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Cell Growth & Differentiation Vol. 10, 255-262, April 1999
© 1999 American Association for Cancer Research

Cell Type and Gene-specific Activity of the Retinoid Inverse Agonist AGN 193109: Divergent Effects from Agonist at Retinoic Acid Receptor {gamma} in Human Keratinocytes

Scott M. Thacher1, Sunil Nagpal, Elliott S. Klein, Taghreed Arefieg, Glenn Krasinski, Dan DiSepio, Chapla Agarwal, Alan Johnson, Richard L. Eckert and Roshantha A. S. Chandraratna1

Departments of Biology [S. M. T., S. N., E. S. K., T. A., G. K., D. D., R. A. S. C.] and Chemistry [A. J., R. A. S. C.], Retinoid Research, Allergan, Irvine, California 92623, and Department of Physiology and Biophysics, Case Western Reserve, Cleveland, Ohio 44106 [C. A., R. L. E.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Retinoids are important regulators of epithelial differentiation. AGN 193109 is a high-affinity antagonist and inverse agonist for the nuclear retinoic acid receptors (RARs). Paradoxically, both AGN 193109 and retinoid agonists inhibit the expression of the differentiation marker MRP-8 in normal human keratinocytes (NHKs). TTNPB, an RAR agonist, and AGN 193109 mutually antagonize MRP-8 inhibition at both mRNA and protein levels. We find that this antagonism, which is greatest at an AGN 193109:TTNPB ratio of about 10:1, is absent when either compound is in significant excess. The potent RAR{alpha}-specific agonist, AGN 193836, has no effect on MRP-8 regulation. These data indicate that inverse agonists and agonists suppress MRP-8 in NHKs through RAR{gamma} using distinct and mutually inhibitory mechanisms. The activity of AGN 193109 on MRP-8 is cell type specific. In differentiating ECE16-1 cervical cells, TTNPB inhibits while AGN 193109 induces MRP-8 mRNA levels. The effect of AGN 193109 on genes inhibited by retinoid agonists in NHKs is also selective; expression of the differentiation markers transglutaminase 1 and keratin 6 is not down-regulated by AGN 193109 whereas stromelysin-1 expression is suppressed. These results show a complex gene and cell context-specific interplay between agonist and inverse agonist for the regulation of gene expression.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Activation of the DNA-binding nuclear receptors RAR2 {alpha}, ß, and {gamma} (1) has profound effects on morphogenesis and epithelial differentiation. The RARs regulate gene transcription as heterodimers with the related retinoid X receptors as a result of ligand-mediated binding of positive transcriptional accessory cofactors and release of negative cofactors (2, 3, 4) . We have described an RAR-specific retinoid antagonist, AGN 193109, which potently blocks RAR-dependent transcription (5) and antagonizes and inhibits retinoid agonist-induced toxicity in vivo (6 , 7) . AGN 193109 has the additional property of inhibiting basal transcription mediated by the RARs (8) , and it is classified as an inverse agonist by analogy to ligands of the ß-adrenergic receptor, which also suppress basal or spontaneous receptor activity (9 , 10) . In an analogous manner, two androstane metabolites have recently been shown to function as inverse agonists for the orphan nuclear receptor CAR-ß (11) . Other retinoid antagonists, such as AGN 193840, do not affect basal receptor transcription and are referred to as neutral antagonists. Neutral antagonists block the gene regulatory activities of both agonists and inverse agonists (8) .

In differentiating NHK cultures, AGN 193109 regulates gene expression in an autonomous fashion, inhibiting the expression of MRP-8 (calgranulin A; Ref. 8 ). Because MRP-8 expression is also inhibited by retinoid agonists (12) but not by the retinoid neutral antagonist AGN 193840, it appears that MRP-8 regulation in NHKs by agonist and inverse agonist occurs through separate pathways. This hypothesis is supported by the observation that the combination of AGN 193109 and the agonist TTNPB is no longer able to inhibit MRP-8 expression (8) . The finding suggests that there is mutual interference between the two distinct ligand-mediated pathways. Although AGN 193109 antagonizes induction of RAR-ß and inhibition of differentiation markers by retinoid agonist in the papilloma virus-immortalized cervical keratinocyte line ECE16–1, autonomous effects of AGN 193109 have not been described thus far in this cell line (13) .

