Chronic UVR causes increased immunostaining of CD44 and accumulation of hyaluronan in mouse epidermis

Abstract

Chronic intense UV radiation is the main cause of epidermal tumors. Because hyaluronan (HA), a large extracellular polysaccharide, is known to promote malignant growth, hyaluronan expression was studied in a model in which long-term UV radiation (UVR) induces epidermal tumors. Mouse back skin was exposed three times a week for 10.5 months to UVR corresponding to one minimal erythema dose, processed for histology, and stained for hyaluronan and the hyaluronan receptor CD44. This exposure protocol caused epidermal hyperplasia in most of the animals; tumors, mainly squamous cell carcinomas (SCCs), were found in ~20% of the animals. Specimens exposed to UVR showed increased hyaluronan and CD44 staining throughout the epidermal tissue. In hyperplastic areas, hyaluronan and CD44 stainings correlated positively with the degree of hyperplasia. Well-differentiated SCCs showed increased hyaluronan and CD44 staining intensities, whereas poorly differentiated tumors and dysplastic epidermis showed areas where HA and CD44 were locally reduced. The findings indicate that HA and CD44 increase in epidermal keratinocytes in the premalignant hyperplasia induced by UV irradiation and stay elevated in dysplasia and SCC, suggesting that the accumulation of hyaluronan and CD44 is an early marker for malignant transformation and may be a prerequisite for tumor formation.

Figure 1.

Hyaluronan and CD44 in skin following chronic UV-irradiation. Specimens from shaved mouse back were stained for hyaluronan (a, c, e) and CD44 (b, d, f). (a, b) Control mouse. (c–f) Mouse exposed to UVR for 10.5 months, three times per week. Dermal connective tissue is positive for hyaluronan in all specimens, but there is a loss of dermal hyaluronan staining below the basement membrane in samples showing epidermal hyperplasia (arrow in e). Epidermal tissue in control skin is negative for hyaluronan (a) and shows faint CD44-positive staining (b). Both mildly (c and d) and strongly (e and f) hyperplastic epidermal areas of UVR epidermis show strong bHABR and CD44-positive staining that covers most of the interfollicular epidermis and is localized in all living cell layers from basal up to the granular cell layer. Magnification bar 20 µm.

Figure 2.

Hyaluronan and CD44 in dysplastic skin and SCC induced by UVR. (a-c) Dysplastic skin. (d-i) SCC. Dermal connective tissue in all specimens contains cells with a strong positive staining. Hyaluronan and CD44 stainings form a net-like pattern around the epithelial cells. The intensities of the stainings are generally moderate or strong, but in dysplastic (a–c) and some of the SCC (g–i) the staining is inhomogeneous with local loss of hyaluronan and CD44 stainings (arrows in b, c, h, and i). Magnification bar 40 µm in c for a–c and 20 µm in i for d–i.

Figure 3.

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).