Ultraviolet Irradiation Induces the Accumulation of Chondroitin Sulfate, but Not Other Glycosaminoglycans, in Human Skin

Ultraviolet (UV) light alters cutaneous structure and function. Prior work has shown loss of dermal hyaluronan after UV-irradiation of human skin, yet UV exposure increases total glycosaminoglycan (GAG) content in mouse models. To more fully describe UV-induced alterations to cutaneous GAG content, we subjected human volunteers to intermediate-term (5 doses/week for 4 weeks) or single-dose UV exposure. Total dermal uronyl-containing GAGs increased substantially with each of these regimens. We found that UV exposure substantially increased dermal content of chondroitin sulfate (CS), but not hyaluronan, heparan sulfate, or dermatan sulfate. UV induced the accumulation of both the 4-sulfated (C4S) and 6-sulfated (C6S) isoforms of CS, but in distinct distributions. Next, we examined several CS proteoglycan core proteins and found a significant accumulation of dermal and endothelial serglycin, but not of decorin or versican, after UV exposure. To examine regulation in vitro, we found that UVB in combination with IL-1α, a cytokine upregulated by UV radiation, induced serglycin mRNA in cultured dermal fibroblasts, but did not induce the chondroitin sulfate synthases. Overall, our data indicate that intermediate-term and single-dose UVB exposure induces specific GAGs and proteoglycan core proteins in human skin in vivo. These molecules have important biologic functions and contribute to the cutaneous response to UV

Wavelength-specific effects of intermediate-term irradiation on GAGs, HA, and CS.

<p>Normal human volunteers were treated with sham, UVA, UVA1, PUVA, SSR, and UVB on different sections of their backs 5 times weekly for 4 weeks. ImagePro 3 analysis of treatment-specific effects were evaluated for (a) total GAG content with Hale stain, (b) HA, and (c) CS. Open bars represent epidermal stain intensity, solid bars represent dermal stain intensity, and hatched bars represent stain intensity in the basal layer of the epidermis. (***p<.001, **p<.01, *p<.05, n = 5 for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014830#pone-0014830-g004" target="_blank">Figure 4a</a>, n = 4 for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014830#pone-0014830-g004" target="_blank">Figures 4b-c</a>).</p

Localization and quantification of CS in human skin following intermediate-term UVB irradiation.

<p>Skin was (a) sham-irradiated or (b) UVB-irradiated 5 times weekly for 4 weeks. CS was labeled with the CS-56 anti-CS antibody and visualized using a biotin-peroxidase system. The images in this figure are of tissue from the same patient. Stain intensity in the dermis was quantified using ImagePro version 3 and graphed (c). Sham skin is represented by open bars and UVB-irradiated skin by solid bars. (size bars = 62.5 µm, *p<.05).</p

Effect of intermediate-term UVB exposure on dermal HA in vivo.

<p>Skin was (a) sham-irradiated or (b) UVB-irradiated 5 times weekly for 4 weeks. HA was labeled with biotinylated HABP and visualized using a peroxidase system. The images in this figure are of tissue from the same patient. Stain intensity in dermis, epidermis, and the basal layer of the epidermis was quantified using ImagePro version 3 and graphed (c). Sham skin is represented by open bars and UVB-irradiated skin by solid bars. (size bars = 62.5 µm, *p<.05).</p

Time course for changes in CS in human skin following single-dose UVB irradiation.

<p>Normal human volunteers were exposed to 2X MED of UVB. Biopsies were taken 1, 2, and 3 days after irradiation. CS was labeled with the CS-56 anti-CS antibody and visualized using a biotin-peroxidase system. Shown are stains of (a) control tissue and tissue taken (b) 24, (c) 48, and (d) 72 hours post-irradiation. The images in this figure are of tissue from the same patient. (size bars = 62.5 µm).</p

Effect of intermediate-term UVB irradiation on total dermal GAG content of human skin in vivo.

<p>Human volunteers were sham-irradiated or exposed to UVB 5 times weekly for 4 weeks. Biopsies were taken and GAGs were visualized using Hale's stain. Collagen stains red, cytoplasm stains yellow, and GAGs stain blue in (a) control skin and (b) UVB-irradiated skin. The images in this figure are of tissue from the same patient. The GAG stain intensity was quantified using ImagePro version 3 and graphed (c). Sham skin is represented by open bars and UVB-irradiated skin by solid bars. (size bars = 62.5 µm, ***p<.001 versus sham, n = 5).</p

Induction of serglycin in human skin following single-dose UVB irradiation and in normal human fibroblasts following stimulation with UVB +IL-1α.

<p>Normal human volunteers were exposed to 2X MED of UVB. Biopsies were taken 1, 2, and 3 days after irradiation. Following enzymatic digestion of CS, serglycin was labeled with the N13 anti-serglycin antibody and visualized using a biotin-peroxidase system. Shown are stains of (a) control tissue and tissue taken (b) 24, (c) 48, and (d) 72 hours post-irradiation (size bars = 62.5 µm). For each, an example of the endothelium of dermal venules is indicated with an arrow, and an example of a serglycin-positive hematopoietic cell is indicate with an arrow head. The images in this figure are of tissue from the same patient. Human skin fibroblasts were irradiated with UVB (30 mJ/cm²), or sham, ± IL-1α (1 ng/ml). RNA was extracted with trizol, and serglycin mRNA levels were evaluated using real-time PCR (e). (***p<.001, **p<.01).</p