong hyaluronan and CD44 stainings (Fig. 2, e and f), but patches of lower staining intensity made the appearance inhomogeneous in some of the tumors (Fig. 2, h and i). Dermal connective tissue stained for hyaluronan in all samples. In semiquantitative scoring of dermal hyaluronan staining intensity, all samples in control group were graded as moderately stained, whereas in the UVR group 1 of 21 was graded as weakly stained, 2 were graded as strongly stained, and the remaining were graded as moderate (NS). However, in UVR-treated animals showing epidermal hyperplasia, there was a loss of subepidermal hyaluronan staining in 69% of the hyperplastic samples (Fig. 1e). We performed immunostainings with antibodies raised against hyaluronan synthase enzymes 1, 2, and 3 (Fig. 3). In the control skin that was not exposed to UVB (Fig. 3, a, e and i) just a few epidermal cells showed HAS- immunoreactivity above the level of diffuse, low-intensity background signal. The dermal cells stained weakly or with moderate intensity for HAS1, HAS2, and HAS3 (Fig. 3, a, e and i). The staining intensity in specimens exposed to the UVR was clearly higher with all HAS antibodies, in both epidermis and dermis (Fig. 3, b, c, f, g, j and k). Strongest induction of staining intensity was found with the HAS2 antibody (Fig. 3, f and g). The increased HAS immunostainings were seen both in hyperplastic areas (Fig. 3, c, g and k) and in the squamous cell carcinomas (Fig. 3, b, f and j). Fig. 3, d, h and l shows specimens stained with HAS- antibodies preincubated with peptides used for immunization. Most of the staining was lost, indicating the specificity of the signal. The retrieval at high temperature required for the exposure of the epitopes (Rilla et al, unpublished) tended to tear the fragile mouse skin sections and precluded HAS immunostainings of sufficient quality for comprehensive scoring of all specimens. The present study shows for the first time that chronic UV irradiation of skin leads to the accumulation of hyaluronan and its receptor CD44 in the epidermis and that this accumulation correlates with the development of epidermal hyperplasia. Although the effect of chronic UVR on GAG and hyaluronan metabolism in the dermal compartment has been studied previously (Schwartz 1988; Margelin et al. 1996; Koshiishi et al. 1999; Dai et al. 2007), none of the previous publications has reported changes in the epidermal compartment. The reports concerning the response of epidermal cells to acute UVR have been contradictory (Calikoglu et al. 2006; Averbeck et al. 2007; Kakizaki et al. 2008), probably because of differences in the dose and UV source. The single dose used in the present work (20 mJ/cm corresponding one minimum erythema dose) is comparable to the doses that have been reported to stimulate hyaluronan synthesis in in vitro experiments (Averbeck et al. 2007; Kakizaki et al. 2008). However, the doses cannot be directly compared, because under in vitro conditions epidermal cells lack the natural protective mechanisms, e.g., melanosomes and stratum corneum, and are therefore more sensitive to the UVR. The UV source used in the present work simulated solar irradiation, containing a broad spectrum of wavelengths from 310 to 400 nm (Kumlin et al. 1998). The main part (98%) of the physical dose is comprised of UVA; however, about 70% of the biologically effective (erythema-inducing) irradiation is expected to come from the UVB wavelength. UVA may either be ineffective in stimulating hyaluronan synthesis (Kakizaki et al. 2008) or even inhibit it when used at high doses (Calikoglu et al. 2006). Although in the present experiment we cannot differentiate between the effects of UVB and UVA on hyaluronan metabolism, our findings indicate that in a chronic setting, UVR resembling solar irradiation at intensities causing skin erythema (1 MED) causes a dramatic change in epidermal hyaluronan metabolism. In the present material, the increase in HAS- immunostaining intensities in the UVR exposed epidermis suggests that the increased hyaluronan content in UVR- treated epidermis is at least partly explained by increased HAS activity. All HAS isoforms showed increased immunostaining in UVR exposed skin. In line with the previous cell culture experiments (Averbeck et al. 2007), HAS2 showed highest increase due to UVR. However, possible differences in the sensitivity of the antibodies preclude direct conclusions concerning HAS2 as the major target. UVR is known to activate several signaling routes, but there are no comprehensive data showing which of the signals are involved in the UV-induced upregulation of hyaluronan synthesis and HAS expression (Averbeck et al. 2007; Kakizaki et al. 2008). The acute phase upregulation of HAS2 and HAS3 was inhibited by blocking IL-1β, suggesting that an inflammatory pathway is involved (Kakizaki et al. 2008). Chronic UVB has been reported to cause enhanced activation of pathways associated with EGFR and ErbB2, PI3K and AKT, JNK, NFkB, and Stat3 (Sano et al. 2005; Katiyar and Meeran. 2007; Wunderlich et al. 2008), all known to influence HAS2 or HAS3 expression (Pienimäki et al. 2001; Saavalainen et al. 2005; Madson and Hansen. 2007; Han et al. 2008). The strongly elevated level of CD44 is likely to contribute to the increased epidermal content of hyaluronan by binding and immobilizing hyaluronan on the plasma membranes of keratinocytes. The close correlation between CD44 and hyaluronan staining patterns observed in the present work and in previous publications (Pirinen et al. 1998; Hirvikoski et al. 1999; Karvinen et al. 2003; Kosunen et al. 2004) supports this conclusion. However, acute high- dose UVB and UVA irradiation was reported to cause depletion of CD44 ...

