Retinoic acid (RA) ()is an important regulator of normal epidermal cell homeostasis(1) . For many years, RA and synthetic retinoids have been used to treat cystic acne, psoriasis, and certain epithelial malignancies. Recent studies in mice and humans have demonstrated that topical RA improves the wrinkled appearance of sun-damaged skin(2, 3, 4) , as well as post-inflammatory hyperpigmentation (5) . In vitro, RA inhibits differentiation of keratinocytes (KCs)(6, 7) . The biological effects produced by RA are believed to be mediated all or in part by two families of nuclear receptors, retinoic acid (RARs) (8, 9, 10, 11) and retinoid X (RXRs) receptors(12, 13, 14) .

RARs and RXRs are ligand-dependent transcription factors that bind to cis-acting DNA sequences called RAREs, which are composed of directly repeated hexameric half-sites with consensus sequences (5`-PuG(G/T)TCA-3`), within the transcriptional regulatory regions of target genes and thereby regulate the rate of transcription of these genes (for review see (15) and (16) and references therein). RARs and RXRs are members of the steroid/thyroid hormone receptor superfamily, each of which consists of three major functional domains, an N-terminal transactivation domain (AF-1), a central DNA binding/dimerization domain, and a C-terminal multi-functional domain, which is involved in ligand binding, dimerization, and transactivation function (AF-2)(13, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) . The RAR family is composed of RAR, , and and their isoforms(27, 28, 29) , which recognize two natural stereoisomers of RA, all-trans RA (tRA) and 9-cis RA (9cRA)(8, 30, 31) . The RXR family consists of three members, , and (13, 14) , which are activated exclusively by 9cRA(30, 31) . Recently, synthetic retinoids such as SR11237 and SR11217 have also been identified as RXR-selective ligands(32) .

In cell-free systems, RARs form heterodimers with RXRs(13, 33, 34) . On the other hand, RXRs are able to form not only homodimers but also heterodimers with other members of the steroid/thyroid hormone receptor superfamily including peroxisome proliferator-activated receptor, thyroid hormone receptor, vitamin D receptor, apoAI regulatory protein, and chicken ovalbumin upstream transcription factor(15, 18, 19, 35, 36, 37, 38) . Interestingly, RAREs with different spacing between half-sites have been found to be selectively activated by heterodimers and/or homodimers formed among overexpressed RARs and RXRs. For example, in vitro, RARE present in both human and mouse RAR2 gene promoters(39, 40) , which is a DR5 core motif composed of two half-sites separated by 5 base pairs, binds RARRXR heterodimers efficiently in a ligand-independent manner (13, 33, 34, 41) and in vitro translation-derived and 9cRA-activated RXR homodimers to a certain degree(35) . In contrast, retinoid X response element (RXRE) in the rCRBPII gene promoter(42) , which is a DR1 core motif consisting of five consensus half-sites spaced from each other by one nucleotide, interacts efficiently with not only RARRXR heterodimers in a ligand-independent way(35, 38) but also in vitro translation-derived and 9cRA-activated RXR homodimers(26, 35) . A synthetic reporter gene tk-CAT containing RARE was activated by overexpressed RARs and/or RXRs in various cell lines(34, 41) , whereas the same reporter gene containing RXRE was transactivated by only RXRs but not RARs in certain cell types(34, 38, 42, 43) .

However, in living tissues, endogenous levels of the RAR and RXR members and their isoforms are tightly controlled(14, 27, 28, 29) . Recently, Dolléet al.(44, 45, 46, 47) and others (14, 48, 49) have shown that developing mouse embryos display distinct spatio-temporal expression patterns for the transcripts of each RAR and RXR member. The transactivation properties and differential expression pattern of these receptors suggest that the pleiotropic effects of retinoids may be in part due to combinatorial dimerization of various RAR and RXR members whose expression is ultimately controlled in a cell type-specific way(50) . In addition, receptor activity may be further regulated by cell-specific levels of natural retinoids such as tRA and 9cRA, which have been shown recently to be interconverted in mammalian cell lines(30, 31, 51) .

