Retinoid binding proteins and human skin

Pharraac. Ther. Vol. 40, No. 1, pp. 45-54, 1989 Printed in Great Britain. All rights reserved

0163-7258/89 $0.00 + 0.50 Copyright © 1988 Pergamon Press plc

Guest Editor: R. M. MAcKm

RETINOID BINDING PROTEINS AND H U M A N SKIN G. SIEGENTHALER and J.-H. SAURAT Clinique de Dermatologie, Hdpital Cantonal Universitaire, Geneva, Switzerland

INTRODUCTION Retinol-binding protein (RBP) is the specific blood carrier for the transport of retinol from liver stores to the target tissues. When bound to retinol, holo-RBP strongly interacts with transthyretin (TTR), previously called prealbumin. Cellular uptake of retinol has been proposed to be a plasma membrane RBP receptor mediated process (Goodman, 1984). Inside the cell, retinol binds to the cellular retinol-binding protein (CRBP), whereas retinoic acid, the main metabolite of retinol, is transported by the cellular retinoic acid-binding protein (CRABP). These two retinoid-binding proteins appear to be involved in the metabolism and/or function of retinoids (Chytil and Ong, 1984). There are at least three good reasons for studying the retinoid-binding proteins in the skin. Firstly, the skin is a vitamin A target tissue. Secondly, alteration of these binding proteins may be associated with diseases which respond to retinoid therapy, and lastly these binding proteins might be the specific carriers for retinoids used in dermatology. In this article we discuss recent results, obtained with new techniques developed in our laboratory, on the transport by plasma RBP of retinoids used in human therapy, the analysis of the three retinoid-binding proteins, RBP, CRABP and CRBP in normal human skin, dermis and epidermis and in oral mucosa, and the interactions of retinoids used in human therapy with the metabolism of natural retinoids (Siegenthaler and Saurat, 1987d). This review has been written before the nuclear receptors for retinoic acid were identified (Petkovich et aL, 1987; Giguere et al., 1987; Brand et al., 1988; Benbrook et al., 1988). PLASMA TRANSPORT OF RETINOIDS USED IN HUMAN THERAPY Ligand-binding studies have shown that RBP seems to be a relatively non-specific carrier. Spectroscopic studies have demonstrated in vitro that ligands such as retinol, retinaldehyde and many of their isomers, retinoic acid (RA), and fl-ionone can bind to RBP (Horwitz and Heller, 1973). In contrast to that of retinol, the transport of retinoic acid in the plasma is not well understood. The difficulties are mainly due to the low level of retinoic acid measured in plasma (about 150-fold less than retinol, Napoli et aL, 1985; De Leenheer et al., 1982). Smith et al. (1973) had suggested that albumin could be the carrier protein for RA because tritiated RA was found bound to albumin when vitamin A deficient rats were fed with labeled RA. The amount of natural RA in the human diet, however, is very low or absent and therefore such experiments are more pharmacological than physiological. The formation of retinoic acid from retinol probably occurs mainly in target tissues or cells. Chen and Heller (1977), however, have demonstrated that RA can enter the retinal pigment epithelium cells only when bound to RBP. For these reasons plasma RBP may be, in fact, a more appropriate and specific carrier for retinoic acid than albumin. Retinoids used in human therapy or, rather, their main active metabolites, are more or less analogous to RA; they possess a free carboxyl group. When compared to the natural analogs, the chemical modifications are on the ring for acitretin (TMMP, Neotegison the J.PT. 40 I--D

45

46

G. SIEGENTHALERand J.-H. SAURAT COOH CH30~

c

O

C

O

O

~ ~

H

Ro 12-7310

CHaO~

C

O

O

H

2

H

s

COOC2Hs

A.rotmoidester, Ro 13-6298 SO2C2Hs

Arofinoidmethylsu~one, Ro 15-1570

A¢itretin, Ro 10 - 1670

~

C

Etretinate, Ro 10 - 9359

Retinoicacid

HO~

O

COOH

Ro 15 - 0778 Arotinoidacid, Ro 13-7410 FIG. 1. Structures of one natural (retinoic acid) and some synthetic retinoids.