Context 5

... with Mayer’s hematoxylin for 1 min, washed, dehydrated, and mounted in DPX. Treatment of primary antibodies with corresponding peptides (sc-34021 P for HAS1, sc-34067 P for HAS2, and sc-34204 P for HAS3; Santa Cruz Biotechnology) was used as control. The stainings were analyzed blind regarding the experimental group. General morphological features of the sections were analyzed and scored for epidermal hyperplasia (no hyperplasia, mild, moderate, strong hyperplasia). In addition, the sections were scored for the presence of tumors, dysplasia, and squamous cell carcinomas. The area of HA and CD44 stainings in the epidermis was estimated with a four-level scoring from 0 to 3. Score 0 was given when no staining or only minimal staining around 1–2 hair follicles was detected. Score 1 was given when less than 33% of the interfollicular area was stained, score 2 when 33–66% of the interfollicular area was stained, and score 3 when more than 66% of the interfollicular area was stained. The intensity of epidermal staining was estimated with a four-level scoring from 0 to 3. The homogeneity of the staining was recorded as well as the intensity of dermal staining and the staining of skin areas showing abnormal morphology. The statistical calculations were performed using the SPSS program for MacIntosh (version 11.0, SPSS, Chicago, IL). The Kruskal-Wallis and Mann-Whitney U-tests were used to compare the extent and intensity of the stainings between treatments. The amount of epidermal hyperplasia was correlated to the HA and CD44 parameters using Kendall’s test. Probability values less than 0.05 were considered significant. In the present work, mouse back skin was exposed to 1 MED of UVR three times a week for 10.5 months. Histological evaluation indicated that in most of the treated animals the epidermal tissue showed marked, approximately 3–4-fold thickening (Fig. 1, e and f) compared with control skin (Fig. 1, a and b, Table 1). One fifth of the UVR-exposed animals developed squamous cell carcinomas (SCC, Fig. 2, d–i, and Table 1), and one third showed dysplastic changes (Fig. 2, a–c). Only one of the control animals showed mild hyperplasia, whereas dysplasia or SCC was not detected in any of the control animals (Table 1). In normal, untreated skin, dermal connective tissue was intensely stained for hyaluronan, whereas most of the epidermis either was negative (Fig. 1a) or showed a weak positive signal around the orifices of hair follicles (Table 1), as previously described in mouse ear and tail (Tammi et al. 2005). Although CD44 immunostaining in control skin was generally weak, some CD44-positive cells were found throughout the whole interfollicular epidermis, in both the basal and suprabasal layers (Fig. 1b, Table 2). In UVR-treated animals, hyaluronan-positive cells cov- ered more than two thirds of the epidermal area in most of the specimens (Table 2), and the intensity of epidermal hyaluronan staining was significantly increased compared with untreated epidermis (Table 2, Fig. 1 and 2). CD44 immunostaining also showed significantly increased epidermal staining intensity and coverage in UVR-treated animals (Figs. 1 and 2, Table 2). Epidermal areas showing benign hyperplasia showed moderate or strong hyaluronan and CD44 stainings, even in the case of moderate hyperplasia (Fig. 1, c and d); however, the number of positive layers was increased when the degree of hyperplasia increased (Fig. 1, e and f), with all vital cell layers except granular cells being positive for hyaluronan and CD44. Epidermal hyperplasia showed a significant positive correlation with both intensity and coverage of hyaluronan staining (Kendall’s τ-b test, p <0.01). Similar correlation was also observed between epidermal hyperplasia and CD44 staining intensity and coverage (Kendall’s τ-b test, p <0.01 and 0.05, respectively). Epidermal areas containing dysplastic cells were generally moderately or intensely positive for hyaluronan and CD44; however, they often contained patches with reduced staining or even no staining at all (Fig. 2, b and c). Similarly, invasive squamous cell carcinomas showed generally moderate or strong hyaluronan and CD44 stainings (Fig. 2, e and f), but patches of lower staining intensity made the appearance inhomogeneous in some of the tumors (Fig. 2, h and i). Dermal connective tissue stained for hyaluronan in all samples. In semiquantitative scoring of dermal hyaluronan staining intensity, all samples in control group were graded as moderately stained, whereas in the UVR group 1 of 21 was graded as weakly stained, 2 were graded as strongly stained, and the remaining were graded as moderate (NS). However, in UVR-treated animals showing epidermal hyperplasia, there was a loss of subepidermal hyaluronan staining in 69% of the hyperplastic samples (Fig. 1e). We performed immunostainings with antibodies raised against hyaluronan synthase enzymes 1, 2, and 3 (Fig. 3). In the control skin that was not exposed to