Nicotinic Acid Receptor Abnormalities in Human Skin Cancer: Implications for a Role in Epidermal Differentiation

Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through G(i)-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells.Nicotinic acid increases epidermal differentiation in photodamaged human skin as judged by the terminal differentiation markers caspase 14 and filaggrin. Both GPR109A and GPR109B genes are transcribed in human skin and in epidermal keratinocytes, but expression in dermal fibroblasts is below limits of detection. Receptor transcripts are greatly over-expressed in squamous cell cancers. Receptor protein in normal skin is prominent from the basal through granular layers of the epidermis, with cellular localization more dispersive in the basal layer but predominantly localized at the plasma membrane in more differentiated epidermal layers. In normal human primary and immortalized keratinocytes, nicotinic acid receptors show plasma membrane localization and functional G(i)-mediated signaling. In contrast, in a squamous cell carcinoma derived cell line, receptor protein shows a more diffuse cellular localization and the receptors are nearly non-functional.The results of these studies justify future genetic and pharmacological intervention studies to define possible specific role(s) of nicotinic acid receptors in human skin homeostasis

Growth Factor-Mediated Telomerase Activity in Ovarian Cancer Cells

Ovarian cancer is the leading cause of gynecological cancer death in the United States. Even though no single genetic alteration can be attributed to all ovarian cancers, 90% of ovarian tumors express telomerase, a ribonucleoprotein that elongates telomeric (TTAGGG)n repeats de novo. In normal somatic cells, telomerase is absent. In cancer cells, the re-expression of telomerase allows senescence to be bypassed contributing to cellular immortalization, a key step for cellular transformation, making telomerase a potentially important target for therapeutic intervention. Ovarian cancer cells secrete vascular endothelial growth factor (VEGF) and lysophosphatidic acid (LPA) that feedback through their receptors present on ovarian cancer cells to promote cell growth. Since telomerase can be regulated by growth factors, I examined VEGF regulation of telomerase activity and the possible contribution of LPA as an upstream regulator of VEGF-mediated telomerase activity in ovarian cancer. My data reveal that both VEGF and LPA upregulate telomerase activity by ERK 1/2-dependent transcriptional activation within the -976 to the -378 bp hTERT promoter regions where Sp1 is one of the major mediators of VEGF- and LPA-induced transactivation of hTERT. It also identifies telomerase as a novel molecular target of LPA as well as a target of VEGF in non-endothelial cells. In addition I found that, vitamin E, a dietary supplement able to degrade and suppress LPA activity, consistently abrogrates LPA-mediated telomerase activity through transcriptional inhibition of the hTERT -976 to -578 bp promoter regions. Lastly, since epidermal growth factor (EGF) promotes ovarian surface epithelial (OSE) cell growth and EGF receptors are frequently constitutively activated in ovarian cancers, the potential contribution of EGF in the regulation of telomerase activity was also examined. While none of the ovarian cancer cell lines examined produced large amounts of EGF, EGF stimulation of telomerase activity was mediated by Sp1 and c-Myc transcription factors within the hTERT core promoter in an ERK 1/2 /Pyk2-dependent manner. In conclusion, my research shows differential regulation of telomerase activity by growth factor and/or anti-oxidant nutraceuticals. In the future, these factors may be exploited as adjuvant therapy for improved chemotherapeutic benefit to decrease the mortality associated with ovarian cancer

Nicotinic acid promotes epidermal differentiation in photodamaged human skin.

<p>Tissue arrays of skin biopsy samples from a clinical study of the effects of myristyl nicotinate (MN) in human subjects with photodamaged skin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020487#pone.0020487-Jacobson1" target="_blank">[5]</a> were stained for the terminal differentiation markers caspase 14 and filaggrin. <b>Panel A:</b> An example of a biopsy sample at baseline and 12 weeks of MN treatment stained with H&E, and immunostaining for caspase 14 or filaggrin. <b>Panel B:</b> Quantification of staining for the placebo (n = 27) and MN treated (n = 31) groups for caspase 14 and filaggrin. Students t-test was used to compare placebo and MN treated groups and p values are shown.</p

Localization of GPR109A/B protein expression in human skin.

<p>Immunohistochemistry (IHC) analyses were performed on paraffin-embedded human skin sections utilizing antibody against GPR109A/B. Panels A and B utilized Streptavidin Quantum Dot 605 Conjugates for detection. Panels C and D used FITC Goat Anti-Rabbit IgG for detection. <b>Panel A:</b> Representative immunostaining sample shown at 200× magnification. <b>Panel B:</b> Representative IHC sample shown at 400× magnification. <b>Panels C and D:</b> Representative IHC samples shown at 400× magnification in the absence (Panel C) or presence (Panel D) of competition with peptide used to generate the antibody. Abbreviations: SC, stratum corneum; SG, stratum granulosum; SS, stratum spinousum; SB, stratum basale. Size marker represents 2 microns.</p

GPR109A and GPR109B are functional in normal human keratinocytes but are defective in malignant cells.

<p><b>Panel A:</b> NHEK, HaCaT, Ras-transformed HaCaT, A-431, SCC-25, and CF3 cells were treated in the presence of 10 µM forskolin (open columns) or 10 µM forskolin and 100 µM nicotinic acid (grey columns) for 1 h followed by measurement of intracellular cAMP levels. Data are from three independent experiments and show forskolin-induced cAMP production relative to cellular protein. <b>Panel B:</b> The percent inhibition by nicotinic acid is shown for each cell line. For both panels A and B, Students t-test was used to compare cAMP produced without nicotinic acid to that produced with nicotinic acid, * p≤0.05.</p

Effect of increasing concentrations of nicotinic acid on forskolin stimulated cAMP levels.

<p>NHEK (squares), HaCaT (circles), and SCC-25 cells (triangles) were treated with different concentrations of nicotinic acid the effects on cAMP levels were determined. Representative data are shown with curve fitting lines using a two site-binding model. The R² values for the curve fitting were NHEK (0.985), HaCaT (0.988) and SCC-25 (0.987). EC₅₀ values for GPR109A and GPR109B, respectively, calculated by curve fitting of data from multiple experiments were: NHEK (6.9 nM and 25 µM); HaCaT (72 nM and 17 µM); SCC-25 (36 nM and 22 µM).</p