To further explore the properties of AGN 193109, we investigated its effects on the expression of additional retinoid-sensitive markers in NHKs. Some markers, like stromelysin-1 (matrix metalloproteinase 3) (14) , are inhibited by both retinoid agonists and AGN 193109, whereas others, such as TGase 1 and K6 (15 , 16) , are regulated only by retinoid agonists but not by AGN 193109. On the basis of the effects of RAR receptor subtype-selective retinoids and the dose-responsive antagonism between agonist and inverse agonist, RAR{gamma} appears to be the critical target for both agonist and inverse agonist regulation of MRP-8 in NHKs. We also show that MRP-8 expression in ECE16-1 cells is inhibited by TTNPB and is surprisingly induced by AGN 193109. These data indicate that regulation of gene expression by the inverse agonist appears to be dependent on cell type as well as promoter context.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Retinoid Ligand Specificity of MRP-8 Down-Regulation in NHKs.
Dose response studies have shown that AGN 193109 (8) and tazarotene (12) are equally potent in suppression of MRP-8 levels and that TTNPB is {approx}10-fold more potent (Table 1)Citation . As reported previously (8) , the neutral antagonist AGN 193840 does not suppress MRP-8 expression (the average inhibition at 1 µM AGN 193840 in two experiments was 21 ± 7%). The absence of MRP-8 regulation is not explained by the lower affinity of AGN 193840 for the RARs, which is only 5-fold less than that of AGN 193109 (8) . If AGN 193840 does bind to the RARs in NHKs, it would be expected to antagonize both retinoid agonist (TTNPB) and inverse agonist (AGN 193109) effects on inhibition of MRP-8 expression. As shown in Fig. 1Citation , AGN 193840 reverses MRP-8 inhibition by both retinoids in a dose-responsive manner. Inhibition by both AGN 193109 and TTNPB is completely reversed by a 10-fold excess of AGN 193840.


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Table 1 Inhibition of MRP-8 by receptor- and function-selective retinoids in differentiated NHKsa

 


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Fig. 1. AGN 193840 is an antagonist of TTNPB and AGN 193109 down-regulation of MRP-8 expression. NHKs were treated for 5 days with 0.1 µM TTNPB or 0.1 µM AGN 193109. Increasing concentrations of AGN 193840 were added simultaneously. The percentage of inhibition of MRP-8 level determined by immunoblotting (mean; bars, SD; n = 3) is compared to untreated control cultures.

 
The receptor specificity of MRP-8 down-regulation in NHKs was also investigated. The ratio of RAR{gamma}:RAR{alpha} protein levels in human epidermis is about 5:1, whereas RARß protein is not detectable (17) . In cultured human keratinocytes, RARß protein is also undetectable, and mRNA levels are extremely low or negligible while, as in skin, RAR{gamma} appears to be more highly expressed than RAR{alpha} (18, 19, 20, 21) . NHKs were exposed to the RAR{alpha}-selective agonist AGN 193836 during induction of differentiation by serum. AGN 193836 binds to RAR{alpha} with high affinity and has a greater than 1000-fold selectivity for RAR{alpha} relative to RARß or RAR{gamma} in assays of receptor binding affinity (22) . Inhibition of MRP-8 by 1 µM AGN 193836 was negligible (<10%; Table 1Citation ). We also observed that 1 µM AGN 193836 does not interfere with down-regulation of MRP-8 protein levels by either 10 nM TTNPB or 100 nM AGN 193109 (Table 2)Citation . These data suggest that retinoid regulation of MRP-8 in NHKs requires binding to RAR{gamma}.