In adult human epidermis and cultured KCs, mRNAs for RAR and and RXR and were found, with RAR and RXR being the predominant species(14, 52, 53, 54, 55) . To date, very little is known about the DNA binding and transactivation properties of these endogenous RAR and RXR proteins in adult human epidermal KCs, although induction of RARE activity by retinoids was observed in human foreskin KCs (56) and transformed epidermal KCs(57) . In this study, we took advantage of the special features of two distinct RAREs mentioned above, RARE (DR5) and RXRE (DR1), to determine how at physiological concentrations RARs and RXRs act to regulate transcription in cultured normal KCs from adult human skin in response to various natural and synthetic retinoids.

EXPERIMENTAL PROCEDURES

Ligands

Reporter Gene Plasmids, Expression Vectors for RAR and , RXR, and Dominant Negative RAR and RXR Mutants

Human Skin Epidermal Biopsies

Keratinocyte Culture

Preparation of Nuclear Extracts

Overexpression of RAR and RXR in Cultured Keratinocytes

Gel Mobility Shift Assays

Transfection of Keratinocytes and CAT Assay

RESULTS

Activation of the hRAR2 Retinoic Acid Response Element (RARE) but not the rCRBPII Retinoid X Response Element (RXRE) by both tRA and 9cRA via Endogenous Retinoid Receptors in Adult Human Epidermal Keratinocytes

Figure 1: Ligand-dependent transactivation of retinoid-responsive reporter genes, RARE-tk-CAT and RARE-tk-CAT, but not RXRE-tk-CAT, by endogenous retinoid receptors in KCs. The y axis represents relative CAT activity, which is the average value from four independent experiments (n = 4), and the x axis shows types of ligands used to treat cells and reporter genes transfected. Standard errors are shown as verticalbars. Cultured KCs were transfected with reporter gene DNA, 3 µg for RARE-tk-CAT and 2 µg for RARE-tk-CAT and RXRE-tk-CAT. After transfection, cells were treated with vehicle (0.1% ethanol) or 0.1 µM tRA or 0.1 µM 9cRA for 48 h.

Keratinocyte Endogenous RAR and RXRs Bind in Vitro to RARE as RARRXR Heterodimers but not Homodimers

Figure 2: Gel electrophoretic mobility shift analysis of endogenous RAR and RXRs from human epidermis and cultured KCs and of RAR and RXR overexpressed in KCs using RARE. Resolved complexes are indicated by labeledtriangles along both sides of the gels. Antibodies used (1.2 µl for RAR Ab and 0.8 µl for RXR Ab unless indicated differently) in postincubation are shown immediately above the gels. 1 µM 9cRA was included in all binding reactions unless indicated differently immediately below gels. A, competition for formation of specific RARE-bound A complexes by wild type RARE but not that containing mutations in half-sites. Types of P-labeled probes are shown below the gel, and types and amounts of unlabeled competitor DNA (equivalent to 2.5-, 10-, 50-, and 100-fold excess) are shown on the top. In vitro binding reactions were performed with 8 µg of nuclear extracts from cultured KCs. Similar or identical results were obtained in the absence of 9cRA (data not shown). B, endogenous RAR and RXRs in nuclear extracts from human epidermis and cultured KCs bind to RARE as heterodimers but not RAR nor RXR homodimers. Types of P-labeled probes are indicated at the top, and those of nuclear extracts are indicated at the bottom. Amount of nuclear extracts used in binding reactions was 8 µg for human epidermis (lanes1-5) and cultured KCs (lanes6-10) and 2 µg for KCs containing co-overexpressed RAR and RXR (lanes 11-15). Similar or identical results were obtained in the presence of vehicle (10% ethanol) or 1 µM tRA (data not shown). Photographs containing lanes1-5, 6-10, and 11-15 are derived from autoradiography of the same gel for 90, 180, and 30 min, respectively. The lowerpanel (lanes 1`-15`) shows autoradiography of the same gel for 30 min. C, antibody titration shows that the A1 complexes are derived from two types of RXR-containing complexes interacting with RARE. Types of P-labeled probes are indicated at the top. 8 µg of nuclear extracts from human epidermis were used. D, RAR overexpressed in KCs binds to RARE preferentially as RARRXR heterodimers but not RAR homodimers, whereas RXR overexpressed alone binds to RARE as the RARRXR heterodimers and RXR homodimers with the later being the minor species (E). Types of P-labeled probes are indicated at the top, and those of nuclear extracts from transfected KCs (2 µg) are indicated at the bottom.