UVB (Fig. 3, a, e and i) just a few epidermal cells showed HAS- immunoreactivity above the level of diffuse, low-intensity background signal. The dermal cells stained weakly or with moderate intensity for HAS1, HAS2, and HAS3 (Fig. 3, a, e and i). The staining intensity in specimens exposed to the UVR was clearly higher with all HAS antibodies, in both epidermis and dermis (Fig. 3, b, c, f, g, j and k). Strongest induction of staining intensity was found with the HAS2 antibody (Fig. 3, f and g). The increased HAS immunostainings were seen both in hyperplastic areas (Fig. 3, c, g and k) and in the squamous cell carcinomas (Fig. 3, b, f and j). Fig. 3, d, h and l shows specimens stained with HAS- antibodies preincubated with peptides used for immunization. Most of the staining was lost, indicating the specificity of the signal. The retrieval at high temperature required for the exposure of the epitopes (Rilla et al, unpublished) tended to tear the fragile mouse skin sections and precluded HAS immunostainings of sufficient quality for comprehensive scoring of all specimens. The present study shows for the first time that chronic UV irradiation of skin leads to the accumulation of hyaluronan and its receptor CD44 in the epidermis and that this accumulation correlates with the development of epidermal hyperplasia. Although the effect of chronic UVR on GAG and hyaluronan metabolism in the dermal compartment has been studied previously (Schwartz 1988; Margelin et al. 1996; Koshiishi et al. 1999; Dai et al. 2007), none of the previous publications has reported changes in the epidermal compartment. The reports concerning the response of epidermal cells to acute UVR have been contradictory (Calikoglu et al. 2006; Averbeck et al. 2007; Kakizaki et al. 2008), probably because of differences in the dose and UV source. The single dose used in the present work (20 mJ/cm corresponding one minimum erythema dose) is comparable to the doses that have been reported to stimulate hyaluronan synthesis in in vitro experiments (Averbeck et al. 2007; Kakizaki et al. 2008). However, the doses cannot be directly compared, because under in vitro conditions epidermal cells lack the natural protective mechanisms, e.g., melanosomes and stratum corneum, and are therefore more sensitive to the UVR. The UV source used in the present work simulated solar irradiation, containing a broad spectrum of wavelengths from 310 to 400 nm (Kumlin et al. 1998). The main part (98%) of the physical dose is comprised of UVA; however, about 70% of the biologically effective (erythema-inducing) irradiation is expected to come from the UVB wavelength. UVA may either be ineffective in stimulating hyaluronan synthesis (Kakizaki et al. 2008) or even inhibit it when used at high doses (Calikoglu et al. 2006). Although in the present experiment we cannot differentiate between the effects of UVB and UVA on hyaluronan metabolism, our findings indicate that in a chronic setting, UVR resembling solar irradiation at intensities causing skin erythema (1 MED) causes a dramatic change in epidermal hyaluronan metabolism. In the present material, the increase in HAS- immunostaining intensities in the UVR exposed epidermis suggests that the increased hyaluronan content in UVR- treated epidermis is at least partly explained by increased HAS activity. All HAS isoforms showed increased immunostaining in UVR exposed skin. In line with the previous cell culture experiments (Averbeck et al. 2007), HAS2 showed highest increase due to UVR. However, possible differences in the sensitivity of the antibodies preclude direct conclusions concerning HAS2 as the major target. UVR is known to activate several signaling routes, but there are no comprehensive data showing which of the signals are involved in the UV-induced upregulation of hyaluronan synthesis and HAS expression (Averbeck et al. 2007; Kakizaki et al. 2008). The acute phase upregulation of HAS2 and HAS3 was inhibited by blocking IL-1β, suggesting that an inflammatory pathway is involved (Kakizaki et al. 2008). Chronic UVB has been reported to cause enhanced activation of pathways associated with EGFR and ErbB2, PI3K and AKT, JNK, NFkB, and Stat3 (Sano et al. 2005; Katiyar and Meeran. 2007; Wunderlich et al. 2008), all known to influence HAS2 or HAS3 expression (Pienimäki et al. 2001; Saavalainen et al. 2005; Madson and Hansen. 2007; Han et al. 2008). The strongly elevated level of CD44 is likely to contribute to the increased epidermal content of hyaluronan by binding and immobilizing hyaluronan on the plasma membranes of keratinocytes. The close correlation between CD44 and hyaluronan staining patterns observed in the present work and in previous publications (Pirinen et al. 1998; Hirvikoski et al. 1999; Karvinen et al. 2003; Kosunen et al. 2004) supports this conclusion. However, acute high- dose UVB and UVA irradiation was reported to cause depletion of CD44 (Calikoglu et al. 2006), and our own data in cultured keratinocytes ...

