Materials and methods

Materials

HaCaT cells were generously provided by Dr. N.E. Fusenig (German Cancer Research Center, Heidelberg, Germany). Dulbecco's modified Eagle's medium (DMEM), heat-inactivated fetal bovine serum, Dulbecco's phosphate-buffered saline (PBS), and trypsin 2.5% were obtained from Gibco-BRL (Grand Island, NY). All-trans RA, 13-cis RA, cycloheximide, Actinomycin D, tolbutamide, phenylmethylsulfonyl fluoride, aprotinin, leupeptin, NADPH, glycerol, and butylated hydroxytoluene were obtained from Sigma (St. Louis, MO). [³H]All-trans RA was purchased from DuPont NEN (Boston, MA). Extracti-Gel D and Coomassie Plus Protein Assay reagent were from Pierce (Rockford, IL). RA metabolite standards, 4-hydroxy all-trans RA, 4-oxo all-trans RA, 18-hydroxy all-trans RA, and 5,6-epoxy all-trans RA were gifts from Drs. M. Rosenberger and P.F. Sorter of Hoffmann-La Roche (Nutley, NJ). 9-cis RA, [³H]9-cis RA, and [³H]13-cis RA were gifts from Drs. J. Grippo, A. Levin, and P.F. Sorter of Hoffmann-La Roche. Dr. Brahm Shroot of CIRD (Galderma, France) generously provided CD367 [4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl) benzoic acid] and CD2665 [4-(6-methoxyethoxymethoxy-7-adamantyl-2-naphthyl) benzoic acid]. Caffeine and -naphthoflavone were purchased from Aldrich (Milwaukee, WI). Liarozole fumarate and ketoconazole were kindly provided by Gert Cauwenburgh of Janssen Research Foundation (Turnhoutseweg, Belgium). Cyclosporine A and s-mephenytoin were gifts from Novartis (Basel, Switzerland). High performance liquid chromatography grade solvents were obtained from Burdick and Jackson Laboratories (Romulus, MI). Spherisorb ODS-1 columns were obtained from Phase Separations (Norwalk, CT).

Culture and treatment of HaCaT cells

HaCaT cells were cultured in DMEM containing 10% fetal bovine serum. Cells were treated at confluency with retinoids (1000-fold concentrated stock solutions in ethanol) or vehicle under reduced lighting. At appropriate times after addition of retinoids, cells were washed twice with PBS, treated with 0.025% trypsin for 15 min at 37°C, and harvested. Cells were washed three times with PBS, and immediately used for assays, or suspended in a storage medium (PBS, 50% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 g leupeptin per ml, and 0.04 units aprotinin per ml) at a cell density of 10⁸ cells per ml, snap-frozen in liquid nitrogen, and stored at –80°C.

Preparation of microsomal fraction

HaCaT cells (5–15 mg whole cell protein per ml) were suspended in ice-cold homogenization medium [10 mM Tris-HCl (pH 7.4), 1 mM ethylenediamine tetraacetic acid, 0.1 M sucrose, 1 mM phenylmethylsulfonyl fluoride, 0.1 g leupeptin per ml, and 0.04 units aprotinin per ml] and subjected to 20 strokes in a Dounce homogenizer (40 ml), followed by sonication (30 pulses using a microprobe sonicator) at 0–4°C. The homogenate was centrifuged at 10,000 g for 10 min over a sucrose cushion (0.5 M sucrose in homogenization buffer). The pellet was homogenized and centrifuged as above. Pooled supernatants were then centrifuged at 10,000 g for 15 min. The supernatant was centrifuged at 100,000 g for 60 min. The resulting microsomal pellet was suspended in storage medium (10 mg microsomal protein per ml storage medium), snap-frozen in liquid nitrogen, and stored at –80°C.

Protein was measured with the Coomassie Plus Protein Assay reagent in a total volume of 2 ml with bovine serum albumin as standard (^{Bradford 1976}).