The protein content of the A complexes was further examined by immunological gel mobility shift assays (Fig. 2B). Postincubation of binding reaction mixtures with mouse monoclonal antibodies specific to either RAR (RAR Ab) or that recognize all three members of the RXR family (, , and ) (RXR Ab) resulted in formation of supershifted complexes B (lane8) and C (lane9), respectively, and reduction of fast migrating complexes A1 but not complexes A2 in the A complexes (in comparison with lane7), indicating that the A1 complexes contain RAR and/or RXR. The A1 and A2 complexes are too close to be completely separated from each other under our experimental conditions. Complexes B and C were also obtained with KC nuclear extracts (2 µg) containing co-overexpressed RAR and RXR, respectively (lanes13 and 14), using the same antibodies. The nature of the A2 complexes, which also specifically recognize RARE, is not known. Note that under the assay conditions (lower amounts of extracts and shorter exposure time) the A2 complexes were absent or less visible with nuclear extracts from KCs transfected with RAR and/or RXR. Interestingly, addition of RAR Ab to the RXR Ab-containing reaction mixture (lane9) further supershifted a portion of the C complexes while resulting in double-supershifted D complexes (lane10) whose amount is approximately equal to that of B in lane8. Thus, formation of the D complexes identifies RARRXR heterodimers, which were also observed with co-overexpressed RAR and RXR (lane15). Interestingly, the amount of the B complexes (lane8) is less than that of C (lane9). Furthermore, the sum of the amount of double-supershifted D complexes and that of single-supershifted C complexes migrating faster than D in lane10 almost equals that of the C complexes in lane9. These observations suggest that the C complexes in lane10 may possibly be heterodimers formed between RXRs and unidentified dimerization partners, which were supershifted by RXR Ab but not RAR Ab. Similar supershift patterns were obtained with nuclear extracts (8 µg) prepared from human epidermis (lanes1-5), although levels of RAR and RXRs bound to RARE in epidermis (lanes 1`-5`) are relatively higher than those in cultured KCs (lanes 5`-10`). Complexes analogous to A2 were not detectable in epidermis (lanes1-5). No significant differences were observed among experiments performed in the presence of vehicle (ethanol) or 1 µM 9cRA or tRA (data not shown).

To exclude the possibility that both A1 (lane3) and C (lane5) complexes not supershifted by RAR Ab result from insufficient amounts of this antibody used, an antibody titration experiment was performed with nuclear extracts from epidermis that contain higher levels of the endogenous receptors than those from cultured KCs (Fig. 2C). As little as 0.4 µl of RXR Ab completely supershifted the A1 complexes (lane4). On the other hand, increasing amounts of RAR Ab above and beyond 1.2 µl did not significantly increase the amount of the B complexes nor reduce that of A1 (compare lane9 with lane12). The simultaneous decreases of the C and D complexes in lanes10 and 14, respectively, were most likely caused by an excessive quantity of protein contained within the combined RAR and RXR antibody fluids. Similar titration data were obtained with nuclear extracts from cultured KCs.

RAR and were not detected in nuclear extracts from cultured KCs by the same assays using appropriate antibodies (data not shown), although under the same conditions RAR present at very low levels but not RAR was readily detectable in extracts from epidermis(68) . No complexes corresponding to endogenous RAR or RXR homodimers were observed with both extracts, which would have been double-supershifted by monoclonal RAR Ab or RXR Ab alone (Fig. 2B, lanes3, 4, 8, and 9).

To know whether RAR at higher concentrations is able to bind to RARE as RAR homodimers, nuclear extracts from KCs transfected with RAR and/or RXR were analyzed by gel mobility shift assays (Fig. 2D). In comparison with KCs transfected with parental expression vector pSG5 (lanes1-3), transfection of KCs with RAR alone increased levels of RARE-bound A1 complexes in the absence (lane4) or presence of tRA (lane5). Mutations affecting both half-sites in RARE abolished formation of these complexes (lane6). The increased A1 complexes apparently correspond to RARRXR heterodimers formed between overexpressed RAR and endogenous RXRs, since these complexes were completely supershifted by RAR Ab and/or RXR Ab (lanes7-9). Similar binding took place with KCs transfected with RXR alone (lanes 10-12). No complexes corresponding to RAR homodimers were observed, which would have been double-supershifted by RAR Ab alone. Combining the extracts containing overexpressed RAR (lanes4 and 5) with those containing overexpressed RXR (lanes10 and 11) synergistically increased the amount of the A1 complexes (lanes13 and 14), similar to the case with extracts containing co-overexpressed RAR and RXR (lanes16 and 17), indicating that KCs transfected with single receptors did contain high levels of RAR or RXR, respectively. Thus, RXR is required for RAR to efficiently bind to RARE, confirming the previous finding(13, 33, 34) .

