pone-0020487-g008: Effect of increasing concentrations of nicotinic acid on forskolin stimulated cAMP levels.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).

Mentions: To characterize receptor function further, we compared dose-dependent inhibition by nicotinic acid of forskolin-induced cAMP production in NHEK, HaCaT, and SCC-25 cells. Fig. 8 shows representative data and curves that fit the data to a two-site binding model predicted for binding to high affinity and low affinity receptors. Data from multiple experiments were fit to a two-site model to calculate EC50 values for receptor affinity and relative receptor abundance was assessed by the magnitude of reduction of cAMP generation by nicotinic acid. The data was a close fit to a two-site model for each cell line with R2 values for fit to the data points ranging from 0.985 to 0.988. NHEK cells showed evidence of a significant abundance of both GPR109A and GPR109B with EC50 values of 6.9 nM and 25 µM, respectively (R2 value of 0.985). Compared to NHEK, HaCaT cells showed a reduced abundance of GPR109A with lower affinity (EC50 of 72 nM) but a similar abundance and affinity of GPR109B (EC50 of 17 µM) (R2 value of 0.988). Compared to NHEK, SCC-25 cells showed a greatly reduced abundance of GPR109A and GPR109B, with calculated EC50 values of 36 nM and 22 µM (R2 value of 0.987). The EC50 value for the endogenous GPR109A in NHEK is lower than that reported previously but the other values for both GPR109A and GPR109B are similar to the previously reported EC50 values for GPR109A and GPR109B in cells transfected with the recombinant receptors or from in vitro studies reported to be approximately 100 nM and 100 µM, respectively [9]. These data further support the conclusion that nicotinic acid receptors have greatly reduced functionality in the tumor-derived SCC-25 cells.

Bermudez Y, Benavente CA, Meyer RG, Coyle WR, Jacobson MK, Jacobson EL - PLoS ONE (2011)

Bottom Line: Receptor transcripts are greatly over-expressed in squamous cell cancers.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.

Affiliation: Arizona Cancer Center and Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona, United States of America.

Abstract: 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.