RA hydroxylase assay

Whole HaCaT cells (0.1–0.25 mg whole cell protein) or microsomal preparations (0.1 mg microsomal protein) were incubated for 30 min at 37°C with 60 nM [³H]all-trans RA, [³H]9-cis RA, or [³H]13-cis RA, and 2 mM NADPH in reaction buffer [0.1 M Tris-HCl (pH 7.4), 20 mM sodium phosphate buffer (pH 7.4), 5 mM MgCl₂, 0.15 M KCl, and 10% glycerol] in a volume of 300 l. In some experiments, microsomes were incubated with 60 nM [³H]all-trans RA in the presence of 1000 nM unlabeled all-trans RA, 9-cis RA, or 13-cis RA. Reactions were halted by addition of 600 l chloroform:methanol (2:1, vol/vol), vortexed, and centrifuged at 500 g for 10 min. The lower organic fraction containing retinoids was collected, evaporated with nitrogen in the dark, resuspended in methanol, and analyzed using reverse-phase high performance liquid chromatography connected to a flow-through scintillation counter (^{Duell et al. 1992}). RA 4-hyroxylase activity was calculated as the sum of 4-hydroxy RA plus 4-oxo RA, which is formed from 4-hydroxy RA. 18-hydroxy RA was also detected and was 50% of 4-hydroxy RA.

Transfection of HaCaT cells and chloramphenicol acetyl transferase (CAT) reporter gene assays

HaCaT cells were plated at 50% confluency in 35 mm 6 well tissue culture plates in DMEM medium containing 10% charcoal-stripped bovine calf serum. Cells were transfected overnight with 1 g each of RARE₃-tk-CAT (^{Xiao et al. 1995}) and pSV--galactosidase plasmid DNA by the calcium phosphate method (^{Cullen 1987}). RARE₃-tk-CAT contains three copies of the RA response element from the mouse RAR-2 promoter, the thymidine kinase minimal promoter, which regulates 5' expression of CAT. pSV--galactosidase is a constitutively active expression vector for the enzyme -galactosidase. Following transfection, cells were washed twice with DMEM, replenished with complete DMEM-10% charcoal-striped bovine calf serum, and where indicated pretreated for 60 min with ketoconazole and/or CD-2665. All-trans RA was added for 24 h. Cells were washed twice with PBS, lyzed by freeze-thawing at –80°C in 0.2 ml of 0.1 M Tris buffer (pH 8.0) containing 0.1% Triton X-100. Cell lysates were centrifuged at 14,000 g for 10 min. Cell supernatants were assayed for CAT (^{Neumann et al. 1987}) and -galactosidase activity (^{Miller 1972}), and CAT activity was normalized to -galactosidase activity. All transfection experiments were performed in triplicate wells.

RNA extraction and northern analysis

Total RNA was isolated from HaCaT cells by the guanidinium phenol extraction method (^{Fisher et al. 1995}). RA 4-hydroxylase (hP450RAI;^{White et al. 1997}) and 36B4 (a ribosomal protein used as an internal control;^{Masiakowski et al. 1982}) mRNA were determined by northern analysis, using random-primed ³²P-labeled cDNA probes, as previously described (^{Fisher et al. 1995}). Filters were washed, visualized, and quantitated by PhosphoImager.

Results and discussion

Control cells and cells treated with CD367 were incubated separately with [³H]all-trans RA, and reverse phase HPLC was used to separate resulting retinoids. Chromatograms show a difference in RA metabolites for untreated control cells (Figure 1a) versus cells treated with CD367 (Figure 1b). Only CD367-treated cells produced 4-hydroxyl RA, 18-hydroxyl RA, and 4-oxo RA.

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 M 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 K_m and maximum rate of reaction (V_max). Data are expressed as mean SEM of three separate experiments.

Full figure and legend (46K)

In the absence of added retinoids, RA 4-hydroxylase activity was not detectable in HaCaT cells. Addition of all-trans RA, or the nuclear RA receptor-specific ligand CD367 (^{Fisher et al. 1994}), resulted in induction of RA 4-hydroxylase activity (Figure 1c). CD367 was 6-fold more potent than all-trans RA in inducing RA 4-hydroxylase activity. Fractionation of HaCaT cells revealed that retinoid-inducible RA 4-hydroxylase activity was localized in the microsomal (high-speed pellet) fraction (Figure 1c). The percentage of total cellular RA 4-hydroxylase activity recovered in the microsomal fraction was 50%.