Taken together, our experiments demonstrate that endogenous RAR and RXR proteins in human epidermis and cultured KCs bind to RARE in vitro almost exclusively as RARRXR heterodimers but not RAR homodimers nor RXR homodimers. Similar binding can occur when RAR is overexpressed alone or co-overexpressed with RXR. In addition, more RXR proteins versus RAR most likely bind to RARE as heterodimers formed between RXR and as yet unidentified dimerization partners. In the case with the extracts from epidermis, RAR proteins most likely contribute in part to the formation of such heterodimers(68) .

Differential Regulation of RXRE by Overexpressed RAR, RAR, and RXR

Figure 3: Differential regulation of reporter gene RXRE-tk-CAT by overexpressed RXR, RAR, and RAR in KCs. The y axis shows average CAT activity expressed as % maximal induction, and the x axis shows types of expression vectors cotransfected. Data are presented as average values derived from three independent experiments (n = 3). The open and filledboxes represent cells treated with vehicle (0.1% ethanol) or 0.1 µM 9cRA, respectively. KCs were transfected with 2 µg of reporter RXRE-tk-CAT alone or together with parental expression vector pSG5 or expression vectors for RXR, RAR, and RAR.

Endogenous and Co-overexpressed RAR and RXR Bind in Vitro to RXRE as Heterodimers but not Homodimers

Figure 4: Gel electrophoretic mobility shift analysis of endogenous RAR and RXRs from human epidermis and cultured KCs and of RAR and RXR overexpressed in KCs using RXRE. Resolved complexes are indicated by labeledtriangles along both sides of the gels. N, nonspecific complexes. Antibodies used (1.2 µl for RAR Ab and 0.8 µl for RXR Ab unless indicated differently) in postincubation are shown immediately above the gels. 1 µM 9cRA was included in all binding reactions unless indicated differently immediately below gels. A, competition for formation of specific RXRE-bound A1 complexes in KC nuclear extracts by wild type RXRE and RARE but not those containing mutations in half-sites (RXREm and RAREm2). Types of P-labeled probes are shown below the gel, and types and amounts in pmoles of unlabeled competitor DNA (equivalent to 10- and 50-fold excess) are shown on the top. In vitro binding reactions were performed with 8 µg of nuclear extracts from cultured KCs. B, endogenous and co-overexpressed RAR and RXR in KCs bind to RXRE as heterodimers but not RAR homodimers nor RXR homodimers. Types of P-labeled probes are indicated at the top, and those of nuclear extracts are indicated at the bottom. Amounts of nuclear extracts used in binding reactions were 4 µg for human epidermis (lanes1-5), 8 µg for cultured KCs (lanes 6-10), and 2 µg for mock-transfected KCs (lane 11) and transfected KCs containing overexpressed RXR (lanes 12-18) or co-overexpressed RAR and RXR (lanes 19-23). Gels including lanes1-10 were subjected to autoradiography for a duration at least four times longer than those comprising lanes 11-23, due to relatively lower levels of endogenous RAR and RXRs versus overexpressed receptors. C, competition and antibody titration analysis of overexpressed RXR bound to RXRE. Types of P-labeled probes are indicated at the bottom, and those of nuclear extracts and competitor DNA are indicated at the top. Amounts of nuclear extracts used in binding reactions were 2 µg for both mock-transfected KCs (lanes1 and 2) and transfected KCs containing overexpressed RXR (lanes3-12). Lanes1` and 2` on the left were obtained from autoradiography of lanes1 and 2 for a longer duration.

