Soluble adenylyl cyclase defines a nuclear cAMP microdomain in keratinocyte hyperproliferative skin diseases

Abstract

Cyclic adenosine monophosphate (cAMP) is a nearly ubiquitous signaling molecule important for numerous signaling pathways in human skin. We studied a novel class of mammalian adenylyl cyclase, the soluble adenylyl cyclase (sAC). We examined sAC localization in normal human skin and found it to be present in keratinocytes, melanocytes, mononuclear cells, eccrine ducts, and nerves. In normal skin, sAC keratinocyte staining was evenly distributed throughout the cell. However, in certain hyperproliferative disorders of the skin, including psoriasis, verruca vulgaris, and SCCIS on sun-damaged skin, sAC keratinocyte staining was predominantly nuclear. In contrast, in other hyperproliferative disorders, such as basal cell carcinoma, sAC staining was similar to normal human skin. Using a model of epithelial differentiation, we established that sAC migrates into the nucleus when differentiated cells are induced to reenter the cell cycle. Previous work had determined that nuclear sAC activates the cAMP-response-element-binding (CREB) transcription factor, and we found that in psoriasis lesions, nuclear sAC occurs concomitantly with activation of CREB. Hence, sAC may play a role in the pathogenesis of certain hyperproliferative skin disorders via modulation of gene expression.

Figure 1. Immunostaining of sAC in normal skin

(a) Hematoxylin (blue) and eosin (red) staining of normal human skin. (b) Normal human skin immunostained with R21 (red) and hematoxylin (blue). (c) Normal human skin immunostained with R21+ blocking peptide (red) and hematoxylin (blue). (d) Normal epidermis immunostained with R21 (red), Melan-A (brown), and hematoxylin (blue). (e) Normal human skin eccrine duct immunostained with R21 (red) and hematoxylin (blue). (f) Normal human skin cutaneous nerve immunostained with R21 (red), PGP9.5 (brown), and hematoxylin (blue). Bars = 50 μm (a–c) and 10μm (d–f).

Figure 2. Immunostaining of sAC in viral infections of the epidermis

(a) Hematoxylin (blue) and eosin (red) staining of a verruca vulgaris skin lesion. (b) Verruca vulgaris skin lesion immunostained with R21 (red) and hematoxylin (blue). (c) Verruca vulgaris skin lesion immunostained with R21 (red) and hematoxylin (blue). The black arrow demonstrates a nucleus negative for sAC. The gray arrow demonstrates a nucleus postive for sAC. (d) Hematoxylin (blue) and eosin (red) staining of a molluscum contagiosum skin lesion. (e) Molluscum contagiosum skin lesion immunostained with R21 (red) and hematoxylin (blue). (f) Molluscum contagiosum skin lesion immunostained with R21 (red) and hematoxylin (blue). Bars = 100 μm (a, b, d, e) and 10 mm (c, f).

Figure 3. sAC is present in the nucleus when epithelial cells are proliferating and not when epithelial cells are differentiating

(a) Differentiated MDCK cells stained with DAPI (left), anti-N-term sAC antibody (center), and overlay of DAPI and anti-N-term (right). (b) Proliferating undifferentiated MDCK cells stained with DAPI (left), anti-N-term sAC antibody (center), and overlay of DAPI and anti-N-term (right). Bar = 10 μm.

Figure 4. Immunostaining of sAC in AK and SCC

(a) Hematoxylin (red) and eosin (blue) staining of AK. (b) AK immunostained with R21 (red) and hematoxylin (blue); inset, magnified view of the area in panel b. (c) Hematoxylin (red) and eosin (blue) staining of SCCIS. (d) SCCIS immunostained with R21 (red) and hematoxylin (blue); inset, magnified view of the area in panel d. (e) Hematoxylin (red) and eosin (blue) staining of SCC. (f) SCC immunostained with R21 (red) and hematoxylin (blue). (g) Hematoxylin (red) and eosin (blue) staining of SCC. (h) SCC immunostained with R21 (red) and hematoxylin (blue). Bars = 50 μm.

Figure 5. Nuclear sAC is enhanced in psoriatic skin and is associated with phosphorylated CREB

(a) Hematoxylin (red) and eosin (blue) staining of psoriatic lesional skin. (b) Psoriatic lesional skin immunostained with R21 (red) and hematoxylin (blue). The inset is a magnified view of a portion of the epidermis demonstrating that some nuclei are positive for sAC (white arrow) and some are negative for sAC (black arrow). (c) Psoriatic lesional skin immunostained with R21 (red) and an antibody against phosphorylated CREB (light blue). Panel c and inset: Nuclei positive for both sAC and phosphorylated CREB appear dark blue to purple (gray arrows). White arrows indicate nuclei positive for phosphorylated CREB only (light blue). Black arrows indicate nuclei positive for sAC only (red). Bars = 50 μm.