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Table 2 TTNPB and AGN 193109 regulation of MRP-8 and TGase 1 activity is unaffected by RAR{alpha}-specific retinoid in NHKs

 
We next addressed the question of whether the mutual antagonism of AGN 193109 and TTNPB on MRP-8 inhibition is consistent with the action of both retinoids through a single receptor such as RAR{gamma}. If the regulation of MRP-8 by AGN 193109 and TTNPB takes place through a single receptor, then the mutual antagonism observed should occur when there is partial receptor occupancy by both compounds, whereas a substantial excess of either agonist or inverse agonist should lead to complete inhibition of MRP-8 without interference by the other. As demonstrated in Fig. 2aCitation , the inhibition of MRP-8 by 10 nM TTNPB is completely reversed by 10 and 100 nM AGN 193109 to a level similar to untreated control cells. When the ratio of inverse agonist to agonist is 100-fold, at 1 µM AGN 193109, MRP-8 levels are once again suppressed. Similarly, 10 and 100 nM TTNPB interfere with the inhibition of MRP-8 by 100 nM AGN 193109, and if TTNPB is added in 10-fold excess over AGN 193109, the interference is no longer observed. Replicates of these experiments were quantitated and are presented as percentage of inhibition relative to untreated control (Fig. 2,b and c)Citation . The mutual antagonism of agonist and inverse agonist also occurs at the level of MRP-8 mRNA expression in NHKs. Expression of MRP-8 was measured by quantitative analysis of the amplification of a PCR fragment covering almost the entire coding region of MRP-8. MRP-8 gives rise to only a single band on Northern blots in cultured keratinocytes (12) as well as in human lung and liver,3 and thus the technique records the levels of a single mRNA species. Although TTNPB and AGN 193109 both reduce MRP-8 mRNA levels as determined by quantitative RT-PCR, the combination of the two retinoids (at 10 and 100 nM, respectively) is not significantly different from untreated controls (Fig. 3)Citation .



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Fig. 2. Mutual antagonism of TTNPB and AGN 193109 in MRP-8 down-regulation. Five day serum-induced NHKs were treated with retinoid combinations as indicated. A, Western Blot analysis of MRP-8 levels shows the effect of an increasing concentration of AGN 193109 on MRP-8 regulation by 10 nM TTNPB (top panel). The lower panel shows the effect of increasing TTNPB in the presence of 100 nM AGN 193109. MRP-8 band intensities from multiple blots were compared: B, dose-responsive antagonism of 10 nM TTNPB by AGN 193109 and C, antagonism of 100 nM AGN 193109 by TTNPB. Shown are means; bars, SE (n = 3–6). *, P < 0.05; **, P < 0.01 with respect to one retinoid alone (10 nM TTNPB or 100 nM AGN 193109, respectively) in each series.

 


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Fig. 3. Mutual antagonism of RAR agonist and inverse agonist on inhibition of MRP-8 mRNA levels. NHKs were induced to differentiate by 10% serum and simultaneously treated for 5 days with solvent control or 100 nM AGN 193109 in the presence or absence of 10 nM TTNPB. MRP-8 mRNA levels relative to GAPDH were determined by real-time RT-PCR. Values are means for replicate (n = 2–4) PCR analyses of individual treated cultures; bars, SD.

 
Regulation of MRP-8 by AGN 193109 Is Cell Context Dependent.
Regulation of MRP-8 expression at the mRNA level by AGN 193109 was tested in a second epithelial cell type, the papilloma virus-immortalized ECE16-1 cervical keratinocyte (13) . Compared with undifferentiated controls, MRP-8 mRNA is elevated by about 5-fold in ECE16-1 keratinocytes induced to differentiate in serum-free medium.4 MRP-8 is also induced by induction of differentiation in NHKs (23) . We also find that, as in NHKs, 0.1 µM TTNPB significantly suppresses MRP-8 mRNA levels to 8% of control values (Fig. 4)Citation . However, in the same experiment, in contrast to its effect in NHKs, 1 µM AGN 193109 increased MRP-8 mRNA in ECE16-1 cells by a factor of 8. The average induction of MRP-8 mRNA in two experiments was 6-fold. In a separate experiment (not shown), we also found that the half-maximal response to AGN 193109 was in the range of 1–10 nM. The neutral antagonist AGN 193840, by contrast, has little effect on MRP-8 mRNA levels by itself (Fig. 4)Citation . Finally, if the inverse agonist or neutral antagonist is added at a 10-fold excess over TTNPB, then the action of TTNPB is blocked and MRP-8 is expressed at the level determined by the competing ligand (e.g., AGN 193109 or AGN 193840).