The protein content of the A1 and A3 complexes bound to RXREwt was further analyzed by immunological gel mobility shift assays. As shown in Fig. 4B, the A1 complexes (lane7) found with KC nuclear extracts were also formed with co-overexpressed RAR and RXR (lane20). Note that the A3 complexes were present at very low levels in nuclear extracts from human epidermis or KCs transfected with RAR and/or RXR under the assay conditions (lower amount of extracts and/or shorter exposure time). Postincubation of binding reaction mixtures with antibodies specific to either RAR or RXRs significantly supershifted the A1 but not the A3 complexes, resulting in complexes B (lane8) and C (lane9), respectively. Note that the amount of the C complexes is higher than that of B, similar to the case with RARE. Formation of the B complexes identifies RAR-containing heterodimers and that of the C complexes, RXR-containing heterodimers. Thus, the A1 complexes apparently correspond to RXREwt-bound endogenous RAR and/or RXRs. Complexes B and C were also obtained with co-overexpressed RAR and RXR using the same antibodies (lanes21 and 22). During postincubation, addition of RAR Ab into binding reaction together with RXR Ab further supershifted a portion of the C complexes, causing formation of D complexes (lane10), which were also obtained with co-overexpressed RAR and RXR (lane23). Formation of the D complexes identifies RARRXR heterodimers. The remaining C complexes (lane10) not double-supershifted by RAR Ab may possibly correspond to RXR Ab-associated heterodimers formed by RXRs and unidentified dimerization partners other than RAR. Binding similar to that with cultured KCs occurred with nuclear extracts prepared from epidermis (lanes1-5) although unlike cultured KCs, the C complexes in human epidermis were almost completely double-supershifted by RAR Ab (lane5). In these three types of extracts, no complexes corresponding to the RAR and RXR homodimers were found, which would have been double-supershifted by RAR Ab or RXR Ab alone. Similar results were obtained in the presence of vehicle (ethanol) or 1 µM 9cRA. The nature of the A3 complexes is not known. Whether the A3 complexes are formed by nuclear proteins involved in formation of the A2 complexes over RARE remains to be determined.

To know whether, at relatively higher levels versus RARs and other dimerization partners, RXRs are able to form RXR homodimers, RXR overexpressed alone in KCs was analyzed (Fig. 4B, lanes11-18). In the presence of vehicle, incubation of nuclear extracts containing overexpressed RXR with RXREwt gave rise to E complexes (lane12), which is absent in a control reaction performed with the same amount of nuclear extracts from mock-transfected KCs (lane11). Addition of 9cRA into the binding reaction resulted in formation of F complexes (lane15) whose mobility is lower than that of the RARRXR heterodimers (A1 in lane20). Mutations in the half-sites of RXRE (RXREm) abolished both E and F complexes (lane14). The specificity of these two complexes were confirmed by the competition experiment shown in Fig. 4C, since RXREwt (lane6) but not RXREm (lane7) specifically competed both complexes. As shown in Fig. 4B, the F complexes apparently correspond to the RXR homodimers bound to RXRE, since RXR Ab (lane17) but not RAR Ab (lane16) completely supershifted the F complexes while resulting in formation of G complexes. The mobility of the G complexes is close to that of the double-supershifted RARRXR heterodimers (D in lane23). The observation that the RXR proteins overexpressed in KCs form homodimers in a 9cRA-dependent way is consistent with the previous finding with in vitro transcribed-translated RXR (35) . The nature of the E complexes is yet unknown. These complexes found only in KCs transfected with RXR alone are not related to RAR because they were not supershifted by RAR Ab (lane16). However, as shown in Fig. 4C, although RXR Ab reduced the intensity of E to a certain extent, adding excessive amounts of RXR Ab did not completely supershift the E complexes (lanes10-12) while the F complexes were completely double-supershifted by the lowest amount (0.2 µl) of the antibody (lane10).

To determine whether RXRs overexpressed alone in KCs are also capable of binding to RARE as homodimers in addition to RARRXR heterodimers, nuclear extracts from KCs transfected with RXR alone were analyzed. As shown in Fig. 2D, overexpression of RXR increased the amount of the A1 complexes (compare lanes10 and 11 with lanes1 and 2) and resulted in E complexes (lane10 in Fig. 2D and lane1 in Fig. 2E) when RAREwt was used as a probe. The presence of tRA (Fig. 2D, lane11) or 9cRA (Fig. 2E, lane2) did not significantly alter the binding patterns. As shown in Fig. 2E, mutations in both half-sites of RARE (RAREm2) abolished formation of both A1 and E complexes (lane3). RAR Ab supershifted only a portion of the A1 complexes resulting in B complexes (lane4), while RXR Ab almost completely supershifted the A1 complexes to give C complexes (lane5). In addition, G complexes corresponding to RXR homodimers double-supershifted by RXR Ab were also detected with RARE (lane5), as in the case with RXRE. The fact that the quantity of the G complexes is very low suggests that complexes analogous to F (RXRRXR) free of RXR Ab may be masked by the E complexes and/or background signals (lane2). Addition of RAR Ab together with RXR Ab into binding reaction further supershifted a portion of the C complexes to give D complexes (RARRXR) (lane6), which co-migrate with G (RXRRXR) (lane5). In this case, C complexes not double-supershifted by RAR Ab were found again as expected (lane6). These data indicate that RXRs bind to RARE as homodimers only when its levels are much higher than other dimerization partners and that RARRXR and other RXR-containing heterodimers have higher affinity for this element than the RXR homodimers do. The idea that the RXR homodimers have lower affinity for RARE than for RXRE was further confirmed by the competition experiment shown in Fig. 4C. RAREwt (lane8) but not RAREm2 (lane9) competed specifically but less efficiently for formation of the F complexes than did RXRE (lane6).