main metabolite of Tigason), on the side chain for 13-c/s-retinoic acid (Accutane, Roaccutane) and on both cycle and chain for arotinoid acid (TTNPB) (Fig. 1). Previous reports have shown that the major part of etretin, now called acitretin (Vahlquist et al., 1984) and of 13-cis-retinoic acid (Paravicini and Busslinger, 1983) was bound on serum albumin when patients were treated by these acidic retinoids. No information about the binding of analogs of retinoic acid on RBP is provided in the literature. We wondered whether retinoids used in human therapy, and particularly those with a carboxyl group, could also be transported by plasma RBP. Indeed this might give some information about either pharmacological effects or side effects of these compounds. We studied the binding of several retinoids by incubating them with delipidized human serum (Fig. 2). The proteins were then separated on a non-denaturating polyacrylamide gel electrophoresis (PAGE) in slab form. This technique separates the complex retinol-RBP (holo-RBP) from the RBP without its ligand (apo-RBP). The separated proteins were then transferred onto a nitrocellulose sheet (protein blotting) and RBP bands were revealed with an anti-human RBP serum (Siegenthaler and Saurat, 1987a and b). Holo-RBP reconstituted with retinol, 3-dehydroretinol (vitamin A2), and retinaldehyde have identical migration positions. These results were confirmed by isoelectrofocusing (IEF) and gel filtration techniques. Retinoic acid-RBP complexes migrated faster than apoand holo-RBP in the PAGE system, but at the same isoelectric point (pI) as retinol and 3-dehydroretinol in the IEF system (Siegenthaler and Saurat, 1987b). We have suggested from these observations that retinoic acid induces major conformation changes of the tertiary structure of the protein, very different from that of retinol-RBP complex. These conformational changes are not due, as previously suggested by others (Horwitz and Heller, 1973), to the interaction with the solution of the carboxyl group of the ligand. Rather a decrease of steric hindrance, or hydrophobic interactions with polyacrylamide gel, might explain the increased mobility of the RA-RBP. Incubation of delipidized

Retinoid carriers

1

2

3

4

47

5

6

7

Fro. 2. Retinoid-binding study on delipidized human serum by PAGE-immunoblotting. Samples (5 #1) of delipidized serum were incubated overnight at 4°C with or without 10/~M of different retinoids. Lane 1 delipidized serum; lane 2 with retinol. Reconstitution of holo-RBP; lane 3 with retinoic acid. RBP-RA migrates faster than holo-RBP; lane 4 with 13-cis-retinoic acid; lane 5, with acitretin; lane 6 with arotinoic acid; lane 7 with Ro 12-7310. No holo-RBP or RBP-RA reconstitution in lanes 4-7.

serum with acitretin (Ro 10-1670), 13-cis-retinoic acid or arotinoid acid (Ro 13-7410) did not produce any modification of the electrophoretic mobility of apo-RBP. This strongly suggests that these synthetic retinoids do not react with apo-RBP similarly to the natural retinoids. Because of their low affinity for RBP, synthetic retinoids used in human therapy cannot therefore be transported by RBP and be selectively delivered to the target cells. In patients treated with 13-cis-retinoic acid about 10% of the cis-isomer is transformed to the trans-isomer. Preliminary PAGE studies on the presence of all-trans-retinoic acid coupled to RBP in the serum of such patients was negative. Finally, it is interesting to note that RBP has no significant affinity for retinoic acid analogs whereas human epidermal CRABP was found to bind most of the acidic retinoids tested (Siegenthaler and Saurat, 1986a). The specificity of RBP appears to be for the retinoyl moieties and not for the polar group of the retinoid, whereas CRABP shows more specificity for the polar group than the retinoyl moieties (Wolf, 1984).

