1. Retinoic Acid Receptors Regulate Expression of Retinoic Acid 4-Hydroxylase that Specifically Inactivates All-Trans Retinoic Acid in Human Keratinocyte HaCaT Cells 
Yasmin Marikar, ZengQuan Wang, Martin Petkovich, John J. Voorhees, Gary J. Fisher 
Journal of Investigative Dermatology 
Volume 111, Issue 3, Pages (September 1998)
DOI: /j x
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
All-trans RA and the synthetic RA receptor-specific retinoid CD367 induce microsomal RA 4-hydroxylase in HaCaT cells. A chromatogram of reverse-phase high performance liquid chromatography separation of retinoids after incubation of [3H]all-trans RA with (a) control cells or (b) cells treated with CD367. (c) HaCaT cells were treated with 0.1% ethanol (vehicle control), or the indicated concentrations of all-trans RA (t-RA) or CD367 for 24 h. RA 4-hydroxylase activity was determined in whole cell suspensions or microsomal fractions, as described in Materials and Methods. Results are means ± SEM of 3–9 experiments. (d) Microsomes prepared from HaCaT cells stimulated with 0.1 μM CD-367 for 18 h were incubated with various concentrations of [3H]-all-trans RA (x axis). The insert shows the Lineweaver–Burk plot, and the Km and maximum rate of reaction (Vmax). Data are expressed as mean ± SEM of three separate experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
All-trans RA induction of RA 4-hydroxylase requires mRNA and protein synthesis. (a) HaCaT cells were incubated with 1 μM all-trans RA (t-RA) for the indicated times, and RA 4-hydroxylase (RA 4-OHase) mRNA (□, n = 3), and activity in the microsomal fraction (, n = 2), were quantitated as described in Materials and Methods. Inset shows representative northern blot of RA 4-hydroxylase and 36B4 (internal control). Results are expressed as means ± SEM. (b) HaCaT cells were treated with 0.1% ethanol (control, CTRL), with 1.0 μM all-trans RA alone, or with 1.0 μg Actinomycin D (ACT D) per ml or 1.0 μg cycloheximide (CHX) per ml for 18 h. RA 4-hydroxylase mRNA levels (□, n = 3), and activity in the microsomal fraction (, n = 2), were determined as described in Materials and Methods. Inset shows a representative northern blot of RA 4-hydroxylase (RA 4-OHase) and 36B4 (internal control). Results are expressed as means ± SEM.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 2
All-trans RA induction of RA 4-hydroxylase requires mRNA and protein synthesis. (a) HaCaT cells were incubated with 1 μM all-trans RA (t-RA) for the indicated times, and RA 4-hydroxylase (RA 4-OHase) mRNA (□, n = 3), and activity in the microsomal fraction (, n = 2), were quantitated as described in Materials and Methods. Inset shows representative northern blot of RA 4-hydroxylase and 36B4 (internal control). Results are expressed as means ± SEM. (b) HaCaT cells were treated with 0.1% ethanol (control, CTRL), with 1.0 μM all-trans RA alone, or with 1.0 μg Actinomycin D (ACT D) per ml or 1.0 μg cycloheximide (CHX) per ml for 18 h. RA 4-hydroxylase mRNA levels (□, n = 3), and activity in the microsomal fraction (, n = 2), were determined as described in Materials and Methods. Inset shows a representative northern blot of RA 4-hydroxylase (RA 4-OHase) and 36B4 (internal control). Results are expressed as means ± SEM.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 2
All-trans RA induction of RA 4-hydroxylase requires mRNA and protein synthesis. (a) HaCaT cells were incubated with 1 μM all-trans RA (t-RA) for the indicated times, and RA 4-hydroxylase (RA 4-OHase) mRNA (□, n = 3), and activity in the microsomal fraction (, n = 2), were quantitated as described in Materials and Methods. Inset shows representative northern blot of RA 4-hydroxylase and 36B4 (internal control). Results are expressed as means ± SEM. (b) HaCaT cells were treated with 0.1% ethanol (control, CTRL), with 1.0 μM all-trans RA alone, or with 1.0 μg Actinomycin D (ACT D) per ml or 1.0 μg cycloheximide (CHX) per ml for 18 h. RA 4-hydroxylase mRNA levels (□, n = 3), and activity in the microsomal fraction (, n = 2), were determined as described in Materials and Methods. Inset shows a representative northern blot of RA 4-hydroxylase (RA 4-OHase) and 36B4 (internal control). Results are expressed as means ± SEM.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 3
All-trans RA induction of RA 4-hydroxylase is mediated by RA receptors. (a) Synthetic retinoid CD2665 inhibits RA receptor-dependent reporter gene expression in HaCaT cells. HaCaT cells were transfected with the RA receptor-dependent reporter plasmid (βRARE3-tk-CAT), and β-galactosidase expression vector (pSV-β-galactosidase). Following transfection, cells were treated with 10 μM CD2665, 0.1 μM of all-trans RA (t-RA), or all-trans RA plus CD2665 for 24 h. CAT activity was determined and normalized as described in Materials and Methods. Results are mean ± SEM of three separate experiments. (b) CD2665 blocks all-trans RA induction of RA 4-hydroxylase. HaCaT cells were treated with the indicated concentrations of CD2665 alone or with 0.1 μM all-trans RA (t-RA) for 18 h. RA 4-hydroxylase activity in the microsomal fraction was determined as described in Materials and Methods. Results are expressed as means ± SEM of three separate experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 4
HaCaT cell RA 4-hydroxylase activity is specific for all-trans RA versus 9-cis or 13-cis RA. HaCaT cells were treated with 0.1 μM CD367 for 18 h to induce RA 4-hydroxylase activity. RA 4-hydroxylase activity in the microsomal fraction was assayed with [3H]all-trans RA (Duell et al. 1996), [3H]9-cis RA, or [3H]13-cis RA as substrate. Where indicated, a 16.6-fold molar excess of unlabeled all-trans RA was included in the reactions. [3H]4-hydroxy RA (4-OH RA) formed was quantitated by high-performance liquid chromatography as described in Materials and Methods.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

8. Figure 5
RA 4-hydroxylase limits RA receptor-dependent gene expression in HaCaT cells. (a) Ketoconazole inhibits RA 4-hydroxylase activity in HaCaT cells. RA 4-hydroxylase (RA 4-OHase) activity was induced by treatment of HaCaT cells with 1.0 μM all-trans RA for 18 h. RA 4-hydroxylase activity in the microsomal fraction was determined in the presence of the indicated concentrations of ketoconazole. (b) HaCaT cells were transfected with the RA receptor-dependent reporter plasmid (βRARE3-tk-CAT) and β-galactosidase expression vector (pSV-β-galactosidase). Following transfection, cells were treated with 0.1% ethanol (vehicle control), 50 μM ketoconazole (KETO), 0.1 μM all-trans RA (tRA) alone, all-trans RA plus ketoconazole, or all-trans RA plus ketoconazole plus the RA receptor antagonist CD2665, for 24 h. CAT activity was determined and normalized to β-galactosidase activity as described in Materials and Methods. The control (CTRL) bar represents percentage CAT activity in HaCaT cells that received no treatment. Results are means ± SEM of three separate experiments.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions