Activation of the PI3K/Akt/mTOR and MAPK Signaling Pathways in Response to Acute Solar-Simulated Light Exposure of Human Skin

Abstract

The incidence of skin cancer is higher than all other cancers and continues to increase, with an average annual cost over $8 billion in the United States. As a result, identifying molecular pathway alterations that occur with UV exposure to strategize more effective preventive and therapeutic approaches is essential. To that end, we evaluated phosphorylation of proteins within the PI3K/Akt and MAPK pathways by immunohistochemistry in sun-protected skin after acute doses of physiologically relevant solar-simulated ultraviolet light (SSL) in 24 volunteers. Biopsies were performed at baseline, 5 minutes, 1, 5, and 24 hours after SSL irradiation. Within the PI3K/Akt pathway, we found activation of Akt (serine 473) to be significantly increased at 5 hours while mTOR (serine 2448) was strongly activated early and was sustained over 24 hours after SSL. Downstream, we observed a marked and sustained increase in phospho-S6 (serine 235/S236), whereas phospho-4E-BP1 (threonines 37/46) was increased only at 24 hours. Within the MAPK pathway, SSL-induced expression of phospho-p38 (threonine 180/tyrosine 182) peaked at 1 to 5 hours. ERK 1/2 was observed to be immediate and sustained after SSL irradiation. Phosphorylation of histone H3 (serine 10), a core structural protein of the nucleosome, peaked at 5 hours after SSL irradiation. The expression of both p53 and COX-2 was increased at 5 hours and was maximal at 24 hours after SSL irradiation. Apoptosis was significantly increased at 24 hours as expected and indicative of a sunburn-type response to SSL. Understanding the timing of key protein expression changes in response to SSL will aid in development of mechanistic-based approaches for the prevention and control of skin cancers.

Figure 1

Examples of immunohistochemically stained proteins in sun-protected skin after 2–3 MED of SSL-irradiation. Phospho-Akt (Serine 473), at baseline (a), 1 hour (b), 5 hours (c), and 24 hours (d). Phospho-mTOR (Serine 2448) expression at baseline (e), 1 hour (f), 5 hours (g), and 24 hours (h). Images are at a magnification of 400X and the scale bar represents 50 μm. Red nuclear and cytoplasmic staining is positive and blue is negative.

Figure 2

Examples of immunohistochemically stained proteins in sun-protected skin after 2–3 MED of SSL-irradiation. Phospho-S6 (Serines 235/236) expression at baseline (a), 1 hour (b), 5 hours (c), and 24 hours (d). Phospho-4E-BP1 (Threonines 37/46) expression at baseline (e), 1 hour (f), 5 hours (g), and 24 hours (h). Images are at a magnification of 400X and the scale bar represents 50 μm. Red nuclear and cytoplasmic staining is positive and blue is negative.

Figure 3

Examples of immunohistochemically stained proteins in sun-protected skin after 2–3 MED of SSL-irradiation. Phospho-p38 (Threonine 180/Tyrosine 182) expression at baseline (a), 1 hour (b), 5 hours (c), and 24 hours (d). Phospho-ERK 1/2 (Threonine 202/Tyrosine 204) expression at baseline (e), 1 hour (f), 5 hours (g), and 24 hours (h). Phospho-histone H3 (Serine 10) expression at baseline (i), 1 hour (j), 5 hours (k), and 24 hours (l). Images are at a magnification of 400X and the scale bar represents 50 μm. Red (phospho-ERK 1/2 and phospho-histone H3) or brown (phospho-p38) nuclear and cytoplasmic stains represent positive and blue stain represents negative staining.

Figure 4

Examples of immunohistochemically stained proteins in sun-protected skin after 2–3 MED of SSL-irradiation. COX-2 expression at baseline (a), 1 hour (b), 5 hours (c), and 24 hours (d). p53 expression at baseline (e), 1 hour (f), 5 hours (g), and 24 hours (h). Images are at a magnification of 400X and the scale bar represents 50 μm. Brown nuclear and cytoplasmic stains represent positive and blue stain represents negative staining.

Figure 5

Examples of proliferation and apoptosis in sun-protected skin after 2–3 MED of SSL-irradiation. PCNA expression at baseline (a), 1 hour (b), 5 hours (c), and 24 hours (d) post SSL. Cleaved caspase 3 at baseline (a) and 24 hours (b). Apoptotic cells on H&E stained slides at baseline (c) and at 24 hours with inset in the lower left at a magnification of 1000X (d). Images of are at a magnification of 400X the scale bar represents 50 μm. Brown nuclear stain is positive for PCNA (a–d). Red stain is positive and blue is negative for cleaved caspase 3 (e and f). Arrows point to apoptotic cells present at 24 hours post SSL.