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Fig. 4. Opposite regulation of MRP-8 by agonist and inverse agonist in ECE16-1 cells. ECE16-1 cells were induced to differentiate by removal of serum for 2 days, as described in "Materials and Methods." MRP-8 mRNA levels, analyzed as in Fig. 3Citation , are presented relative to solvent control. The effect of 0.1 µM TTNPB was compared with 1 µM AGN 193109 and 1 µM AGN 193840. The combination of 0.1 µM TTNPB with 1 µM AGN 193109 or 1 µM AGN 193840 was also tested.

 
AGN 193109 Suppresses Some but not All Genes Inhibited by Retinoid Agonist in NHKs.
MRP-8 expression in cultured NHKs is linked to the induction of cell differentiation by serum and IFN-{gamma} (12 , 23) . We therefore wished to investigate whether AGN 193109, like the retinoid agonists, might be a global inhibitor of cultured NHK differentiation. TGase 1 is induced during keratinocyte differentiation in vivo and in culture (15 , 24) . TGase 1 activity is predominantly expressed in the particulate fraction of cultured keratinocytes, and serum-induced particulate TGase activity is potently inhibited (IC50 <10 nM) by the retinoid agonists TTNPB, tazarotene, and AGN 190121 in NHKs (12) . As shown in Fig. 5Citation , AGN 193109 has no inhibitory effect on detergent-extractable cell particulate TGase 1 activity at a concentration up to 1 µM. However, MRP-8 protein levels in the cytosolic fraction of these same cell lysates were sharply reduced as predicted (data not shown). AGN 193109 also blocks the inhibitory effect of TTNPB on particulate TGase activity when added in excess (Fig. 5)Citation . We confirmed that the enzymatic assay for TGase measured exclusively TGase1 in this experiment. Immunoprecipitation with the specific monoclonal antibody B.C1 eliminated {approx}90% of enzyme activity from extracts of control and TTNPB- and AGN 193109-treated cell cultures. The distinct effects of agonist and inverse agonist on MRP-8 and TGase 1 expression may, in principle, be a consequence of differential involvement of RAR{alpha} and RAR{gamma} in regulation of these two markers. However, as in the case of MRP-8, inhibition of TGase 1 appeared to occur exclusively through RAR{gamma} because AGN 193836 had no effect on TGase 1 regulation (Table 2)Citation .



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Fig. 5. AGN 193109 blocks TTNPB inhibition of NHK cell particulate TGase activity. NHKs, induced to differentiate by removal of hydrocortisone and induction by 10% serum, were treated simultaneously with TTNPB, AGN 193109, or combination as shown. TGase activity in detergent extracts of the cell particulate fraction is presented relative to untreated control. Activities are means for duplicate cultures; bars, SD.

 
K6 is up-regulated in the differentiated layers of hyperproliferative epidermis, as occurs during wound healing or in psoriasis (25) , and is down-regulated by retinoid treatment in keratinocyte culture (16) . Levels of K6 mRNA were compared semiquantitatively according to the number of PCR cycles required to produce a specific K6 product from total cDNA. Fig. 6Citation shows that the level of K6 mRNA is significantly inhibited by TTNPB, whereas AGN 193109 by itself has no effect. AGN 193109 instead acts strictly as an antagonist of the TTNPB effect on K6 mRNA levels (Fig. 6)Citation . The level of GAPDH mRNA, also determined by RT-PCR, was unaffected by retinoid treatment. In contrast, stromelysin-1, which is negatively regulated by retinoid agonists in NHKs (14) , was inhibited by both TTNPB and AGN 193109 according to similar RT-PCR quantitation (not shown).