The substrate dependence of all-trans RA-induced microsomal RA 4-hydroxylase activity is illustrated in Figure 1(d). Analysis of the data using a Lineweaver–Burk plot (Figure 1d, inset) indicated that the concentration of all-trans RA required for half-maximal activity (K_m) was 37 9 nM, and that the maximum rate of reaction (V_max) was 205 5 pg per min per mg protein (Figure 1d).

Induction of RA 4-hydroxylase by all-trans RA requires new mRNA and protein synthesis

The time course of induction of RA 4-hydroxylase mRNA and activity by all-trans RA is shown in Figure 2(a). RA 4-hydroxylase mRNA was induced within 2 h, and reached a maximal level within 8 h after addition of all-trans RA. Two RA 4-hydroxylase transcripts were detected, a major band of 1.9 kb and a minor band of 2.3 kb. Induction of RA 4-hydroxylase activity followed induction of its mRNA, first becoming detectable 4 h after addition of all-trans RA. Activity continued to increase, reaching a maximal level 24 h after addition of all-trans RA. Induction of RA 4-hydroxylase activity by all-trans RA was relatively transient, however, returning to near-baseline levels by 48 h (data not shown). This disappearance of RA 4-hydroxylase activity coincided with catabolism of all-trans RA, and was substantially accelerated by removal of all-trans RA from the culture medium. Replacement of the culture media 20 h after addition of all-trans RA, with fresh media lacking exogenous all-trans RA, resulted in complete loss of RA 4-hydroxylase activity within 6 h (data not shown). These data indicate that the continued presence of all-trans RA is required to maintain RA 4-hydroxylase activity, and that the half-life of RA 4-hydroxylase activity is relatively short (less than 6 h) in HaCaT cells.

Figure 2.

All-trans RA induction of RA 4-hydroxylase requires mRNA and protein synthesis. (a) HaCaT cells were incubated with 1 M 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 M all-trans RA alone, or with 1.0 g Actinomycin D (ACT D) per ml or 1.0 g 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.

Full figure and legend (49K)

In contrast, RA 4-hydroxylase activity remained maximally elevated for at least 48 h, following induction by CD367. In addition, RA 4-hydroxylase activity remained fully induced for at least 24 h following removal of CD367 from the culture media (data not shown). This long-lived induction of RA 4-hydroxylase activity was likely attributable to the lack of catabolism of CD367 by HaCaT cells. Unlike all-trans RA, which was rapidly catabolized by HaCaT cells, CD367 was metabolically inert (data not shown).

A recent report showed that induced RA 4-hydroxylase mRNA levels remained elevated for up to 96 h in the murine F9 teratocarcinoma cell line (^{Abu-Abed et al. 1998}). This difference in expression of RA 4-hydroxylase mRNA after induction may be due to lower RA 4-hydroxylase-mediated catabolism of RA by F9 cells relative to HaCaT cells. Lower catabolism in F9 cells would allow sustained elevation of cellular RA, thus maintaining induced levels of RA 4-hydroxylase activity, as we observed in CD367-treated HaCaT cells. Alternatively, differences in the stability of RA 4-hydroxylase mRNA may exist between F9 and HaCaT cells.

The rapid induction of RA 4-hydroxylase mRNA by all-trans RA (Figure 2a) suggests that RA 4-hydroxylase is transcriptionally regulated. We therefore examined the effect of transcription and translation inhibitors on RA 4-hydroxylase induction. Inhibition of transcription by actinomycin D completely blocked induction of RA 4-hydroxylase mRNA and activity (Figure 2b). Inhibition of translation by cycloheximide caused super-induction of RA 4-hydroxylase mRNA, and complete inhibition of induction of RA 4-hydroxylase activity (Figure 2b). Super-induction of inducible transcripts by cycloheximide is commonly observed, and results from increased mRNA stability (^{Edwards & Mahadevan 1992;Schuetz et al. 1995}). These data demonstrate that all-trans RA induction of RA 4-hydroxylase is dependent on mRNA and protein synthesis.