Context 6

... analyzed and scored for epidermal hyperplasia (no hyperplasia, mild, moderate, strong hyperplasia). In addition, the sections were scored for the presence of tumors, dysplasia, and squamous cell carcinomas. The area of HA and CD44 stainings in the epidermis was estimated with a four-level scoring from 0 to 3. Score 0 was given when no staining or only minimal staining around 1–2 hair follicles was detected. Score 1 was given when less than 33% of the interfollicular area was stained, score 2 when 33–66% of the interfollicular area was stained, and score 3 when more than 66% of the interfollicular area was stained. The intensity of epidermal staining was estimated with a four-level scoring from 0 to 3. The homogeneity of the staining was recorded as well as the intensity of dermal staining and the staining of skin areas showing abnormal morphology. The statistical calculations were performed using the SPSS program for MacIntosh (version 11.0, SPSS, Chicago, IL). The Kruskal-Wallis and Mann-Whitney U-tests were used to compare the extent and intensity of the stainings between treatments. The amount of epidermal hyperplasia was correlated to the HA and CD44 parameters using Kendall’s test. Probability values less than 0.05 were considered significant. In the present work, mouse back skin was exposed to 1 MED of UVR three times a week for 10.5 months. Histological evaluation indicated that in most of the treated animals the epidermal tissue showed marked, approximately 3–4-fold thickening (Fig. 1, e and f) compared with control skin (Fig. 1, a and b, Table 1). One fifth of the UVR-exposed animals developed squamous cell carcinomas (SCC, Fig. 2, d–i, and Table 1), and one third showed dysplastic changes (Fig. 2, a–c). Only one of the control animals showed mild hyperplasia, whereas dysplasia or SCC was not detected in any of the control animals (Table 1). In normal, untreated skin, dermal connective tissue was intensely stained for hyaluronan, whereas most of the epidermis either was negative (Fig. 1a) or showed a weak positive signal around the orifices of hair follicles (Table 1), as previously described in mouse ear and tail (Tammi et al. 2005). Although CD44 immunostaining in control skin was generally weak, some CD44-positive cells were found throughout the whole interfollicular epidermis, in both the basal and suprabasal layers (Fig. 1b, Table 2). In UVR-treated animals, hyaluronan-positive cells cov- ered more than two thirds of the epidermal area in most of the specimens (Table 2), and the intensity of epidermal hyaluronan staining was significantly increased compared with untreated epidermis (Table 2, Fig. 1 and 2). CD44 immunostaining also showed significantly increased epidermal staining intensity and coverage in UVR-treated animals (Figs. 1 and 2, Table 2). Epidermal areas showing benign hyperplasia showed moderate or strong hyaluronan and CD44 stainings, even in the case of moderate hyperplasia (Fig. 1, c and d); however, the number of positive layers was increased when the degree of hyperplasia increased (Fig. 1, e and f), with all vital cell layers except granular cells being positive for hyaluronan and CD44. Epidermal hyperplasia showed a significant positive correlation with both intensity and coverage of hyaluronan staining (Kendall’s τ-b test, p <0.01). Similar correlation was also observed between epidermal hyperplasia and CD44 staining intensity and coverage (Kendall’s τ-b test, p <0.01 and 0.05, respectively). Epidermal areas containing dysplastic cells were generally moderately or intensely positive for hyaluronan and CD44; however, they often contained patches with reduced staining or even no staining at all (Fig. 2, b and c). Similarly, invasive squamous cell carcinomas showed generally moderate or strong hyaluronan and CD44 stainings (Fig. 2, e and f), but patches of lower staining intensity made the appearance inhomogeneous in some of the tumors (Fig. 2, h and i). Dermal connective tissue stained for hyaluronan in all samples. In semiquantitative scoring of dermal hyaluronan staining intensity, all samples in control group were graded as moderately stained, whereas in the UVR group 1 of 21 was graded as weakly stained, 2 were graded as strongly stained, and the remaining were graded as moderate (NS). However, in UVR-treated animals showing epidermal hyperplasia, there was a loss of subepidermal hyaluronan staining in 69% of the hyperplastic samples (Fig. 1e). We performed immunostainings with antibodies raised against hyaluronan synthase enzymes 1, 2, and 3 (Fig. 3). In the control skin that was not exposed to UVB (Fig. 3, a, e and i) just a few epidermal cells showed HAS- immunoreactivity above the level of diffuse, low-intensity background signal. The dermal cells stained weakly or with moderate intensity for HAS1, HAS2, and HAS3 (Fig. 3, a, e and i). The staining intensity in specimens exposed to the UVR was clearly higher with all HAS antibodies, in both epidermis and dermis (Fig. 3, b, c, f, g, j and k). Strongest induction of staining intensity was found with the HAS2 antibody (Fig. 3, f and g). The increased HAS immunostainings were seen both in hyperplastic areas (Fig. 3, c, g and k) and in the squamous cell carcinomas (Fig. 3, b, f and j). Fig. 3, d, h and l shows specimens stained with HAS- antibodies preincubated with peptides used for immunization. Most of the staining was lost, indicating the specificity of the signal. The retrieval at high temperature required for the exposure of the epitopes (Rilla et al, unpublished) tended to tear the fragile mouse skin sections and precluded HAS immunostainings of sufficient quality for comprehensive scoring of all specimens. The present study shows for the first time that chronic UV irradiation of skin leads to the accumulation of hyaluronan and its receptor CD44 in the epidermis and that this accumulation correlates with the development of epidermal hyperplasia. Although the effect of chronic UVR on GAG and hyaluronan metabolism in the dermal compartment has been studied previously (Schwartz 1988; Margelin et al. 1996; Koshiishi et al. 1999; Dai et al. 2007), none of the previous publications has reported changes in the epidermal compartment. The reports concerning the response of epidermal cells to acute UVR have been contradictory (Calikoglu et al. 2006; Averbeck et al. 2007; Kakizaki et al. 2008), probably because of differences in the dose and UV source. The single dose used in the present work (20 mJ/cm corresponding one minimum erythema dose) is comparable to the doses that have been reported to stimulate hyaluronan synthesis in in vitro experiments (Averbeck et al. 2007; Kakizaki et al. 2008). However, the doses cannot be directly compared, because under in vitro conditions epidermal cells lack the natural protective mechanisms, e.g., melanosomes and stratum corneum, and are therefore more sensitive to the UVR. The UV source used in the present work simulated solar irradiation, containing a broad spectrum of wavelengths from 310 to 400 nm (Kumlin et al. 1998). The main part (98%) of the physical dose is comprised of UVA; however, about 70% of the biologically effective (erythema-inducing) irradiation is expected to come from the UVB wavelength. UVA may either be ineffective in stimulating hyaluronan synthesis (Kakizaki et al. 2008) or even inhibit it when used at high doses (Calikoglu et al. 2006). Although in the present experiment we cannot differentiate between the effects of UVB and UVA on hyaluronan metabolism, our findings indicate that in a chronic setting, UVR resembling solar irradiation at intensities causing skin erythema (1 MED) causes a dramatic change in epidermal hyaluronan metabolism. In the present material, the increase in HAS- immunostaining intensities in the UVR exposed epidermis suggests that the increased hyaluronan content in UVR- treated epidermis is at least partly explained by increased HAS activity. All HAS isoforms showed increased immunostaining in UVR exposed skin. In line with the previous cell culture experiments (Averbeck et al. 2007), HAS2 showed highest increase due to UVR. However, possible differences in the sensitivity of the antibodies preclude direct conclusions concerning HAS2 as the major target. UVR is known to activate several signaling routes, but there are no comprehensive data showing which of the signals are involved in the UV-induced upregulation of hyaluronan synthesis and HAS expression (Averbeck et al. 2007; Kakizaki et al. 2008). The acute phase upregulation of HAS2 and HAS3 was inhibited by blocking IL-1β, suggesting that an inflammatory pathway is involved (Kakizaki et al. 2008). Chronic UVB has been reported to cause enhanced activation of pathways associated with EGFR and ErbB2, PI3K and AKT, JNK, NFkB, and Stat3 (Sano et al. 2005; Katiyar and Meeran. 2007; Wunderlich et al. 2008), all known to influence HAS2 or HAS3 expression (Pienimäki et al. 2001; Saavalainen et al. 2005; Madson and Hansen. 2007; Han et al. 2008). The strongly elevated level of CD44 is likely to contribute to the increased epidermal content of hyaluronan by binding and immobilizing hyaluronan on the plasma membranes of keratinocytes. The close correlation between CD44 and hyaluronan staining patterns observed in the present work and in previous publications (Pirinen et al. 1998; Hirvikoski et al. 1999; Karvinen et al. 2003; Kosunen et al. 2004) supports this conclusion. However, acute high- dose UVB and UVA irradiation was reported to cause depletion of CD44 (Calikoglu et al. 2006), and our own data in cultured keratinocytes support this finding (Rauhala et al. unpublished), indicating that the CD44 response to UVR is biphasic. Mutations of p53 are typically found in epidermal keratinocytes exposed to solar radiation very early, before the actual tumor (de Gruijl and Rebel. 2008; Klein et al. 2010), and cause upregulation of CD44 expression (Godar et al. 2008), suggesting a plausible ...

Context 7

... addition, the sections were scored for the presence of tumors, dysplasia, and squamous cell carcinomas. The area of HA and CD44 stainings in the epidermis was estimated with a four-level scoring from 0 to 3. Score 0 was given when no staining or only minimal staining around 1–2 hair follicles was detected. Score 1 was given when less than 33% of the interfollicular area was stained, score 2 when 33–66% of the interfollicular area was stained, and score 3 when more than 66% of the interfollicular area was stained. The intensity of epidermal staining was estimated with a four-level scoring from 0 to 3. The homogeneity of the staining was recorded as well as the intensity of dermal staining and the staining of skin areas showing abnormal morphology. The statistical calculations were performed using the SPSS program for MacIntosh (version 11.0, SPSS, Chicago, IL). The Kruskal-Wallis and Mann-Whitney U-tests were used to compare the extent and intensity of the stainings between treatments. The amount of epidermal hyperplasia was correlated to the HA and CD44 parameters using Kendall’s test. Probability values less than 0.05 were considered significant. In the present work, mouse back skin was exposed to 1 MED of UVR three times a week for 10.5 months. Histological evaluation indicated that in most of the treated animals the epidermal tissue showed marked, approximately 3–4-fold thickening (Fig. 1, e and f) compared with control skin (Fig. 1, a and b, Table 1). One fifth of the UVR-exposed animals developed squamous cell carcinomas (SCC, Fig. 2, d–i, and Table 1), and one third showed dysplastic changes (Fig. 2, a–c). Only one of the control animals showed mild hyperplasia, whereas dysplasia or SCC was not detected in any of the control animals (Table 1). In normal, untreated skin, dermal connective tissue was intensely stained for hyaluronan, whereas most of the epidermis either was negative (Fig. 1a) or showed a weak positive signal around the orifices of hair follicles (Table 1), as previously described in mouse ear and tail (Tammi et al. 2005). Although CD44 immunostaining in control skin was generally weak, some CD44-positive cells were found throughout the whole interfollicular epidermis, in both the basal and suprabasal layers (Fig. 1b, Table 2). In UVR-treated animals, hyaluronan-positive cells cov- ered more than two thirds of the epidermal area in most of the specimens (Table 2), and the intensity of epidermal hyaluronan staining was significantly increased compared with untreated epidermis (Table 2, Fig. 1 and 2). CD44 immunostaining also showed significantly increased epidermal staining intensity and coverage in UVR-treated animals (Figs. 1 and 2, Table 2). Epidermal areas showing benign hyperplasia showed moderate or strong hyaluronan and CD44 stainings, even in the case of moderate hyperplasia (Fig. 1, c and d); however, the number of positive layers was increased when the degree of hyperplasia increased (Fig. 1, e and f), with all vital cell layers except granular cells being positive for hyaluronan and CD44. Epidermal hyperplasia showed a significant positive correlation with both intensity and coverage of hyaluronan staining (Kendall’s τ-b test, p <0.01). Similar correlation was also observed between epidermal hyperplasia and CD44 staining intensity and coverage (Kendall’s τ-b test, p <0.01 and 0.05, respectively). Epidermal areas containing dysplastic cells were generally moderately or intensely positive for hyaluronan and CD44; however, they often contained patches with reduced staining or even no staining at all (Fig. 2, b and c). Similarly, invasive squamous cell carcinomas showed generally moderate or strong hyaluronan and CD44 stainings (Fig. 2, e and f), but patches of lower staining intensity made the appearance inhomogeneous in some of the tumors (Fig. 2, h and i). Dermal connective tissue stained for hyaluronan in all samples. In semiquantitative scoring of dermal hyaluronan staining intensity, all samples in control group were graded as moderately stained, whereas in the UVR group 1 of 21 was graded as weakly stained, 2 were graded as strongly stained, and the remaining were graded as moderate (NS). However, in UVR-treated animals showing epidermal hyperplasia, there was a loss of subepidermal hyaluronan staining in 69% of the hyperplastic samples (Fig. 1e). We performed immunostainings with antibodies raised against hyaluronan synthase enzymes 1, 2, and 3 (Fig. 3). In the control skin that was not exposed to UVB (Fig. 3, a, e and i) just a few epidermal cells showed HAS- immunoreactivity above the level of diffuse, low-intensity background signal. The dermal cells stained weakly or with moderate intensity for HAS1, HAS2, and HAS3 (Fig. 3, a, e and i). The staining intensity in specimens exposed to the UVR was clearly higher with all HAS antibodies, in both epidermis and dermis (Fig. 3, b, c, f, g, j and k). Strongest induction of staining intensity was found with the HAS2 antibody (Fig. 3, f and g). The increased HAS immunostainings were seen both in hyperplastic areas (Fig. 3, c, g and k) and in the squamous cell carcinomas (Fig. 3, b, f and j). Fig. 3, d, h and l shows specimens stained with HAS- antibodies preincubated with peptides used for immunization. Most of the staining was lost, indicating the specificity of the signal. The retrieval at high temperature required for the exposure of the epitopes (Rilla et al, unpublished) tended to tear the fragile mouse skin sections and precluded HAS immunostainings of sufficient quality for comprehensive scoring of all specimens. The present study shows for the first time that chronic UV irradiation of skin leads to the accumulation of hyaluronan and its receptor CD44 in the epidermis and that this accumulation correlates with the development of epidermal hyperplasia. Although the effect of chronic UVR on GAG and hyaluronan metabolism in the dermal compartment has been studied previously (Schwartz 1988; Margelin et al. 1996; Koshiishi et al. 1999; Dai et al. 2007), none of the previous publications has reported changes in the epidermal compartment. The reports concerning the response of epidermal cells to acute UVR have been contradictory (Calikoglu et al. 2006; Averbeck et al. 2007; Kakizaki et al. 2008), probably because of differences in the dose and UV source. The single dose used in the present work (20 mJ/cm corresponding one minimum erythema dose) is comparable to the doses that have been reported to stimulate hyaluronan synthesis in in vitro experiments (Averbeck et al. 2007; Kakizaki et al. 2008). However, the doses cannot be directly compared, because under in vitro conditions epidermal cells lack the natural protective mechanisms, e.g., melanosomes and stratum corneum, and are therefore more sensitive to the UVR. The UV source used in the present work simulated solar irradiation, containing a broad spectrum of wavelengths from 310 to 400 nm (Kumlin et al. 1998). The main part (98%) of the physical dose is comprised of UVA; however, about 70% of the biologically effective (erythema-inducing) irradiation is expected to come from the UVB wavelength. UVA may either be ineffective in stimulating hyaluronan synthesis (Kakizaki et al. 2008) or even inhibit it when used at high doses (Calikoglu et al. 2006). Although in the present experiment we cannot differentiate between the effects of UVB and UVA on hyaluronan metabolism, our findings indicate that in a chronic setting, UVR resembling solar irradiation at intensities causing skin erythema (1 MED) causes a dramatic change in epidermal hyaluronan metabolism. In the present material, the increase in HAS- immunostaining intensities in the UVR exposed epidermis suggests that the increased hyaluronan content in UVR- treated epidermis is at least partly explained by increased HAS activity. All HAS isoforms showed increased immunostaining in UVR exposed skin. In line with the previous cell culture experiments (Averbeck et al. 2007), HAS2 showed highest increase due to UVR. However, possible differences in the sensitivity of the antibodies preclude direct conclusions concerning HAS2 as the major target. UVR is known to activate several signaling routes, but there are no comprehensive data showing which of the signals are involved in the UV-induced upregulation of hyaluronan synthesis and HAS expression (Averbeck et al. 2007; Kakizaki et al. 2008). The acute phase upregulation of HAS2 and HAS3 was inhibited by blocking IL-1β, suggesting that an inflammatory pathway is involved (Kakizaki et al. 2008). Chronic UVB has been reported to cause enhanced activation of pathways associated with EGFR and ErbB2, PI3K and AKT, JNK, NFkB, and Stat3 (Sano et al. 2005; Katiyar and Meeran. 2007; Wunderlich et al. 2008), all known to influence HAS2 or HAS3 expression (Pienimäki et al. 2001; Saavalainen et al. 2005; Madson and Hansen. 2007; Han et al. 2008). The strongly elevated level of CD44 is likely to contribute to the increased epidermal content of hyaluronan by binding and immobilizing hyaluronan on the plasma membranes of keratinocytes. The close correlation between CD44 and hyaluronan staining patterns observed in the present work and in previous publications (Pirinen et al. 1998; Hirvikoski et al. 1999; Karvinen et al. 2003; Kosunen et al. 2004) supports this conclusion. However, acute high- dose UVB and UVA irradiation was reported to cause depletion of CD44 (Calikoglu et al. 2006), and our own data in cultured keratinocytes support this finding (Rauhala et al. unpublished), indicating that the CD44 response to UVR is biphasic. Mutations of p53 are typically found in epidermal keratinocytes exposed to solar radiation very early, before the actual tumor (de Gruijl and Rebel. 2008; Klein et al. 2010), and cause upregulation of CD44 expression (Godar et al. 2008), suggesting a plausible mechanism for the increased CD44 levels in our model. The p53-induced effect of CD44 on cell growth and ...