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Fig. 6. AGN 193109 antagonizes the inhibitory effect of TTNPB on K6 expression. The mRNA levels of K6 in cultured NHKs were determined by RT-PCR. NHKs were treated with 10% serum for 3 days as described in "Materials and Methods," and K6 mRNA levels were determined by RT-PCR. Band intensity for K6 and GAPDH is presented with respect to PCR cycle for solvent-treated control or for treatment by 0.1 µM TTNPB, 1.0 µM AGN 193109, or the combination of the two retinoids.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
This study further explores the ability of AGN 193109, which was first identified and characterized as a retinoid antagonist (5 , 6 , 13) , to regulate gene expression in an autonomous fashion consistent with its properties as an inverse agonist (8) . In NHKs, AGN 193109 and TTNPB are mutually antagonistic toward suppression of MRP-8, and our data indicate that this antagonism only occurs when RAR{gamma} is partially occupied by both ligands simultaneously. The neutral antagonist, AGN 193840, has little or no effect on MRP-8 expression in NHKs by itself but blocks inhibition of MRP-8 by AGN 193109 and TTNPB. These data rule out competition for culture-derived retinoic acid as a factor in AGN 193109 regulation of MRP-8 in NHKs. They also indicate that agonist, neutral antagonist, and inverse agonist induce three distinct functional states in RAR{gamma} in NHKs, just as described previously with transfected, chimeric RAR{gamma} (8) . We also observe that the relationship of AGN 193109 activity to retinoid agonists depends on both the particular cell type and gene investigated. For example, whereas TTNPB, a retinoid agonist, suppresses MRP-8 in both NHKs and in ECE16-1 cells, the inverse agonist inhibits MRP-8 expression only in NHKs and induces MRP-8 in ECE16-1 cells. In the case of the expression of the differentiation markers K6 and TGase1 in NHKs, AGN 193109 acts purely as an antagonist of retinoid agonist effects. Therefore, AGN 193109 is not a generalized inhibitor of cultured NHK differentiation but has autonomous effects only on a subset of genes in this cell type.

Our data strongly indicate that the divergent effects of RAR agonist and inverse agonist on MRP-8 expression in NHKs take place through modulation of the conformational state of a single retinoid receptor, RAR{gamma}. RAR{gamma} is more highly expressed than RAR{alpha} in epidermis and cultured keratinocytes. NHK RARß is undetectable at the protein level and is extremely low or undetectable at the mRNA level (17, 18, 19, 20, 21) . Although RARß is highly inducible by retinoids in many tissues and cell types (1) , all-trans retinoic acid treatment had little or no effect on RARß expression in epidermis or cultured epidermal keratinocytes in the same studies. It is unlikely that regulation of gene expression through RARß was functionally significant in our studies of NHKs because AGN 193840 is a partial agonist of RARß (8) but does not inhibit MRP-8 expression in NHKs by itself (Table 1)Citation or in the presence of TTNPB (Fig. 1)Citation . RAR{alpha}-specific retinoids such as AGN 193836 (22) are potent inhibitors of the growth of cultured mammary carcinoma cell lines that express higher levels of RAR{alpha} (26) . However, AGN 193836 has no autonomous or antagonistic effects on MRP-8 and TGase 1 expression in NHKs. RAR{gamma} therefore appears to be the critical target for retinoid agonist and inverse agonist regulation of MRP-8 in NHKs, implying that the mutual antagonism between agonist and inverse agonist is the result of downstream interactions between distinct signaling pathways triggered through a single receptor (i.e., RAR{gamma}).

The dose dependence of mutual antagonism between AGN 193109 and TTNPB on MRP-8 is also consistent with utilization of a single receptor by both compounds. Down-regulation is maximal when the two compounds are at a concentration ratio of about 10:1, a ratio that is roughly similar to the relative potencies of the individual compounds for binding to RAR{gamma} (8) and for MRP-8 inhibition (Table 1)Citation . MRP-8 expression is inhibited, however, when the AGN 193109:TTNPB ratio is substantially greater or smaller than 10, presumably as the result of near-exclusive occupancy of RAR{gamma} by one retinoid or the other. These data do not support a model in which two distinct retinoid receptors, each with relative selectivity for either AGN 193109 or TTNPB, regulate MRP-8 expression in NHKs. Our data support a competitive antagonism between the two compounds at a single receptor rather than a noncompetitive interaction at two separate receptors.