RETINOID-BINDING PROTEINS IN NORMAL H U M A N SKIN Retinoids may act through cellular retinoid-binding proteins (Chytil and Ong, 1984). Functions of such carriers may be the transport of retinoids from one cell compartment to another or to protect retinoids from a degradative system such as cytochrome P-450 (Finnen, M. J., 1987; Leo and Lieber, 1986). Some caution must be taken when measuring retinoid-binding proteins in a target tissue. Free RBP, CRABP and CRBP have low and closely related molecular weights whereas albumin and retinol-RBP-TTR complexes have high closely related molecular weights. Sucrose gradient, gel filtration and charcoal-dextran assays used for the study of these retinoid-binding proteins may not be accurate enough for separating all these proteins (Siegenthaler and Saurat, 1986b, 1985).

48

G. SIEGENTHALERandJ.-H. SAURAT THE PAGE ASSAY

In order to avoid these problems we have developed and improved a PAGE assay for analyzing separately RBP, CRBP and CRABP in tissues and especially in human skin extracts (Siegenthaler and Saurat, 1987c). We used both dermis and epidermis heat-separated or normal skin samples obtained with a keratome set at 180/~m. Incubation of the 100,000-g supernatants was performed with either tritiated retinol or retinoic acid with or without an excess of unlabeled retinoids. The samples thus obtained were applied to each well of the gel. After electrophoresis, the gels were divided into lanes, cut into 2-mm bands, and the radioactivity assessed in a liquid scintillation counter. The specific binding was calculated from the radioactivity recovered with or without the excess of the unlabeled retinoid. Depending on incubation conditions different results were obtained for retinoic acid binding sites in normal epidermis. A high concentration of ligand (500 h i ) and a long incubation time (16hr) produced several radioactive peaks corresponding to CRABP (8.5 pmol/mg prot.), albumin and one unidentified peak. When the incubation was performed at a low concentration of (3H)retinoic acid (50 nM) over a shorter time interval (1 hr; 0°C), only the CRABP peak was detected. These conditions were therefore used for charcoal-dextran assays. Dermal extracts showed three radioactive peaks corresponding to CRABP (1.5 pmol/mg prot.), 3HRA-RBP (0.8 pmol/mg prot.) and albumin complexes. The migration position of these different radioactive peaks corresponded to purified standards from human tissues. When epidermal extracts were analyzed for retinol-binding sites we found that CRBP was present at a level of 0.2 pmol/mg prot. but no binding activity was detected for plasma RBP. This confirmed our previous observations with the PAGE-immunoblotting technique (Siegenthaler and Saurat, 1987a). Recently we reported (Siegenthaler et al., 1988c) that terminal differentiation in cultured human keratinocytes is associated with increased levels of CRABP. In contrast, in dermal extracts we found a CRBP peak (0.12pmol/mg prot.), the presence of (3H)retinol-RBP (0.8 pmol/mg prot.) and albumin peaks. Again the presence of a high concentration of RBP in dermis, about 8 times that of CRBP, confirms our qualitative observations by PAGE-immunoblotting. It remains to be established whether RBP found in the dermis comes only from blood vessels. We have thus demonstrated by gel filtration (Siegenthaler et al., 1984), charcoal-dextran (Siegenthaler and Saurat, 1986a,b), and now by PAGE (Siegenthaler and Saurat, 1987a), that the epidermis shows high levels of CRABP as compared to the dermis. This suggests a distinct metabolism for retinoic acid in the epidermis and might explain why this tissue is a target for retinoids with a carboxyl group. The presence of both CRABP and CRBP in the dermis was not unexpected (Siegenthaler, 1986). Dermis contains several adnexae structures such as hair and sebaceous follicles which respond to retinoids and are derived from keratinocytes which express CRABP and CRBP. Endothelial cells forming the dermal blood vessels may also contribute to the levels of binding proteins found in this tissue. In addition, we have demonstrated by PAGE (Fig. 3a and b) that cultured human fibroblasts have very high levels of CRABP (19.2 pmoles/mg prot.). This qualitatively confirms the results obtained with gel filtration by Lacroix et al. (1981), Oikarinen et al. (1985a) and Gates et al. (1987), and extends them to CRBP, which we found to be at a level of 100 fmoles/mg prot. i.e. nine-fold less than in Gates et al. (1987). This is of interest because retinoids have been shown to suppress the production of collagenase (Bauer et al., 1982; Brinckerhoff et al., 1982) and of procollagen (Oikarinen et al., 1985b) by cultured fibroblasts. At present we have no explanation for the high levels of CRABP found in cultured fibroblasts when compared to tissue fibroblasts. PAGE IMMUNOBLOTTING:PLASMA RETINOL-BINDING PROTEIN IN SKIN AND ORAL MUCOSA