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... was estimated with a four-level scoring from 0 to 3. Score 0 was given when no staining or only minimal staining around 1–2 hair follicles was detected. Score 1 was given when less than 33% of the interfollicular area was stained, score 2 when 33–66% of the interfollicular area was stained, and score 3 when more than 66% of the interfollicular area was stained. The intensity of epidermal staining was estimated with a four-level scoring from 0 to 3. The homogeneity of the staining was recorded as well as the intensity of dermal staining and the staining of skin areas showing abnormal morphology. The statistical calculations were performed using the SPSS program for MacIntosh (version 11.0, SPSS, Chicago, IL). The Kruskal-Wallis and Mann-Whitney U-tests were used to compare the extent and intensity of the stainings between treatments. The amount of epidermal hyperplasia was correlated to the HA and CD44 parameters using Kendall’s test. Probability values less than 0.05 were considered significant. In the present work, mouse back skin was exposed to 1 MED of UVR three times a week for 10.5 months. Histological evaluation indicated that in most of the treated animals the epidermal tissue showed marked, approximately 3–4-fold thickening (Fig. 1, e and f) compared with control skin (Fig. 1, a and b, Table 1). One fifth of the UVR-exposed animals developed squamous cell carcinomas (SCC, Fig. 2, d–i, and Table 1), and one third showed dysplastic changes (Fig. 2, a–c). Only one of the control animals showed mild hyperplasia, whereas dysplasia or SCC was not detected in any of the control animals (Table 1). In normal, untreated skin, dermal connective tissue was intensely stained for hyaluronan, whereas most of the epidermis either was negative (Fig. 1a) or showed a weak positive signal around the orifices of hair follicles (Table 1), as previously described in mouse ear and tail (Tammi et al. 2005). Although CD44 immunostaining in control skin was generally weak, some CD44-positive cells were found throughout the whole interfollicular epidermis, in both the basal and suprabasal layers (Fig. 1b, Table 2). In UVR-treated animals, hyaluronan-positive cells cov- ered more than two thirds of the epidermal area in most of the specimens (Table 2), and the intensity of epidermal hyaluronan staining was significantly increased compared with untreated epidermis (Table 2, Fig. 1 and 2). CD44 immunostaining also showed significantly increased epidermal staining intensity and coverage in UVR-treated animals (Figs. 1 and 2, Table 2). Epidermal areas showing benign hyperplasia showed moderate or strong hyaluronan and CD44 stainings, even in the case of moderate hyperplasia (Fig. 1, c and d); however, the number of positive layers was increased when the degree of hyperplasia increased (Fig. 1, e and f), with all vital cell layers except granular cells being positive for hyaluronan and CD44. Epidermal hyperplasia showed a significant positive correlation with both intensity and coverage of hyaluronan staining (Kendall’s τ-b test, p <0.01). Similar correlation was also observed between epidermal hyperplasia and CD44 staining intensity and coverage (Kendall’s τ-b test, p <0.01 and 0.05, respectively). Epidermal areas containing dysplastic cells were generally moderately or intensely positive for hyaluronan and CD44; however, they often contained patches with reduced staining or even no staining at all (Fig. 2, b and c). Similarly, invasive squamous cell carcinomas showed generally moderate or strong hyaluronan and CD44 stainings (Fig. 2, e and f), but patches of lower staining intensity made the appearance inhomogeneous in some of the tumors (Fig. 2, h and i). Dermal connective tissue stained for hyaluronan in all samples. In semiquantitative scoring of dermal hyaluronan staining intensity, all samples in control group were graded as moderately stained, whereas in the UVR group 1 of 21 was graded as weakly stained, 2 were graded as strongly stained, and the remaining were graded as moderate (NS). However, in UVR-treated animals showing epidermal hyperplasia, there was a loss of subepidermal hyaluronan staining in 69% of the hyperplastic samples (Fig. 1e). We performed immunostainings with antibodies raised against hyaluronan synthase enzymes 1, 2, and 3 (Fig. 3). In the control skin that was not exposed to UVB (Fig. 3, a, e and i) just a few epidermal cells showed HAS- immunoreactivity above the level of diffuse, low-intensity background signal. The dermal cells stained weakly or with moderate intensity for HAS1, HAS2, and HAS3 (Fig. 3, a, e and i). The staining intensity in specimens exposed to the UVR was clearly higher with all HAS antibodies, in both epidermis and dermis (Fig. 3, b, c, f, g, j and k). Strongest induction of staining intensity was found with the HAS2 antibody (Fig. 3, f and g). The increased HAS immunostainings were seen both in hyperplastic areas (Fig. 3, c, g and k) and in the squamous cell carcinomas (Fig. 3, b, f and j). Fig. 3, d, h and l shows specimens stained with HAS- antibodies preincubated with peptides used for immunization. Most of the staining was lost, indicating the specificity of the signal. The retrieval at high temperature required for the exposure of the epitopes (Rilla et al, unpublished) tended to tear the fragile mouse skin sections and precluded HAS immunostainings of sufficient quality for comprehensive scoring of all specimens. The present study shows for the first time that chronic UV irradiation of skin leads to the accumulation of hyaluronan and its receptor CD44 in the epidermis and that this accumulation correlates with the development of epidermal hyperplasia. Although the effect of chronic UVR on GAG and hyaluronan metabolism in the dermal compartment has been studied previously (Schwartz 1988; Margelin et al. 1996; Koshiishi et al. 1999; Dai et al. 2007), none of the previous publications has reported changes in the epidermal compartment. The reports concerning the response of epidermal cells to acute UVR have been contradictory (Calikoglu et al. 2006; Averbeck et al. 2007; Kakizaki et al. 2008), probably because of differences in the dose and UV source. The single dose used in the present work (20 mJ/cm corresponding one minimum erythema dose) is comparable to the doses that have been reported to stimulate hyaluronan synthesis in in vitro experiments (Averbeck et al. 2007; Kakizaki et al. 2008). However, the doses cannot be directly compared, because under in vitro conditions epidermal cells lack the natural protective mechanisms, e.g., melanosomes and stratum corneum, and are therefore more sensitive to the UVR. The UV source used in the present work simulated solar irradiation, containing a broad spectrum of wavelengths from 310 to 400 nm (Kumlin et al. 1998). The main part (98%) of the physical dose is comprised of UVA; however, about 70% of the biologically effective (erythema-inducing) irradiation is expected to come from the UVB wavelength. UVA may either be ineffective in stimulating hyaluronan synthesis (Kakizaki et al. 2008) or even inhibit it when used at high doses (Calikoglu et al. 2006). Although in the present experiment we cannot differentiate between the effects of UVB and UVA on hyaluronan metabolism, our findings indicate that in a chronic setting, UVR resembling solar irradiation at intensities causing skin erythema (1 MED) causes a dramatic change in epidermal hyaluronan metabolism. In the present material, the increase in HAS- immunostaining intensities in the UVR exposed epidermis suggests that the increased hyaluronan content in UVR- treated epidermis is at least partly explained by increased HAS activity. All HAS isoforms showed increased immunostaining in UVR exposed skin. In line with the previous cell culture experiments (Averbeck et al. 2007), HAS2 showed highest increase due to UVR. However, possible differences in the sensitivity of the antibodies preclude direct conclusions concerning HAS2 as the major target. UVR is known to activate several signaling routes, but there are no comprehensive data showing which of the signals are involved in the UV-induced upregulation of hyaluronan synthesis and HAS expression (Averbeck et al. 2007; Kakizaki et al. 2008). The acute phase upregulation of HAS2 and HAS3 was inhibited by blocking IL-1β, suggesting that an inflammatory pathway is involved (Kakizaki et al. 2008). Chronic UVB has been reported to cause enhanced activation of pathways associated with EGFR and ErbB2, PI3K and AKT, JNK, NFkB, and Stat3 (Sano et al. 2005; Katiyar and Meeran. 2007; Wunderlich et al. 2008), all known to influence HAS2 or HAS3 expression (Pienimäki et al. 2001; Saavalainen et al. 2005; Madson and Hansen. 2007; Han et al. 2008). The strongly elevated level of CD44 is likely to contribute to the increased epidermal content of hyaluronan by binding and immobilizing hyaluronan on the plasma membranes of keratinocytes. The close correlation between CD44 and hyaluronan staining patterns observed in the present work and in previous publications (Pirinen et al. 1998; Hirvikoski et al. 1999; Karvinen et al. 2003; Kosunen et al. 2004) supports this conclusion. However, acute high- dose UVB and UVA irradiation was reported to cause depletion of CD44 (Calikoglu et al. 2006), and our own data in cultured keratinocytes support this finding (Rauhala et al. unpublished), indicating that the CD44 response to UVR is biphasic. Mutations of p53 are typically found in epidermal keratinocytes exposed to solar radiation very early, before the actual tumor (de Gruijl and Rebel. 2008; Klein et al. 2010), and cause upregulation of CD44 expression (Godar et al. 2008), suggesting a plausible mechanism for the increased CD44 levels in our model. The p53-induced effect of CD44 on cell growth and survival was mediated by EGFR (Godar et al. 2008). In our material the epidermal hyperplasia strongly correlated with the levels of epidermal CD44 and ...