Regulation of epidermal homeostasis through P2Y2 receptors

Abstract

1. Previous studies have indicated a role for extracellular ATP in the regulation of epidermal homeostasis. Here we have investigated the expression of P2Y2 receptors by human keratinocytes, the cells which comprise the epidermis. 2. Reverse transcriptase-polymerase chain reaction (RT - PCR) revealed expression of mRNA for the G-protein-coupled, P2Y2 receptor in primary cultured human keratinocytes. 3. In situ hybridization studies of skin sections revealed that P2Y2 receptor transcripts were expressed in the native tissue. These studies demonstrated a striking pattern of localization of P2Y2 receptor transcripts to the basal layer of the epidermis, the site of cell proliferation. 4. Increases in intracellular free Ca2+ concentration ([Ca2+]i) in keratinocytes stimulated with ATP or UTP demonstrated the presence of functional P2Y receptors. 5. In proliferation studies based on the incorporation of bromodeoxyuridine (BrdU), ATP, UTP and ATPgammaS were found to stimulate the proliferation of keratinocytes. 6. Using a real-time firefly luciferase and luciferin assay we have shown that under static conditions cultured human keratinocytes release ATP. 7. These findings indicate that P2Y2 receptors play a major role in epidermal homeostasis, and may provide novel targets for therapy of proliferative disorders of the epidermis, including psoriasis.

Figure 1

Expression of P2Y₂ receptor transcripts in primary cultured human keratinocytes and osteoclasts. (A) PCR amplification of a specific 362 bp fragment of the human P2Y₂ receptor using cDNA from primary cultured human keratinocytes. cDNA from a sorted human osteoclast population was included as a positive control. (B) Amplification of a specific 519 bp GAPDH product confirmed the integrity of the cDNA used. This result is typical of three analyses using keratinocytes derived from skin samples from three individual patients.

Figure 1

Expression of P2Y₂ receptor transcripts in primary cultured human keratinocytes and osteoclasts. (A) PCR amplification of a specific 362 bp fragment of the human P2Y₂ receptor using cDNA from primary cultured human keratinocytes. cDNA from a sorted human osteoclast population was included as a positive control. (B) Amplification of a specific 519 bp GAPDH product confirmed the integrity of the cDNA used. This result is typical of three analyses using keratinocytes derived from skin samples from three individual patients.

Figure 2

In situ localization of P2Y₂ receptor transcripts in sections of human skin using ³²P-dUTP-labelled probes. (A) Low power photomicrograph of hybridization with a sense probe (negative control) showing no specific granular localization. (B) Similar section hybridized with an anti-sense probe and exposed for 15 days. Note the localization over the basal (proliferative) layer of the epidermis and in the glandular structures deep in the dermis. (C) High magnification view of a skin section hybridized with a sense probe (negative control) and exposed for 5 days. Note the absence of signal. (D and E) High magnification detail of skin sections hybridized with anti-sense probe and exposed for 5 days and 15 days respectively. Note the intense specific localization of signal over the basal (proliferative) layer of the epidermis. (F) High magnification view showing specific hybridization of the anti-sense probe over sweat glands.

Figure 3

Increases in [Ca²⁺]_i induced by ATP and UTP in cultured human keratinocytes. Cultured keratinocytes were loaded with fura-2 by incubation with 5 μM fura-2 AM. Groups of 8–10 cells were excited with light at 340 and 380 nm and the emission at 510 nm recorded. The application of ATP or UTP led to increases in [Ca²⁺]_i as indicated by the change in the ratio of fluorescence at 340 and 380 nm.

Figure 4

Proliferation of keratinocytes in response to (A) increasing concentrations of ATP and (B) 100 nM ATP, UTP and ATPγS was assessed using a Biotrak cell proliferation ELISA system. All data are represented as mean±s.e.mean (n=16 wells). ^* denotes significant difference at P<0.01. This result is typical of four separate experiments.

Figure 4

Proliferation of keratinocytes in response to (A) increasing concentrations of ATP and (B) 100 nM ATP, UTP and ATPγS was assessed using a Biotrak cell proliferation ELISA system. All data are represented as mean±s.e.mean (n=16 wells). ^* denotes significant difference at P<0.01. This result is typical of four separate experiments.

Figure 5

ATP release from human keratinocytes. The chamber of the photon-counting luminometer was filled with firefly luciferase (10 μg ml⁻¹) and luciferin (10 μM) in HEPES buffer and the background signal recorded. Introduction of a coverslip of keratinocytes at confluency resulted in an increase in signal to approximately 500 c.p.s. The signal was calibrated by measuring the luciferase luminescence over a range of ATP concentrations as shown on the right hand axis. This result is typical of five coverslips although considerable variability was seen in the signals recorded from individual coverslips.