Pharmacological doses of nicotinic acid have long been known to profoundly affect blood lipoproteins by decreasing low-density lipoproteins (LDL) and very-low density lipoproteins (triglycerides) and increasing high-density lipoproteins (HDL) 
. More recent studies have demonstrated pharmacological effects of nicotinic acid in skin. Nicotinic acid can prevent both UV-induced skin tumors and immune suppression in mice 
. In humans, nicotinic acid promotes epidermal differentiation in photodamaged skin as assessed by increased epidermal and stratum corneum thickness, enhanced barrier function, and increased minimal erythemal dose 
. We report here additional evidence that nicotinic acid enhances epidermal differentiation as measured by it effect on the terminal differentiation markers caspase 14 and filaggrin ().
Studies of the pharmacological effects of nicotinic acid have focused on nicotinic acid receptors GPR109A/B in humans 
. These receptors have been mainly studied in the context of effects on blood lipoproteins and associated skin flushing side effects of oral nicotinic acid therapy 
. While prior studies have surveyed human tissues or cell lines for GPR109A mRNA expression including skin 
, we describe here a more detailed characterization of the nicotinic acid receptors in human skin and skin-derived cells using qRT-PCR, Western blot, immunochemistry, and pharmacological analyses. Our studies show that GPR109A/B genes are transcribed and translated in skin keratinocytes and human skin epidermis (, , and ) but that expression in dermal fibroblasts is below limits of detection.
Studies reported here provide several pieces of evidence that support an in depth investigation of the hypothesis that GPR109A/B is involved in the differentiation-promoting effects of nicotinic acid in photodamaged human skin. First, the pattern of GPR109A/B expression in the epidermal layer of skin shows a more diffuse cellular localization in basal keratinocytes and a more peripheral membrane localization in areas of the epidermis undergoing active differentiation (), supporting the possibility that the receptor is brought to the cell surface as keratinocytes undergo differentiation. Similar results are seen for normal keratinocytes in culture where isolated cells show a more diffuse localization but “cobblestone” clusters of cells beginning to differentiate show a more peripheral localization ().
Second, endogenous GPR109A/B is functional in normal human keratinocytes in culture (, , ). Most other studies that have examined nicotinic acid receptors have used transfected cells not inherently expressing GPR109A/B, although a previous study has reported that the epidermoid cell line A-431 highly expresses GPR109A and GPR109B receptors 
. Even though this cell line is not of skin origin, we included it in the studies described here (). Our results corroborate the over-expression data of the previous study. Our studies indicate that the endogenous GPR109A has a higher affinity for nicotinic acid in NHEK cells compared to results of other investigators using transfected cells and isolated membranes but the affinity of GPR109A in HaCaT and GPR109B in both NHEK and HaCaT is similar to that reported both for transfected cells and isolated membranes 
. For SCC-25 cells, the affinity of GPR109A is reduced compared to NHEK and the abundance of both receptors is greatly reduced.
Third, our results indicate that abnormalities in GPR109A/B transcription and function develop as progressive skin damage leads to differentiation defects seen in squamous cell skin cancers. The degree of over-expression of the GPR109A gene increases as a function of disease progression in skin () and cultured SCC-25 cells also significantly over-express the genes encoding the receptors compared with normal keratinocytes (). Further, SCC-25 cells show an abnormal cellular localization () and contain a nearly non-functional receptor (, , ). The severe reduction of functional cell surface receptors in SCC-25 cells may be related to the observed over expression of the genes, where increased mRNA levels represent an attempt to compensate for the lack of functional protein on the cell surface. The epidermoid carcinoma cell line, A-431, also shows reduced receptor function.
Our report of the loss of functional nicotinic acid receptors in SCC-25 cells is of interest with regard to a previous study of the potential role of GPR109A in colon cancer. Thangaraju et al
. have characterized the expression of GPR109A in colon tissues and found expression in the lumen-facing apical membrane of colonic and intestinal epithelial cells. However the receptor is silenced in human colon cancers, in a mouse model of colon cancer, and in colon cancer cell lines 
. They also show that GPR109A mediates the tumor suppressive effects of butyrate, a putative ligand of GPR109A in the colon, suggesting that the GPR109A acts as a tumor suppressor in the colon 
. These data, taken together with our data, suggest that a functional GPR109A/B may be important in maintaining a differentiated state in epithelial cells and that cancer cells may utilize different mechanisms to generate a non-functional nicotinic acid receptor to avoid differentiation signals mediated by the receptor. While butyrate is proposed as a putative endogenous ligand of the receptor in the colon, the ketone body ß-hydroxybutryate has been proposed as an endogenous ligand in tissues 
. ß-hydroxybutyrate would represent a logical ligand in skin as it accumulates under conditions such as fasting or starvation, where it could function to support skin barrier function under stress conditions.
In summary, the increased epidermal differentiation in nicotinic acid-treated human skin may involve nicotinic acid receptor-mediated signaling pathways, although other mechanisms may contribute to the effects of nicotinic acid on photodamaged skin. Both forms of the receptor are expressed in human skin, appearing mainly in the epidermis but are not present in the dermis. The altered expression of the genes encoding GPR109A/B, the abnormal pattern of cellular distribution, and impaired functionality of the receptor in squamous cell carcinoma cells suggest that progression of skin damage leading to receptor defects could provide a mechanism for squamous cell cancers to avoid differentiation signals. However, the observation that poorly differentiated keratinocytes can be driven to differentiate in the presence of sufficient numbers of normal keratinocytes 
raises the possibility that treatments that could promote epidermal differentiation in photodamaged skin could restore the balance of proliferation and differentiation required for maintaining skin homeostasis and thus may counteract the development of AK lesions and SCC. In that context, nicotinic acid receptors could be potential targets for skin cancer prevention. The studies reported here provide sufficient evidence to justify genetic and pharmacologic interventions to define whether and how the nicotinic acid receptor family may function in promoting differentiation of photodamaged skin.