The concept that RBP is responsible for the delivery of retinol from liver to the extrahepatic action sites of vitamin A is generally accepted (Goodman, 1984). Although

Retinoid carriers

49

80 15

CRABP

60

!

x

c=)

=o

i ~ m

20

10

o -

c BP

5

ii

?-

(A) 0 0

'I

I

20

40

Number of band (2 mm)

0

I

I

20

40

(B)

Number of band (2 mr,)

FIG. 3. PAGE analysis of [3H]retinoid-binding proteins from human cultivated fibroblasts. Cytosolic fractionwas incubated with 600 nM [3H]retinoic acid (A) or [3H]retinol (B) for 16hr at 4°C in absence or in presence of a 200-fold molar excess of unlabeled retinoids.

no strong evidence has been reported that RBP leaves the blood and diffuses throughout dermis to bring retinol to epidermis, the detection of RBP in dermis and epidermis makes this concept acceptable, even if the synthesis of mRNA for RBP in tissues other than liver has been reported (Soprano et al., 1986). Interestingly, when the PAGE-immunoblotting technique was used on skin or oral mucosa extracts (Siegenthaler et al., 1988a), we found that human dermis (the mesenchymal part of the skin) contained intact RBP having the ability to bind retinol, whereas epidermis (the epithelial part of skin) showed a high content of RBP degradation products (Siegenthaler and Saurat, 1987a). These results confirm with another technique those found with the radiobinding assay (see above). It seems that degradation of RBP is very low if not absent in dermis whereas it is high in the epidermis and oral mucosa. Indeed, RBP does not diffuse throughout the epidermis in its intact holo-form, but is rather inactivated, or loses affinity, in the lowest layer of the epidermis, where RBP might give up retinol and itself become chemically modified with subsequent loss of affinity. The availability of less retinol is thought to be involved in the terminal differentiation (cornification) of the epidermis; this might be related to the lack of a functionally active form of RBP diffusing throughout the epidermis. This hypothesis is strengthened by the fact that oral mucosa epithelium, which is a non cornifying epithelium, contains a high quantity of functional RBP (Siegenthaler and Saurat, 1987a; Siegenthaler et al., 1988a). It is commonly accepted, but not proven, that after tissue delivery of retinol, RBP as the apo-form, returns in the blood vessels before its glomerular filtration. If so, both the non-functional apo-RBP and its degradation products present in the epidermis should be found in the dermis. This is not the case since dermis contains only functional RBP. It is likely that RBP remains in the epidermis where it is degraded. From these different analyses of RBP in epidermis and dermis, it appears that the detection of a binding protein depends on the type of technique used. For instance the immunoblotting technique showed the presence of immunoreactive bands of RBP in epidermis, whereas the radiobinding assay failed to detect functional RBP. This again stresses the importance of the choice of the technique used for the analysis of binding proteins. The immunoblotting technique has shown immunoreacting bands of RBP in epidermis as would have a radioimmunoassay. Only the radiobinding assay can measure the functionally active form of the binding protein and exclude all inactive forms. Both techniques are complementary.

