Georges Siegenthaler

Endothelial Cells of the Human Microvasculature Express Epidermal Fatty Acid–Binding Protein

Epidermal fatty acid-binding protein (E-FABP), previously characterized in human keratinocytes, is a cytoplasmic protein of 15 kD that specifically binds fatty acids (FAs). Previous PAGE-immunoblotting studies indicated that several human tissues display an immunoreactive band with an electrophoretic mobility identical to that of E-FABP. The aim of this study was to determine in which cells, other than keratinocytes, E-FABP might be expressed. By immunohistochemistry, we show that E-FABP is expressed in endothelial cells of the microvasculature of the placenta, heart, skeletal muscle, small intestine, lung, and renal medulla. Interestingly, in lung, a tissue of endodermal origin, E-FABP staining was also localized to secretory cells, ie, Clara cells, goblet cells, and probably a subpopulation of pneumocytes. RNA isolated from cultured human umbilical vein and normal human dermal microvascular endothelial cells was analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR). Southern blotting and sequencing of the cloned RT-PCR products demonstrate that endothelial E-FABP is identical to keratinocyte E-FABP. These data suggest that E-FABP-mediated FA transport occurs at the level of the microvasculature in several FA target organs.

Terminal differentiation in cultured human keratinocytes is associated with increased levels of cellular retinoic acid-binding protein

The control of normal epithelial cell growth and differentiation by retinoids (vitamin A and its analogs) may involve, at least in part, cellular retinoid-binding proteins. In the present study the levels of cellular retinoic acid (CRABP)- and retinol (CRBP)-binding proteins were measured in cultured normal human epidermal keratinocytes. To this end cells were cultured under either low calcium (nondifferentiating) or normal calcium (differentiating) conditions; in the latter culture the cells were also separated into nondifferentiated, attached cells, and differentiating, shed, cell populations. Two different techniques [gel filtration and polyacrylamide gel electrophoresis (PAGE)] were used, for both the qualitative and the quantitative determinations of retinoid-binding proteins. Gel filtration analysis on Sephadex G75 columns showed the presence of high-molecular-weight binding sites for retinol and retinoic acid in both nondifferentiated and the differentiating keratinocytes; the nature of these binding proteins is unclear. Free CRABP was found in the cytosol of differentiating cells, but was undetectable in nondifferentiated cells. Using a more sensitive PAGE technique, very low levels of CRABP and CRBP could be detected in nondifferentiated keratinocytes. By this technique it was possible to demonstrate very high levels of CRABP only in differentiating keratinocytes; the CRBP levels were found to be very low in differentiating cells and comparable to the amount found in nondifferentiated cells. On the basis of molecular weight determinations by gel exclusion and by electrophoretic mobility, the CRABP and CRBP of cultured keratinocytes were found to be identical to CRBP and CRABP from human epidermis.

Epidermal Fatty-Acid-Binding Protein in Psoriasis, Basal and Squamous Cell Carcinomas: An Immunohistological Study

BACKGROUND In human keratinocytes, we have recently characterized a low-molecular-weight cytosolic protein of 15 kD that specifically binds fatty acids (FAs) with high affinity, the epidermal FA-binding protein (E-FABP). The distribution of E-FABP in skin diseases is not known. OBJECTIVE To localize by immunohistochemistry the expression of E-FABP in psoriasis, basal and squamous cell carcinomas in order to obtain indirect information, at the cellular level, on the transport of the FAs. RESULTS E-FABP was localized in the upper stratum spinosum and stratum granulosum in normal and non-lesional psoriatic skin. In contrast, lesional psoriatic epidermis strongly expressed E-FABP in all suprabasal layers, like nonkeratinized oral mucosa. The basal layer did not express E-FABP reactivity in any of these samples. Accordingly, basal cell carcinomas were E-FABP negative whereas only well-differentiated cells of squamous cell carcinomas expressed E-FABP. CONCLUSION It is unlikely that E-FABP plays a significant role in FA uptake by basal cells. Our data rather indicate that E-FABP expression is related to the commitment of keratinocyte differentiation and that the putative role of E-FABP should not be restricted to the formation of the skin lipid barrier. Since the pattern of E-FABP expression mimics cellular FA transport, our results suggest that lesional psoriatic skin and oral mucosa have a higher metabolism/transport for FAs than normal and non-lesional psoriatic epidermis.

Probable interaction between S100A7 and E-FABP in the cytosol of human keratinocytes from psoriatic scales

The overexpression of E-FABP and S100A7 in lesional psoriatic skin suggests a possible link with this hyperproliferative skin disease. In order to investigate a role for the proteins in this disease, the purifications for both proteins were re-analyzed. Moreover, a specific antiserum directed against purified human S100A7 was generated. By SDS-PAGE immunoblotting we show that E-FABP and S100A7 are expressed in cultured human differentiating keratinocytes and confirm their overexpression in psoriatic scales. Gel filtration and non-denaturing PAGE revealed that S100A7 co-purified with E-FABP, indicating an association between the two proteins. Ion-exchange chromatography resulted in the dissociation of the complex. Finally, immunoprecipitations using antiserum against E-FABP revealed that S100A7 co-immunoprecipitated with E-FABP from protein extracts of psoriatic scales. These data indicate that E-FABP and S100A7 might form a complex in the cytosol of human keratinocytes.

Isotretinoin differs from other synthetic retinoids in its modulation of human cellular retinoic acid binding protein (CRABP)

Isotretinoin differs from acitretin and R0137410 by its striking sebostatic effect in acne after oral, but not topical, administration. The reason for this is not yet understood.

