Regulated proenkephalin expression in human skin and cultured skin cells

Abstract

Skin responds to environmental stressors via coordinated actions of the local neuroimmunoendocrine system. Although some of these responses involve opioid receptors, little is known about cutaneous proenkephalin expression, its environmental regulation, and alterations in pathology. The objective of this study was to assess regulated expression of proenkephalin in normal and pathological skin and in isolated melanocytes, keratinocytes, fibroblasts, and melanoma cells. The proenkephalin gene and protein were expressed in skin and cultured cells, with significant expression in fibroblasts and keratinocytes. Mass spectroscopy confirmed Leu- and Met-enkephalin in skin. UVR, Toll-like receptor (TLR)4, and TLR2 agonists stimulated proenkephalin gene expression in melanocytes and keratinocytes in a time- and dose-dependent manner. In situ Met/Leu-enkephalin peptides were expressed in differentiating keratinocytes of the epidermis in the outer root sheath of the hair follicle, in myoepithelial cells of the eccrine gland, and in the basement membrane/basal lamina separating epithelial and mesenchymal components. Met/Leu-enkephalin expression was altered in pathological skin, increasing in psoriasis and decreasing in melanocytic tumors. Not only does human skin express proenkephalin, but this expression is upregulated by stressful stimuli and can be altered by pathological conditions.

Figure 1. Expression of proenkephalin (PENK) in human skin

(a) RT-PCR detection of PENK mRNA. HaCaT keratinocytes (1), melanocytes (2), adult epidermal keratinocytes (3), dermal fibroblasts (4), and WM98 (5), WM164 (6), and WM1341 (7) melanomas; markers (M). (b) Western blot detection of PENK (left, arrow), and negative control incubated with nonimmune serum (right). The dividing line in the left panel separates the HaCaT sample from molecular markers, which, in the right panel, shows the normal skin cells from melanomas. (c) PENK immunoreactivity in HaCaT keratinocytes (left panel), neonatal epidermal melanocytes (middle panel), and negative control (HaCaT keratinocytes) (right panel). Bar=50 μm. (d, e) Time-of-flight liquid chromatography mass spectrometry identification of Leu-enkephalin (d) and Met-enkephalin (e) in skin (sample). Peptide standard (STD). Left: retention times; right: m/z of peptides with C₁₃ isotopes. (f, g) Relative expression of PENK mRNA in comparison with melanoma (=1) (f) and Leu-enkephalin (g) was measured using Taqman technology and ELISA, respectively. Data are means±SEM (n=4–8). FIB, dermal fibroblasts; KER, epidermal keratinocytes.

Figure 2. Localization of proenkephalin (PENK)-derived Met- and Leu-enkephalin peptides in normal human scalp skin

(a, b) PENK expression in suprabasal keratinocytes (SB), base membrane zone (arrow), and dermis (D, asterisk) and its absence in basal keratinocytes (B, asterisk). (c) Negative control for PENK (peptide block) in the skin, i.e., pre-absorption of primary antibody with 1:1 mix of Leu/Met-enkephalin (see Materials and Methods). (d–f) PENK peptides in the outer root sheath (ORS) (arrow, d, e) of anagen VI hair follicle (HF), basal lamina (arrow, e), fibroblasts of the dermal sheath (asterisk, d, arrow, f), and follicular dermal papilla (DP, e). (g) Negative control for PENK (peptide block) in HF. (h, i) PENK peptides in myoepithelial cells (arrows) of the eccrine glands. (a) Bar=60 μm; (b) bar=20 μm; (c) bar=60 μm; (d) bar=100 μm; (e, f) bar=50 μm; (g) bar=100 μm; (h, i) bar=20 μm.

Figure 3. Localization of proenkephalin (PENK) antigens in pathological human skin

Immunolocalization of PENK in granuloma annulare (a), lichenoid keratosis (b), dermatofibroma (c), melanocytic nevus (d), psoriasis (e), melanoma in situ (f), and nodular malignant melanoma (g). Negative control represents skin with psoriasis (see e) in which primary antibodies were omitted (h). Arrows indicate PENK staining in the suprabasal layer, arrowheads indicate negative PENK staining in the basal layer, double arrowheads show basal PENK staining; asterisks indicate weak stain in the intradermal nevocytes and melanoma cells. Skin sections were stained with anti-PENK antibody, followed by secondary antibody linked to fluorescein. Insets show high magnification of cells indicated by squares. Bar=50 μm.

Figure 4. Lipopolysaccharide (LPS) stimulates expression of proenkephalin (PENK) in normal human epidermal keratinocytes

LPS-induced time (at 1 μgml⁻¹) (a) and dose (at 1 hour) (b) responses of PENK mRNA expression were measured using Taqman technology. Data are means±SEM (n=3). **P<0.005 versus control. Flow cytometry detection of LPS- (1 μgml⁻¹) stimulated expression of intracellular PENK protein measured (c). The results are representative of three independent experiments. PAM3CSK4-induced time (at 1 μgml ⁻¹) (d) and dose (at 1 hour) (e) responses of PENK mRNA. Data are means±SEM (n=3). *P<0.05 versus control. TLR2, Toll-like receptor 2.

Figure 5. UVB stimulates expression of proenkephalin (PENK) mRNA in normal human epidermal keratinocytes and melanocytes

Keratinocytes: time (at 25 mJ cm⁻²) (a) and dose (at 1 hour) (b) responses of PENK mRNA expression were measured using Taqman technology. Melanocytes: time (at 100 mJ cm⁻²) (c) and dose (at 6 hours) (d) responses of PENK mRNA levels were measured using Sybr Green technology. Data are means±SEM (n=3). *P<0.05 versus control. (e) PENK-luc reporter plasmid (lower panel) containing PENK promoter sequence (−772 to +11 bp) and pLINTER cloning vector coding promoterless firefly luciferase (upper panel). (f) UVB stimulates the PENK promoter in AtT-20 cells. After transfection with pPENK-luc, cells were treated with UVB at 25, 50, 75, and 100mJ cm⁻². Results represent a fold change in luciferase activity in comparison with control.