Induction of RA 4-hydroxylase by all-trans RA is mediated by nuclear RA receptors

The above data demonstrate that all-trans RA induction of RA 4-hydroxylase is transcriptionally regulated. The biologic effects of all-trans RA are mediated by nuclear RA receptors (^{Fisher & Voorhees 1996}). These receptors are activated through direct binding of all-trans RA and function to regulate transcription of specific target genes. We therefore investigated the involvement of nuclear RA receptors in induction of RA 4-hydroxylase gene expression by all-trans RA. To do this, we utilized the nuclear RA receptor antagonist CD2665 (^{Sun et al. 1997}). This synthetic retinoid effectively inhibited all-trans RA induction of nuclear RA receptor-dependent reporter gene expression in HaCaT cells (Figure 3a). CD2665 also inhibited all-trans RA induction of RA 4-hydroxylase activity, in a concentration-dependent manner (Figure 3b). At a ratio of 1:100 (all-trans RA:CD2665) inhibition was nearly complete. These data indicate that all-trans RA induction of RA 4-hydroxylase is mediated by nuclear RA receptors. Recent studies with transformed cell lines have also provided evidence for involvement of nuclear RA receptors in induction of all-trans RA metabolism (^{van der Leede et al. 1997;Isogai et al. 1997;Abu-Abed et al. 1998}).

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 (RARE₃-tk-CAT), and -galactosidase expression vector (pSV--galactosidase). Following transfection, cells were treated with 10 M CD2665, 0.1 M 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 M 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.

Full figure and legend (18K)

RA 4-hydroxylase activity is specific for all-trans RA versus 9-cis RA or 13-cis RA

Recent studies employing molecular cloning techniques have demonstrated that RA 4-hydroxylase is a member of the cytochrome P-450 supergene family (^{White et al. 1996, 1997;Ray et al. 1997}). This multigene family encodes a large number of P-450 isoenzymes that have varying degrees of substrate specificity, and act on a wide range of different substrates (^{Coon et al. 1996}). To examine the substrate specificity of RA 4-hydroxylase in HaCaT cells, we determined the effect of a series of known substrates for cytochrome P-450 subfamilies on RA 4-hydroxylase activity. Each substrate was added separately at 2-fold, 20-fold, and 100-fold higher concentrations than [³H]all-trans RA in the RA 4-hydroxylase assay. The 11 compounds utilized, and the cytochrome P-450 subfamilies for which they are substrates, are listed in Table I. Any compound tested that was a substrate for, or interacted with, RA 4-hydroxylase would be expected to alter the amount of [³H]4-hydroxy RA product generated in the assay; however, none of the compounds examined had any significant effect on RA 4-hydroxylase activity (data not shown). This finding is consistent with molecular cloning data indicating that RA 4-hydroxylase is unique, and that it defines a new cytochrome P-450 subfamily (^{White et al. 1996, 1997;Ray et al. 1997}).

Table I - Cytochrome P-450 substrates tested for their effect on HaCaT cell RA 4-hydroxylase activity.

Full table

We next examined the specificity of RA 4-hydroxylase towards the three naturally occurring isomers of RA: all-trans RA, 9-cis RA, and 13-cis RA. The amount of 4-hydroxy RA formed by HaCaT cell microsomes incubated with [³H]9-cis RA and [³H]13-cis RA was 1% and 5%, respectively, of that formed from all-trans RA (Figure 4). Because 9-cis RA and 13-cis RA spontaneously isomerize to all-trans RA (^{Duell et al. 1996;Randolph & Simon 1997}), it is likely that the low levels of [³H]4-hydroxy RA formed in incubations containing [³H]9-cis RA or [³H]13-cis RA were generated from [³H]all-trans RA. Our preparations of 9-cis and 13-cis RA contained 4% and 18% all-trans RA, respectively. Addition of excess unlabeled all-trans RA to incubations containing either [³H]9-cis RA or [³H]13-cis RA substantially reduced the amount of [³H]4-hydroxy RA product formed (Figure 4), supporting this supposition.^{White et al. (1997)} have reported that 9-cis RA served as a good substrate for RA 4-hydroxylase; however, the degree of isomerization of the 9-cis RA used as substrate to all-trans RA was not assessed. Taken together, the above data indicate that microsomal RA 4-hydroxylase activity in HaCaT cells is highly specific for the all-trans isomer of RA.^{Krekels et al. (1997)} have also reported that all-trans RA is the preferred substrate for RA 4-hydroxylase in MCF-7 breast cancer cells.