Thus, results from these experiments clearly indicate that 1) endogenous RXRs bind to RXRE as RARRXR heterodimers but not homodimers due to the presence of RAR and other unidentified dimerization partners (at least in the case of cultured KCs), 2) RXRs are able to form RXRE-bound homodimers only when their concentrations are much higher than other dimerization partners and in the presence of 9cRA, and 3) RARE has much lower affinity for RXR homodimers than RXRE does.

Specificity and Potency of tRA, 9cRA, CD367, and SR11237 in Regulation of Transactivation of RXRE by Overexpressed RXR in KCs

Figure 5: Selective and dose-dependent activation of RA-responsive reporter genes, RXRE-tk-CAT via overexpressed RXR homodimers (A) and RARE-tk-CAT via endogenous RARRXR heterodimers (B), by tRA, 9cRA, CD367, and SR11237 in cultured KCs. The y axis represents average CAT activity expressed as % maximal response, and the x axis represents concentrations of ligand in a log scale. Labels corresponding to tRA, 9cRA, CD367, and SR11237 are indicated on the top. n refers to number of human subjects from whom KCs were prepared and analyzed independently. Standard errors are shown as verticalbars. KCs were transfected with 2 µg of RXRE-tk-CAT together with 400 ng of the RXR expression vector (A) or 3 µg of RARE-tk-CAT alone (B).

Specificity and Potency of tRA, 9cRA, CD367, and SR11237 in Regulation of Endogenous Retinoid Receptor-mediated Transactivation of RARE in KCs

RAR and RXR-derived Dominant Negative Mutants Repressed Transactivation of RARE-tk-CAT by Endogenous Retinoid Receptors in KCs

Figure 6: Suppression of endogenous RARRXR heterodimer-mediated transactivation of RARE in KCs by the RAR and RXR dominant negative mutants. The y axis shows average CAT activity in % maximal induction, and the x axis shows types of expression vectors cotransfected. dnRAR and dnRXR refer to expression vectors for the RAR and RXR dominant negative mutants, respectively. KCs were transfected with 2 µg of RARE-tk-CAT alone or together with 400 ng of expression vectors. Results were expressed as average values derived from three independent experiments (n = 3). The open and different filledboxes represent cells treated with vehicle (0.1% ethanol) or 0.1 µM tRA or 0.1 µM 9cRA, respectively. The standard errors are represented by verticalbars.

DISCUSSION

In this study, we demonstrated that natural retinoids including tRA and 9cRA strongly induced RARE (DR5) activity but not that of RXRE (DR1) in cultured KCs. This result suggests that KCs contain functional endogenous retinoid receptors that specifically activate RARE but not RXRE. Availability of monoclonal antibodies and synthetic ligands specific to RAR or RXR allowed us to identify roles of endogenous RARs and RXRs in this restricted regulation. Several lines of evidence indicate that RARRXR heterodimers but not RXR homodimers nor RAR homodimers are the major regulators of RARE and RXRE in cultured KCs.