AGN 193109 was originally characterized as an inverse agonist because of its inhibitory effects on basal gene transcription from chimeric nuclear receptors containing the RAR{gamma} ligand binding domain (8) . Mechanistic studies of the RARs and other members of the steroid/thyroid/retinoid superfamily of nuclear receptors demonstrate that ligand-dependent interactions with coactivator and corepressor complexes are central to control of gene transcription. RAR associates most strongly with positive transcriptional coactivators in the presence of agonist and overexpression of nuclear corepressors, such as N-CoR and SMRT, inhibits basal transcription through the RARs (3 , 4 , 27 , 28) . The coactivators and corepressors are linked in turn to histone acetylase and deacetylase enzymatic activity, respectively, suggesting that derepression of chromatin structure is part of the mechanism by which RARs elevate gene transcription (29) . It is attractive to speculate that retinoid inverse agonists autonomously regulate gene expression through enhanced binding of nuclear corepressors, although ligand-mediated release of coactivators is also a possible mechanism as in the case of CAR-ß (11) . Indeed, our ongoing investigations into AGN 193109 receptor interactions show that it enhances N-CoR binding to RAR{gamma}.5

The antiestrogenic activity of tamoxifen has been shown to require N-CoR activity, providing direct evidence for the importance of corepressors in antagonist activity by nuclear receptor ligands (4) . As in the case of MRP-8 induction by AGN 193109 in ECE16–1 cells, there are also examples of the induction of gene expression by antiestrogens. The ER receptor antagonist raloxifene is a potent and active inducer of transforming growth factor ß3 expression in the osteosarcoma cell MG63, whereas estradiol (E2) is weakly active and antagonizes the raloxifene effect (30) . A similar reversal of ligand effects, in which estrogen antagonists induce gene expression but agonists do not, has been reported for the quinone reductase gene A (31) . Promoters have been defined for both genes that convey reversed pharmacology, but neither has a direct binding site for ER as found in E2-inducible genes (31 , 32) , suggesting that the reversed pharmacology observed may involve indirect regulation of other transcriptional factors by ER. In the case of the TR, interaction of N-CoR and SMRT with TR has been shown to induce expression of a group of genes that are normally suppressed by T3, and thyroid hormone-resistant TR binds N-CoR and SMRT more tightly and induces this same group of genes more effectively (33 , 34) . Conceivably, a TR inverse agonist would have the same inductive effect.

NF-IL6 (C/EBPß), which is strongly expressed during epidermal keratinocyte differentiation (35 , 36) , was shown recently to induce gene expression through binding sites in the 1.3-kb promoter for MRP-8, and RAR agonists appear to inhibit NF-IL6 activity and suppress transcription from the MRP-8 promoter (37) . We propose that under certain circumstances, such as we observe in ECE16–1 cells, inverse agonist binding to RAR may actually induce NF-IL6 activity by sequestering receptor in the corepressor bound form. The mechanism by which inverse agonist suppresses MRP-8 expression in NHKs could be more complex. In the first place, MRP-8 mRNA levels in NHKs are about two orders of magnitude higher than in ECE16-1 cells relative to GAPDH,4 suggesting that the enhancer proteins involved in NHKs differ significantly from ECE16-1 cells. We find that the 1.3-kb MRP-8 promoter does not respond directly to AGN 193109 when tested in HeLa cells,6 and it is therefore also possible that additional promoter sequences may be required to fully replicate inverse agonist effects in NHKs. As an example, if RAR{gamma} binds elsewhere on the MRP-8 promoter, it clearly would have the potential to suppress gene expression in the presence of inverse agonist by recruitment of negative coactivators. This inhibition might only be significant at the high level of gene expression observed in differentiating NHKs. In any case, the more complex regulation of MRP-8 in NHKs indicates that RARs could regulate MRP-8 expression through more than one site on the MRP-8 promoter.