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 M 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.

Full figure and legend (9K)

RA 4-hydroxylase functions to limit all-trans RA-dependent gene expression

Finally, we examined the effect of RA 4-hydroxylase on nuclear RA receptor-dependent gene expression. For these functional studies we utilized ketoconazole, which completely inhibits RA 4-hydroxylase activity in HaCaT cells when used at concentrations in excess of 10 M (Figure 5a). HaCaT cells were transfected with a nuclear RA receptor-dependent reporter gene, and then treated with either all-trans RA (0.1 M) alone, or in combination with ketoconazole (50 M). Reporter gene expression was nearly 4-fold greater in the presence of all-trans RA plus ketoconazole, compared with all-trans RA alone (Figure 5b). Activation of the reporter gene by either all-trans RA alone or all-trans RA plus ketoconazole, was inhibited by 90% by the nuclear RA receptor antagonist CD2665, confirming that reporter gene activity was dependent on nuclear RA receptors. These data demonstrate that by conversion of all-trans RA to 4-hydroxy RA, RA 4-hydroxylase functions to limit induction of nuclear RA receptor-regulated gene expression in HaCaT cells.

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 M 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 (RARE₃-tk-CAT) and -galactosidase expression vector (pSV--galactosidase). Following transfection, cells were treated with 0.1% ethanol (vehicle control), 50 M ketoconazole (KETO), 0.1 M 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 -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.

Full figure and legend (16K)

Concluding remarks

Previous studies have demonstrated that all-trans RA induces its own catabolism to 4-hydroxy RA in human skin (^{Duell et al. 1996}) and other organs and cells (^{Ray et al. 1997;White et al. 1997}). We have utilized human keratinocyte HaCaT cells to investigate the mechanism of this induction. The data presented above demonstrate that induction of RA 4-hydroxylase is dependent on nuclear RA receptor-mediated gene transcription. This conclusion is in accord with a recent study demonstrating that disruption of RAR- and RAR- gene expression by homologous recombination causes loss of RA induction of RA 4-hydroxylase in murine F9 cells (^{Abu-Abed et al. 1998}). These findings strongly suggest that the RA 4-hydroxylase gene contains a RA response element that is directly regulated by nuclear RA receptors. Cloning and characterization of the RA 4-hydroxylase gene promoter will reveal whether this hypothesis is correct.

4-hydroxy RA and its metabolites are less potent than all-trans RA in activating nuclear RA receptors (^{Siegenthaler et al. 1990;Reddy et al. 1992}), and in eliciting biologic responses (^{Reynolds et al. 1993}). Therefore, by inducing RA 4-hydroxylase activity, all-trans RA limits its own actions in skin. This negative autoregulatory mechanism operates simultaneously with, and continually controls, the pharmacologic actions of all-trans RA. The actions of all-trans retinol are similarly controlled, because it is converted to all-trans RA. In contrast, most synthetic retinoids are not subject to the natural restraining mechanism provided by RA 4-hydroxylase, due to the stringent substrate specificity of the enzyme. This fact raises the possibility that synthetic retinoids may need to have the equivalent of a built-in, self-adjusting brake in order to be safe and effective. A braking mechanism may be designed by exploiting alternative routes of metabolic disposal, and/or activating RA receptors in a manner that is qualitatively distinct from activation by all-trans RA. Design and development of such synthetic retinoids represents a significant challenge.

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Acknowledgments

Supported in part by the Babcock Endowment for Dermatological Research and a grant from the Johnson & Johnson Corporation.