50

G. SIEGENTHALERand J.-H. SAURAT BINDING AFFINITIES AND BIOLOGICAL ACTIVITY

PAGE analysis has shown the optimal conditions for measuring only CRABP with the charcoal-dextran technique in epidermal extracts excluding other possible retinoic acid binding sites (Siegenthaler and Saurat, 1987c). These new conditions of incubation and analysis were then applied for studying the binding of different retinoids on human epidermal CRABP. A lower dissociation constant Kd (13ni) than those previously published by us (Siegenthaler et al., 1984; Siegenthaler et al., 1986a; Siegenthaler and Saurat, 1986a) and others (Kfing et al., 1980; Puhvel and Sakamoto, 1984) was measured. Binding experiments were then designed to test the ability of some analogs of retinoic acid to compete with (3H)retinoic acid for human epidermal CRABP. As we can see from Fig. 4, when measuring the IC50, the decreasing order of competition were retinoic acid (5 x 10-8 M), arotinoid acid (6 × 10-8 M), Ro 12-7310 or trimethylhydroxy-phenyl analog of retinoic acid (7 x 10 -8 u), and acitretin (1.8 x 10 -7 M). AS expected, retinoids with a blocked carboxyl or sulfonyl group and retinoids without a carboxyl group did not compete with human epidermal CRABP. The case of 13-cisretinoic acid was more difficult to investigate because of its isomerization. However, recent experiments have shown that 13-cis-RA is a weak competitor for 3HRA-CRABP (Siegenthaler and Saurat, 1988b). It is interesting, at this stage, to compare the IC50 of these retinoids to their pharmacological effects in man. Of the four retinoids that did not compete with 3HRA binding on CRABP (Fig. 4), two have been shown to induce significant mucocutaneous symptoms characteristic of retinoid activity when given to men. These are etretinate (Ro 10-9359) and arotinoid ethyl ester (Ro 13-6298). Both are esters that are metabolized in vivo to the acidic form, which is thought to be the active compound. This is well established for etretinate--metabolized to acitretin (Paravicini et al., 1984)--and could be extrapolated for arotinoid ethyl ester. The same may apply to arotinoid methyl sulfone, although, to date, this compound has not been found to induce mucocutaneous effects of retinoid activity when given, as is appropriate for an arotinoid, in the dose range of microgrammes per kg/bw. Finally the arotinoid without a carboxyl group, Ro 15-0778, neither competed with [3H]RA binding on CRABP nor induced any sign of retinoid activity when given to human volunteers at a dosage of more than 1 g per day (personal observation). In contrast, three out of the four retinoids that were found to compete in vitro with 3HRA binding, have been given to human subjects. These are retinoic acid, acitretin and arotinoid acid. All were found to induce mucocutaneous symptoms of retinoid activity.

_

5,°,,,,

50

0

i

I

10-9

10-8

l

10-7 Concentration (M)

!

10-6

I

10-5

FIG. 4. Competitive binding of retinoids to [3H]RA-CRABP. The analysis was performed by the charcoal-dextran technique as described previously (Siegenthaler and Saurat, 1987c). (1) retinoic acid; (2) arotinoid acid, TTNPB, Ro 13-7410; (3) TM(OH)P, Ro 12-7310; (4) acitretin, TMMP, Ro 10-1670; (5) Ro 15-0778; (6) arotinoid methyl sulfone, Ro 15-1570; (7) etretinate, Ro 10-9359; (8) arotinoid ester, Ro 13-6298.