On the basis of inhibition of MRP-8 and stromelysin-1 in cultured keratinocytes, retinoid inverse agonists could be useful therapeutically in inflammatory conditions such as psoriasis and arthritis, where these proteins are elevated. Also, retinoid inverse agonists may have a similarly broad diversity and tissue specificity of action as antiestrogens (38) , suggesting that additional therapeutic targets for retinoid inverse agonists remain to be discovered.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Materials.
The CF145 antibody was obtained from V. Van Heyningen (39) . The monoclonal antibody B.C1 (24) to TGase 1 was obtained from Biomedical Technologies (Stoughton, MA). Charcoal-treated FCS was provided by Gemini Bioproducts. Tazarotene, TTNPB, AGN 190121, AGN 193109, AGN 193840, and AGN 193836 were synthesized at Allergan and have been characterized previously (5 , 8 , 14 , 22) .

Cell Culture.
Keratinocyte cultures were initiated from newborn foreskin on feeder layers of mitomycin C (7 µg/ml)-treated 3T3-J2 cells (40) . Epidermis was separated from dermis in 0.5 mg/ml thermolysin and briefly treated in 0.05% trypsin, 0.01% EDTA in PBS (41 , 42) . Cells were maintained in DMEM:F12 (3:1), 5% FCS, 10 ng/ml EGF, 0.18 mM adenine, 10 ng/ml cholera toxin, 0.4 µg/ml hydrocortisone, and 25 µg/ml gentamicin and passaged when preconfluent by trypsinization (40 , 43) . During first-passage culture, the 3T3 feeder layer was removed by incubation with 0.02% EDTA (5 min at 37°C.), and the cells were maintained subsequently in KGM, a low (0.15 mM) calcium defined medium (Clonetics, San Diego). Retinoid treatment was carried out on third-passage cells grown in T-25 flasks. When about 50% confluent, keratinocytes were cultured in KGM lacking hydrocortisone for 24 h before retinoid treatment. For MRP-8 and TGase 1 assays, cultures were treated for 5 more days in hydrocortisone-free medium with the addition of 10% charcoal-treated serum and retinoid (final concentration, 0.1% ethanol). K6 mRNA was measured in NHKs treated similarly with serum and retinoid for 3 days, whereas stromelysin-1 mRNA was determined after 24-h retinoid treatment in the absence of hydrocortisone and serum.

The isolation and culture of papilloma-virus immortalized cervical epithelial ECE16-1 cells has been described previously (44) . Briefly, the cells are maintained in a complete medium containing 5% FCS, DMEM:F12 (3:1), antibiotics, nonessential amino acids, 5 µg/ml bovine transferrin, 2 nM T3, 0.1 nM cholera toxin, 2 mM glutamine, 0.18 mM adenine, and 10 ng/ml EGF. To induce differentiation, the cells were switched to a defined medium modified from the above containing 0.1% BSA, 50 µg/ml ascorbic acid, and 20 ng/ml EGF but no serum or cholera toxin. Cells were treated simultaneously with retinoids dissolved in DMSO, and the concentration of DMSO in the medium did not exceed 0.1%.

Assay of MRP-8 and TGase 1 Levels.
Cultures were harvested and analyzed for the level of MRP-8 protein by Western Blot with the monoclonal antibody CF145 (39) as described (8 , 12) using cytosolic extracts of the cultures containing equal amounts of protein. Individual bands were quantitated by densitometry, and the percentage of inhibition was calculated compared with control cells not treated with retinoid. A detergent extract of the pellet (5 min at 10,000 x g) of the original cell homogenate was obtained by sonication in the presence of 10 mM Tris-Cl (pH 7.5), 1 mM EDTA, 0.3% NP40, 1 mM DTT, and 5 µg/ml each of leupeptin and antipain. TGase activity was assayed by [3H]putrescine incorporation into dimethylated casein after the detergent extract was cleared (10 min at 10,000 x g; Ref. 12 ). TGase activity was immunoprecipitated with the antibody B.C1 (24) bound to Sepharose A beads (Pharmacia). Hybridoma culture medium or a control medium containing DMEM plus 5% FCS was preincubated with Sepharose A beads (1 h at 4°C). The beads were washed, and detergent extracts were incubated with beads for 2 h at 4°C. Beads were removed by centrifugation, and the supernatant was assayed for TGase activity. ANOVA was performed with StatView (Abacus Systems, Berkeley, CA) using the Bonferroni-Dunn post-hoc test for significance where appropriate.