Our in vitro binding studies clearly showed that endogenous RAR and RXR proteins in nuclear extracts from human epidermis and cultured KCs bind to RARE as RARRXR heterodimers but not RAR homodimers nor RXR homodimers. In fact, retinoid receptor-related binding activity observed in epidermis was mainly contributed by RAR and RXR, which represent 90% of total RAR and RXR proteins, respectively(68) , in good correlation with their mRNA levels previously reported(14, 52, 53, 54) . In cultured KCs, RAR was not detected by specific antibodies in gel mobility shift assays due to both its low levels and limitation on amounts of nuclear extracts that could be loaded on gels (data not shown). However, in human epidermis, low levels of RAR proteins were readily detectable by the same assays(68) . RAR was not detected in human epidermis (68) nor in cultured KCs (data not shown), consistent with the absence of the corresponding mRNA in this tissue(52) . On the other hand, RAR overexpressed in KCs also binds to RARE exclusively as RARRXR heterodimers, and this binding is quantitatively regulated by the levels of RXRs available in KCs. These data further support the notion that RXRs, as cofactors for RARs, are required for RARs to efficiently bind to targets such as RARE(13, 33, 34, 41) .

In both epidermis and cultured KCs, endogenous RXRs, which are present in relative excess versus RARs, appear to form heterodimers with unknown dimerization partners besides RARs. In addition to endogenous RARRXR heterodimers, overexpressed RAR proteins bound to RARE exclusively as RARRXR heterodimers but not RARRAR homodimers through heterodimerizing with endogenous RXRs (Fig. 2D), indicating that the unknown dimerization partners can be dissociated from RXRs by RARs. Recently, Baes et al.(71) identified a new orphan receptor, called MB67, which shares high sequence homology with the retinoid receptor families. It binds as MB67RXR heterodimers to consensus half-sites present in only RARE (DR5) but not any other direct repeats, and its activity is not regulated by retinoic acid. Their finding suggests that receptors capable of binding to RARE (DR5) are not limited to receptor dimers formed among the RAR and RXR family members. Whether the endogenous RXR-containing heterodimers other than RARRXR observed in this study correspond to dimers formed between RXR and unidentified orphan receptors present in human epidermis and cultured KCs remains to be further characterized.

The ED values of tRA, 9cRA, and CD367 in induction of RARE activity via endogenous retinoid receptors correlate well with the affinity of these ligands for RARs but not RXRs, with CD367 being the most potent(51, 58, 70, 72) . The fact that RXR-specific ligand SR11237 did not significantly activate RARE through endogenous RXRs excludes the possibility that RAR-unrelated RXR-containing heterodimers or undetectable endogenous RXR homodimers also contribute to transactivation of RARE. This result also indicates that binding of ligands to RXR in RARRXR heterodimers does not confer ligand-dependent transactivation of RARE. In vitro ligand binding assays have showed that CD367 does not interact with RXRs(68) . In this study, we found that this ligand neither induces formation of RXR homodimers in vitro nor activates RXRs in vivo. Therefore, binding of ligands such as CD367 to only RAR in RARRXR heterodimers seems to be sufficient for conferring the ligand inducibility to transactivation of RARE. In other words, occupation of the E domain of RXR by ligands is most likely not required.

Overexpression of dominant negative mutants, dnRAR and dnRXR, drastically repressed endogenous receptor-mediated induction of RARE in cultured KCs. These two mutants have been previously shown to be functional in dimerization and DNA binding but not in ligand-dependent transactivation(62) . The repression we observed here most likely resulted from formation of transactivation-deficient receptor dimers dnRARRXR or RARdnRXR involving endogenous wild type RXRs and RARs. These mutant dimers, which contain only one AF-2 domain with transactivation function preserved, most probably competed the remaining endogenous RARRXR from binding to RARE, but their own binding failed to activate this element. Thus, based on the results from both transactivation in cultured KCs and in vitro binding studies, we conclude that transactivation of RARE in KCs is mainly mediated by endogenous RARRXR heterodimers, in which RXR is required for RAR to efficiently bind to RARE, and the RAR ligand binding domain confers ligand inducibility whereas the RXR AF-2 domain without the need for bound ligands cooperates with the AF-2 domain of liganded RAR in transactivation.

Transactivation of either isolated RXRE or the natural RXRE-containing rCRBPII gene promoter was initially observed only under experimental conditions where RXRs were overexpressed alone(34, 42, 43) . Recently, Nakshatri and Chambon (38) have further demonstrated that whether RXRE is transactivated by overexpressed RXR homodimers or RARRXR heterodimers depends on cell type. In this study, we found that in contrast to RARE (DR5), RXRE (DR1) was not activated by endogenous retinoid receptors in KCs. Endogenous RARRXR heterodimers bound to but failed to transactivate this element in the presence of appropriate ligands. In vitro, when RXRE was used as a probe, 9cRA-dependent RXR homodimers were obtained with RXR overexpressed alone in KCs but not with RXR expressed at endogenous levels in both human epidermis and cultured KCs, similar to the case with in vitro transcribed-translated RXRs(26, 35) . Under the same conditions, RXR interacts with RARE mainly as heterodimers and barely as RXR homodimers. Previous in vitro binding studies have shown that RXR homodimers produced by in vitro transcription-translation were able to bind to a synthetic RARE, called DR1G, but not RXRE in the absence of 9cRA(38) . All of these observations suggest that organization and/or sequences of half-sites in RAREs may also play important roles in ligand-dependent formation of RXR homodimers.