Retinoid carriers

51

However, no strict correlation was found between the binding affinity and the pharmacological potency. In early clinical trials it was noted that mucocutaneous symptoms were seen in the dose range of mg/kg bw/day when RA was given to patients. We observed that arotinoid acid (IC50 6 × 10 -8 M) induced mucocutaneous symptoms of retinoid activity in humans in the dose range of 0.5 #g/kg/day (Saurat et al., 1988), whereas acitretin, (Ro 10-1670, the main metabolite of Tegison) whose IC50 was 1.8 x 10 -7 M, induced similar symptoms in the dose range of 1 mg/kg/bw/day. This shows that the potency of a given retinoid cannot be extrapolated only from the binding affinity to CRABP. Other parameters such as initial metabolism and further clearance and stability of the retinoid towards their catabolism should be considered. As far as the aromatic retinoids are concerned it might be that the benzene ring(s) stabilize the molecule against degradation (Stephens-Jarnagin et al., 1985). CRABP LEVELS IN SKIN DISEASES A N D T H E I R M O D U L A T I O N We have recently found, using the charcoal dextran and gel filtration assay, that CRABP was dramatically increased in psoriatic lesional skin as compared to nonlesional skin, and to the skin of non-psoriatic subjects (Siegenthaler et al., 1986a). We were able to confirm this observation with the more specific P A G E analysis that showed an even higher increase (Table 1). The significance of this increased CRABP is not understood; it might be linked to the proliferative state of the epidermis and does not appear to be confined to psoriatic epidermis because it has been observed in lamellar ichtyosis and pityriasis rubra pilaris (Siegenthaler et al., 1986b). However, preliminary observations made on hyperplastic lichenified eczema, where CRABP was not elevated, suggest that factors other than hyperplasia should be considered. It is tempting to suggest that increased CRABP might be linked to the retinoid responsiveness of a given dermatosis, but this should now be carefully studied (Siegenthaler et al., 1986b). In all these studies, CRBP levels were not increased. An extremely interesting observation was the dramatic increase of CRABP as measured by the charcoal dextran assay in the nonlesional skin of psoriatic patients during therapy with acitretin (Ro 10-1670) (Siegenthaler and Saurat, 1986a). We were able to confirm and extend this observation when we used the more specific P A G E analysis of CRABP. Table 2 shows that an increase of CRABP also occurs during acitretin therapy in the nonlesional skin of patients affected with diseases other than psoriasis. In addition, it will be seen that not only acitretin, but also arotinoid acid, induces the increase of CRABP, showing that this effect is reproducible with several structurally different retinoids. A return to pretreatment levels was observed after cessation of therapy with acitretin or arotinoid acid. Preliminary time course observations suggest that this return TABLE 1. Comparison of Charcoal Dextran Assay with PAGE Assay for Skin CRABP in Identical Samples

Charcoal dextran PAGE (p mol/mg prot.) Psoriatic skin Non lesional 2.8 3.3 (n = 5) ( ± 1.2) ( +__1.5) Lesional 13.0 30.3 (n = 5) (+2.8) (+6.2) % Increase in lesional versus non lesional 364% 809% Five samples of non-lesional and lesional psoriatic skin wereassayedin parallel with charcoal dextran assay (CDA) and polyacrylamide gel electrophoresis assay (PAGE). The increase of CRABP previously detected with charcoal-dextran technique (Siegenthaler et al., 1986a) is well confirmed by the PAGE assay.

52

G. SIEGENTHALERand J.-H. SAURAT TABLE 2. Percentage of lncrease of CRABP in Non-Lesional Skin During Therapy with Synthetic Retinoids (as Compared to Pre- Treatment ) Charcoal dextran (16 hr 4°C) (1 hr 0°C) Acitretin (Ro 10-1670) Psoriasis Lichen planus Lichenified eczema Multiple carcinomas Arotinoid ethyl ester (Ro 13-6298) Psoriasis Arotinoid acid (Ro 13-7410) Psoriasis Prurigo nodularis

PAGE

160% 88% 150% 89%

257 % ND ND ND

415 % ND ND ND

172%

124%

225%

ND ND

187% 99%

ND 635%

The table shows that the increase of CRABP (measured by charcoal dextran assay 16 hr, 4°C) previously reported in non-lesional skin of psoriatic patients during therapy with acitretin (Siegenthaler and Saurat, 1986a) is also observed with other techniques, in other diseases and during therapy with other retinoids. CRBP levels were not modified.