RNA isolation from NHKs was performed as described (12) or by extraction of cells with TRIzol (Life Technologies, Inc., Bethesda, MD). ECE16-1 total RNA was extracted in phenol/chloroform and purified on a 6 M CsCl cushion. Some RNA samples were treated with RNase-free DNase I and repurified on RNeasy columns (Qiagen) before analysis.

PCR Amplification of K6 and Stromelysin-1.
Total RNA from treated cells (1 µg) was reverse transcribed and amplified by PCR. Band intensity following individual PCR cycles was determined by ethidium bromide staining on agarose gels (14) . Primers for K6 (5'-ACA TCA TGA TGT AAT CAC CAC-3' and 5'-ATA CAG GCT TTG TAC ATC ATA-3') amplified a 270-bp fragment, and primers for stromelysin (14) and GAPDH (Stratagene) amplified 546- and 600-bp fragments, respectively. Each result was typical of at least two experiments.

Quantitation of MRP-8 mRNA Levels.
Real time PCR of reverse-transcribed total RNA was performed in the presence of an internal oligonucleotide (Synthetic Genetics, La Jolla, CA) 5'-labeled with 5'-fluorescein phosphoramidite and 3'-labeled with 6-carboxytetramethylrhodamine, succinimidyl ester (TAMRA). Loss of fluorescence quenching due to hydrolysis of the labeled oligonucleotide by 5'-3' endonuclease activity of Taq polymerase (45) was continuously quantitated by an ABI Prism 7700 (Applied Biosystems, Foster City, CA). PCR was performed with AmpliTaq Gold reagents (Perkin-Elmer) with cDNA from 0.2 µg of total RNA in the presence of 5 mM MgCl2, 0.3 µM primer, and 0.1 µM probe. The threshold cycle, at which signal was elevated above background (46) , was used to quantitate relative mRNA levels for MRP-8 and GAPDH based on values for serial 2-fold dilutions of control RNA. Total RNA giving a significant signal due to chromosomal DNA contamination (analysis of sample without reverse transcriptase treatment) was further purified by DNase treatment as described above. MRP-8 mRNA levels were normalized by the GAPDH content of each sample and compared as percentage of RNA levels in untreated controls. The MRP-8 primers, 5'-TAT CAT CGA CGT CTA CCA CAA GTA CTC-3' (forward) and 5'-TAC TCT TTG TGG CTT TCT TCA TGG-3' (reverse), span 245 bp of the 282-bp MRP-8 coding sequence. The labeled oligonucleotide for MRP-8 was 5'-TAC TCT TTG TGG CTT TCT TCA TGG-3'. The GAPDH primers used were: 5'-TGA GCA CAG GGT ACT TTA TTG ATG GTA-3' (forward) and 5'-CCC TCC TCA CAG TTG CCA TGT A-3' (reverse), and the labeled probe was 5'-ACA AGG TGC GGC TCC CTA GGC C-3'.


    Acknowledgments
 
We thank Dr. V. Van Heyningen (Edinburgh) for the CF145 antibody, Dr. H. Green (Harvard) for the 3T3-J2 feeder cell strain, and L. Munoz for help in preparing the manuscript.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Allergan, 2525 Dupont Dr., P.O. Box 19534, Irvine, CA 92623-9534. Phone: (714) 246-4516; Fax: (714) 246-5578. Back

2 The abbreviations used are: RAR, retinoic acid receptor; NHK, normal human keratinocyte; K6, keratin 6; TGase 1, transglutaminase 1; RT-PCR, reverse transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TR, thyroid receptor; ER, estrogen receptor; NF-IL6, nuclear factor-interleukin 6; EGF, epidermal growth factor. Back

3 S. Nagpal and S. Patel, unpublished observations. Back

4 S. M. Thacher, T. Arefieg, C. Agarwal, R. L. Eckert, and R. A. S. Chandraratna, unpublished observations. Back

5 E. S. Klein and R. A. S. Chandraratna, unpublished observations. Back

6 D. DiSepio, R. A. S. Chandraratna, and S. Nagpal, unpublished observations. Back

Received for publication 11/ 6/98. Revision received 1/19/99. Accepted for publication 2/ 2/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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