Retinol-binding protein in human serum: Conformational changes induced by retinoic acid binding

Polyacrylamide gel electrophoresis and isoelectrofocusing followed by immunoblotting technique with an anti-human retinol-binding protein (RBP) serum were used to study holo-RBP and apo-RBP in human plasma. Three observations were made the technique allowed for the first time to directly and quantitatively analyse holo- and apo-RBP. Holo-RBP represented 97.86 +/- 0.78% and apo-RBP 1.94 +/- 0.73% of the total RBP. All-trans-retinoic acid (RA) was found to bind to apo-RBP and to significantly modify the tertiary structure of the protein; this raises the question of RBP involvement in the transport of RA. reconstitution of holo-RBP using apo-RBP from delipidized serum was achieved only after its incubation with natural all-trans-retinoids such as retinol, 3-dehydroretinol and retinoic acid but not with synthetic analogs of retinoic acid (13-cis-retinoic acid, TMMP, 13-cis-TMMP, TTNPB). It appears that RBP has a structure specificity for natural retinoids.

Antibacterial Activity of Retinaldehyde against Propionibacterium acnes

Background: Retinaldehyde has been shown to exert antibacterial activity in vitro. Aim: This study evaluates the effect of retinaldehyde on Propionibacterium acnes both in vivo and in vitro. Methods: Microbial minimal inhibitory concentrations (MICs) of retinaldehyde and retinoic acid were determined on reference strains of P. acnes. In vivo activity of daily topical application of 0.05% retinaldehyde on the P. acnes density was evaluated after application in a single-blind randomised study. Results: MICs of retinaldehyde were 4 mg/l for P. acnes No. CIP179 and CIP53119 and 8 mg/l for P. acnes No. CIP53117. In contrast, the MICs of retinoic acid were superior to 128 mg/l for these three strains. In vivo, retinaldehyde-treated areas displayed a significant decrease in counts of viable P. acnes as compared with the untreated areas with a median decrease of 102 log P. acnes/cm2 after 2 weeks of daily application. Vehicle alone had no effect. Conclusion: The MIC of retinaldehyde against P. acnes suggests a direct antibacterial activity. Daily topical application of 0.05% retinaldehyde is associated with a clear reduction of the P. acnes density.

Overexpression of cellular retinoic acid-binding protein type II (CRABP-II) and down-regulation of CRABP-I in psoriatic skin.

Cellular retinoic acid-binding proteins (CRABPs) might exert a physiological function by controlling the intracellular levels of free retinoic acid. The aim of this study was to analyze the relative expression of CRABP-I and CRABP-II in lesional (LPS) and nonlesional (NLPS) psoriatic skin. CRABP-I and -II proteins were analyzed by a PAGE-autoradioblotting technique, and their respective mRNA were studied by RNA blot and in situ hybridization. We found that CRABP-II levels were 6-fold higher in LPS and 2-fold in NLPS as compared to normal skin, whereas CRABP-I levels were decreased in NLPS and LPS. CRABP-II mRNA were grossly overexpressed in all LPS and some NLPS specimens. These results indicate a switch to the overexpression of CRABP-II mRNA in psoriasis which induces high levels of the protein mainly in LPS; these observations may be relevant to the pathophysiology and therapy of psoriasis as CRABP-I and -II have different ligand-binding affinities.

Skin cellular retinoid-binding proteins and retinoid-responsive dermatoses

We have previously found an important increase of cellular retinoic acid-binding protein (CRABP) in psoriatic plaques whereas the cellular retinol-binding protein (CRBP) was not elevated compared to normal human skin and nonlesional psoriatic skin. In the present study we analyzed CRABP and CRBP levels in a panel of dermatoses in order to address several questions raised by the above findings. Three observations were made: CRBP showed little or no variations whereas CRABP was either normal (seborrheic keratosis, lichenification, nonlesional psoriatic and nonlesional Darier disease skin) or elevated (psoriatic plaques, lamellar ichthyosis, lesional Darier disease, pityriasis rubra pilaris, keratosis pilaris); high levels of CRABP might indicate a greater sensitivity of the lesions to systemic synthetic retinoids with a carboxyl group in the C15 position, and systemic administration of etretin increased the levels of CRABP but not CRBP. These observations suggest that CRABP might be the receptor for synthetic retinoids in the skin and that its analysis might be useful in monitoring retinoid therapy.

Human Skin Retinoid-Binding Proteins and Therapy with Synthetic Retinoids: A Still Unexplained Link

Human skin contains several proteins that could bind retinoids. One is the retinol-binding protein (RBP, the plasma carrier of natural vitamin A, retinol, which was found to also bind retinoic acid (RA) in vitro. A polyacrylamide gel electrophoresis technique followed by protein immunoblot analysis with an antiserum specific against RBP was developed. This technique allowed us to study the binding of several natural and synthetic retinoids to the plasma RBP; it was found that only natural retinoids bind to RBP in vitro; RA binding was found to induce major conformational changes in the protein. The technique was then used for the study of RBP in human epidermal and dermal extracts; this showed that RBP degradation with loss of binding properties for retinol occurred within the epidermis. A polyacrylamide gel electrophoresis binding assay was developed for studying cellular retinol-(CRABP) and retinoic acid-(CRABP) binding protein in human skin cytosolic extracts. CRABP was (1) found at much higher levels in normal epidermis than in the dermis (2); dramatically elevated in psoriatic plaques and in some retinoid-responsive dermatoses, and (3) up-modulated by both topical and systemic administration of natural and synthetic retinoids. Such alterations were not observed for CRBP. From these and other observations presented here, it appears that CRABP should be one of the first candidates to be carefully analyzed in the search of the cellular receptor for the mediation of the synthetic retinoids pharmacological effects.