to pretreatment levels occurs at least 15 days after withdrawal of the drug (Fig. 5). The significance of this up-modulation of epidermal CRABP by systemic administration of synthetic retinoids is not understood. It may be a specific event or it may illustrate the hyperplastic response of the epidermis induced by the retinoids. This is currently under active investigation. Topical application of either the natural ligand retinoic acid, or a synthetic retinoid, acitretin, also caused a significant increase of epidermal CRABP levels in normal volunteers. This suggests that no systemic metabolization is required for inducing this effect (Hirschel-Scholz et al., 1987). CRABP - PAGE A N A L Y S I S - NON LESIONAL SKIN - PRURIGO NODULARIS

3o

! i

Ro 13-7410

II 3smccoyx30

I

Ro 13-7410

I

4omcg/cbyx7

2O

I0

0

30

BEFORE

DURING

WS

30 AFTER

30 days DURING

FIG. 5. CRABP was studied in the non-lesional skin in two patients with prurigo nodularis before, during and after therapy with arotinoid acid, Ro 13-7410.

Retinoid carriers

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SUMMARY N e w techniques for the analysis o f p r o t e i n s with specific b i n d i n g for n a t u r a l retinoids in h u m a n p l a s m a a n d skin extracts have been developed. P o l y a c r y l a m i d e gel e l e c t r o p h o r e sis ( P A G E ) , followed by p r o t e i n b l o t t i n g with an a n t i s e r u m specific to r e t i n o l - b i n d i n g p r o t e i n (RBP), the p l a s m a c a r r i e r o f retinol, showed that: (1) retinoic acid i n d u c e d striking c o n f o r m a t i o n a l changes when b o u n d to RBP, a n d (2) n o n e o f the several synthetic retinoids used in h u m a n t h e r a p y were f o u n d to b i n d to RBP. This directly confirms a n d extends previous indirect o b s e r v a t i o n s t h a t synthetic retinoids are n o t delivered to the target o r g a n s t h r o u g h RBP. H u m a n skin extracts i n c u b a t e d with either [3H]retinol o r [3 H]retinoic acid a n d a n a l y z e d b y P A G E is a novel technique for the study o f cellular r e t i n o l - ( C R B P ) a n d retinoic a c i d - ( C R A B P ) b i n d i n g proteins; it allows one to m o r e specifically analyse these b i n d i n g p r o t e i n s a n d differentiate t h e m f r o m RBP. This technique c o n f i r m e d a n d e x t e n d e d o u r p r e v i o u s observations: (1) C R A B P is present in m u c h higher a m o u n t s in the epidermis t h a n in the dermis, whereas C R B P is d e t e c t a b l e in very low a m o u n t s in b o t h tissues, (2) a d r a m a t i c increase o f C R A B P is f o u n d in psoriatic p l a q u e s a n d (3) there is an u p - m o d u l a t i o n o f e p i d e r m a l C R A B P d u r i n g systemic o r topical synthetic retinoid t h e r a p y . W h e n the ability o f s o m e synthetic a n a l o g s o f retinoic acid to c o m p e t e with [3H]retinoic acid b i n d i n g on h u m a n skin C R A B P was studied, two i m p o r t a n t o b s e r v a t i o n s were m a d e : (1) the a n a l o g s that, when given to h u m a n subjects were p h a r m a c o l o g i c a l l y active, were f o u n d to be g o o d c o m p e t i t o r s a n d vice-versa, (2) no strict c o r r e l a t i o n was f o u n d between the IC50 a n d the p h a r m a c o l o g i c a l p o t e n c y o f the retinoid. Acknowledgements--We thank Mrs F. Jaunin and R. Hotz for expert technical assistance. We thank Drs D.

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