In addition, we found that when RARs were co-overexpressed, high levels of overexpressed RXRs bound to RXRE preferentially as RARRXR heterodimers but not RXR homodimers even in the presence of 9cRA. Thus, ligand-independent interaction between RARs and RXRs appears to be much stronger than ligand-dependent interaction among RXRs themselves. In cultured KCs, RARs overexpressed alone did not significantly activate RXRE. Furthermore, overexpressed RARs were able to suppress transactivation of RXRE mediated by overexpressed RXRs, similar to the case with monkey kidney epithelial cells, CV1(42) . These observations together with those from in vitro binding studies suggest that in KCs formation of RARRXR heterodimers dominate over that of RXR homodimers, and the resulting dominant RARRXR heterodimers, which are poor activators for RXRE, may suppress RXR-mediated activation of this element by competing the remaining RXR homodimers from binding to this element, while their own binding does not produce effective transactivation. Recently, a number of groups (36, 38) have shown that in addition to RARs, chicken ovalbumin upstream transcription factor and apoAI regulatory protein are able to efficiently form heterodimers with RXRs and bind to RXRE. In this study, we also observed that in cultured KCs, unidentified proteins bound to RXRE as RXR-containing heterodimers. Taken together, we predict that in KCs, RXRs would form 9cRA-induced homodimers over RXRE only when their concentrations are much higher than those of RARs and of other dimerization partners.

Finally, our finding that in human epidermal KCs the RARRXR heterodimer-mediated nuclear retinoid signal transduction pathway dominates over that mediated by RXR homodimers in regulating transcription may be physiologically relevant to the recent finding that in contrast to tRA, SR11237 produced no detectable changes when applied on rhino mouse skin(73) . On the other hand, the fact that tRA and 9cRA showed similar potencies in activating RXR homodimers overexpressed in KCs suggests that there is efficient interconversion between these two natural ligands in these cells, similar to that observed with other continuous mammalian cell lines(30, 31, 51) . This idea was further supported by results from HPLC analysis of retinoids extracted from KCs treated with 0.1 µM tRA or 9cRA for 48 h, which revealed the presence of both ligands in either case. ()Unlike natural retinoids tRA and 9cRA, which are subjected to interconversion in living tissues such as human epidermis, synthetic retinoids SR11237 and CD367 are able to independently trigger the RXR homodimer- and RARRXR heterodimer-mediated nuclear signal pathways, respectively. Advantages of these synthetic retinoids over natural retinoids may open an avenue leading to not only tissue targeting for therapeutic purposes but can also be used to address the question of whether an RXR homodimer-mediated signal transduction pathway exists in other human tissues.

FOOTNOTES

*

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

§

Supported in part by the Dermatology Foundation-Albert Kligman Research Fellowship. To whom correspondence should be addressed: Dept. of Dermatology, University of Michigan, Kresge I, Rm. 6558, 1301 E. Catherine St., Ann Arbor, MI 48109-0528. Tel.: 313-936-1910; Fax: 313-747-0076.

()

The abbreviations used are: RA, retinoic acid; 9cRA, 9-cis retinoic acid; Ab, antibody; CAT, chloramphenicol acetyltransferase; DR1, directly repeated consensus hexameric half-sites separated by one base pair; DR5, directly repeated consensus hexameric half-sites separated by five base pairs; hRAR2, human retinoic acid 2; KC, keratinocyte; KGM, keratinocyte growth medium; m, mutant; rCRBPII, rat cellular retinol binding protein II; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response element; RXRE, retinoid X response element; tk, thymidine kinase; tRA, all-trans retinoic acid; wt, wild type; HPLC, high pressure liquid chromatography.

()

E. Duell, J. H. Xiao., and J. J. Voorhees, unpublished results.

ACKNOWLEDGEMENTS

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