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Nicotinic acid receptor abnormalities in human skin cancer: implications for a role in epidermal differentiation.

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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

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Related in: MedlinePlus

GPR109A/B genes are transcribed and translated in human skin cells.Panel A: qRT-PCR was performed on total RNA from normal human epidermal keratinocytes (NHEK), immortalized human epidermal keratinocytes (HaCaT), immortalized Ras-transformed human epidermal keratinocytes (Ras-transformed HaCaT), human epidermoid carcinoma cells (A-431), squamous cell carcinoma cells (SCC-25), and human diploid fibroblasts (CF3), using probes specific for GPR109A (dark grey columns) and GPR109B (light grey columns) receptors. Students t-test was used to compare to NHEK, * p≤0.05. Panels B and C: Protein Expression of GPR109A and GPR109B in Human Skin Cells. Cell extracts from NHEK (lane 1), HaCaT (lane 2), Ras-transformed HaCaT (lane 3), A-431 (lane 4), and SCC-25 (lane 5) were subjected to SDS-PAGE and Western immunoblot analyses using an antibody against GPR109A/B pre-incubated in the absence (panel B) or presence (panel C) of 1000-fold excess peptide against which the antibody was generated relative to purified antibody. β-Actin was used as a loading control for Western immunoblot analyses. One representative blot is shown of three independent experiments. The relative densities were quantified using ImageJ. Graphical representation shows the average densitometric units of three independent experiments. Students t-test was used to compare to NHEK, * p≤0.05.
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pone-0020487-g003: GPR109A/B genes are transcribed and translated in human skin cells.Panel A: qRT-PCR was performed on total RNA from normal human epidermal keratinocytes (NHEK), immortalized human epidermal keratinocytes (HaCaT), immortalized Ras-transformed human epidermal keratinocytes (Ras-transformed HaCaT), human epidermoid carcinoma cells (A-431), squamous cell carcinoma cells (SCC-25), and human diploid fibroblasts (CF3), using probes specific for GPR109A (dark grey columns) and GPR109B (light grey columns) receptors. Students t-test was used to compare to NHEK, * p≤0.05. Panels B and C: Protein Expression of GPR109A and GPR109B in Human Skin Cells. Cell extracts from NHEK (lane 1), HaCaT (lane 2), Ras-transformed HaCaT (lane 3), A-431 (lane 4), and SCC-25 (lane 5) were subjected to SDS-PAGE and Western immunoblot analyses using an antibody against GPR109A/B pre-incubated in the absence (panel B) or presence (panel C) of 1000-fold excess peptide against which the antibody was generated relative to purified antibody. β-Actin was used as a loading control for Western immunoblot analyses. One representative blot is shown of three independent experiments. The relative densities were quantified using ImageJ. Graphical representation shows the average densitometric units of three independent experiments. Students t-test was used to compare to NHEK, * p≤0.05.

Mentions: Various skin cell lines were examined by qRT-PCR for GPR109A/B gene expression and the results were compared relative to normal primary human epidermal keratinocytes (NHEK). Immortalized epidermal keratinocytes (HaCaT cells), tumorigenic epidermal keratinocytes (Ras-transformed HaCaT), an epidermoid vulva carcinoma-derived cell line previously reported to contain nicotinic acid receptors (A-431), and a human squamous cell carcinoma cell line (SCC-25) express mRNA for both receptors (Fig. 3A). Over-expression relative to NHEK is observed up to 40-fold in skin cancer cells as compared to NHEK (Fig. 3A). In contrast, expression of nicotinic acid receptors in dermis-derived normal human diploid fibroblasts (CF3) is undetectable.


Nicotinic acid receptor abnormalities in human skin cancer: implications for a role in epidermal differentiation.

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

GPR109A/B genes are transcribed and translated in human skin cells.Panel A: qRT-PCR was performed on total RNA from normal human epidermal keratinocytes (NHEK), immortalized human epidermal keratinocytes (HaCaT), immortalized Ras-transformed human epidermal keratinocytes (Ras-transformed HaCaT), human epidermoid carcinoma cells (A-431), squamous cell carcinoma cells (SCC-25), and human diploid fibroblasts (CF3), using probes specific for GPR109A (dark grey columns) and GPR109B (light grey columns) receptors. Students t-test was used to compare to NHEK, * p≤0.05. Panels B and C: Protein Expression of GPR109A and GPR109B in Human Skin Cells. Cell extracts from NHEK (lane 1), HaCaT (lane 2), Ras-transformed HaCaT (lane 3), A-431 (lane 4), and SCC-25 (lane 5) were subjected to SDS-PAGE and Western immunoblot analyses using an antibody against GPR109A/B pre-incubated in the absence (panel B) or presence (panel C) of 1000-fold excess peptide against which the antibody was generated relative to purified antibody. β-Actin was used as a loading control for Western immunoblot analyses. One representative blot is shown of three independent experiments. The relative densities were quantified using ImageJ. Graphical representation shows the average densitometric units of three independent experiments. Students t-test was used to compare to NHEK, * p≤0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105069&req=5

pone-0020487-g003: GPR109A/B genes are transcribed and translated in human skin cells.Panel A: qRT-PCR was performed on total RNA from normal human epidermal keratinocytes (NHEK), immortalized human epidermal keratinocytes (HaCaT), immortalized Ras-transformed human epidermal keratinocytes (Ras-transformed HaCaT), human epidermoid carcinoma cells (A-431), squamous cell carcinoma cells (SCC-25), and human diploid fibroblasts (CF3), using probes specific for GPR109A (dark grey columns) and GPR109B (light grey columns) receptors. Students t-test was used to compare to NHEK, * p≤0.05. Panels B and C: Protein Expression of GPR109A and GPR109B in Human Skin Cells. Cell extracts from NHEK (lane 1), HaCaT (lane 2), Ras-transformed HaCaT (lane 3), A-431 (lane 4), and SCC-25 (lane 5) were subjected to SDS-PAGE and Western immunoblot analyses using an antibody against GPR109A/B pre-incubated in the absence (panel B) or presence (panel C) of 1000-fold excess peptide against which the antibody was generated relative to purified antibody. β-Actin was used as a loading control for Western immunoblot analyses. One representative blot is shown of three independent experiments. The relative densities were quantified using ImageJ. Graphical representation shows the average densitometric units of three independent experiments. Students t-test was used to compare to NHEK, * p≤0.05.
Mentions: Various skin cell lines were examined by qRT-PCR for GPR109A/B gene expression and the results were compared relative to normal primary human epidermal keratinocytes (NHEK). Immortalized epidermal keratinocytes (HaCaT cells), tumorigenic epidermal keratinocytes (Ras-transformed HaCaT), an epidermoid vulva carcinoma-derived cell line previously reported to contain nicotinic acid receptors (A-431), and a human squamous cell carcinoma cell line (SCC-25) express mRNA for both receptors (Fig. 3A). Over-expression relative to NHEK is observed up to 40-fold in skin cancer cells as compared to NHEK (Fig. 3A). In contrast, expression of nicotinic acid receptors in dermis-derived normal human diploid fibroblasts (CF3) is undetectable.

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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus