Expression of an alpha7 duplicate nicotinic acetylcholine receptor-related protein in human leukocytes.

We examined the potential expression and function of alpha7 nicotinic acetylcholine receptors (nAChRs) in leukocytes. RT-PCR with alpha7 specific primers revealed the presence of the receptor mRNA in leukocytes. Immunoblotting and immunofluorescence experiments demonstrated the expression of a protein that is recognized by alpha7 specific antibodies. However, nicotine and acetylcholine (ACh) failed to elicit current in leukocytes. Binding experiments with alpha-bungarotoxin rhodamine conjugated were negative, illustrating the absence of a high-affinity binding site. RT-PCR analysis revealed the selective expression of the dupalpha7 mRNA. These data indicate that leukocytes express in their membrane the dupalpha7 protein but its physiological role remains to be identified.

A mammalian H+ channel generated through alternative splicing of the NADPH oxidase homolog NOH-1.

Voltage-gated proton (H+) channels are found in many human and animal tissues and play an important role in cellular defense against acidic stress. However, a molecular identification of these unique ion conductances has so far not been achieved. A 191-amino acid protein is described that, upon heterologous expression, has properties indistinguishable from those of native H+ channels. This protein is generated through alternative splicing of messenger RNA derived from the gene NOH-1 (NADPH oxidase homolog 1, where NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate).

Decreases in retinol and retinol-binding protein during total parenteral nutrition in rats are not due to a vitamin A deficiency.

Perfusion feeding in rats induced a decrease in circulating retinol despite an adequate supply of vitamin A. We studied the effect of total parenteral nutrition (TPN) on the retinol specific carrier in rat, analyzing holo-RBP (bound to retinol) and apo-RBP (without retinol) in serum and in liver. Vitamin A-sufficient (A+) and -deficient (A-) rats were characterized in terms of vitamin A and RBP status and then perfused (TPN-A+ and TPN-A-) or orally pair-fed (O-A+ and O-A-) with vitamin A. In A+ rats, a decrease in serum retinol (2.6-fold) and an increase in apo-RBP was concomitant with a massive accumulation of RBP in the liver. In TPN-A rats, both circulating RBP and liver total RBP were decreased. In TPN-A+ rats, there was a decrease in circulating retinol (2.4-fold) in parallel to a decrease of serum and liver RBP protein and mRNA. We provide evidence that infused retinyl palmitate was not responsible for serum retinol and RBP decrease and that retinol depletion was not due to vitamin A deficiency. Whatever the vitamin A status, TPN may induce in rats a down-regulation of hepatic RBP synthesis, which may, at least partially, explain the alteration of retinol and RBP in serum.

Analysis of normal and truncated holo- and apo-retinol-binding protein (RBP) in human serum: altered ratios in chronic renal failure.

Retinol, the precursor of the retinoic acid hormone, is transported in the serum by a specific carrier, the retinol-binding protein (RBP). Compared to serum of healthy controls, the serum of patients with chronic renal failure (CRF) contains markedly increased levels of the RBP form truncated at the C terminal, des(182Leu-183Leu), (RBP2), which suggests that RBP2 is cleared by the kidney in healthy people but accumulates in serum of CRF patients (Jaconi S, et al. J Lipid Res 1995:36:1247-53). To understand better the mechanism of retinol transport, we have developed a new analytical strategy to analyze the various forms of RBP that circulate in the blood: RBP with and without retinol (holo- and apo-RBP, respectively), RBP bound or not to transthyretin (TTR) and to determine in which of these forms RBP2 circulates. We confirm, but now by direct measurement, that holo-RBP and, to a larger extent, apo-RBP are increased in CRF serum compared to normal serum. We also show that almost all apo-RBP and about 50% of total holo-RBP, corresponding to RBP excess in CRF serum, circulate free and are not complexed to TTR, the remaining 50% being complexed to TTR. This observation suggests that the high levels of free holo-RBP, not bound to TTR, which correspond to the increase in total RBPs measured in CRF serum, may alter the tissue uptake of retinol and be responsible for the signs of hypervitaminosis A observed in these patients. Secondly, we found that the truncation resulting in RBP2 does not alter its binding properties for retinol nor those of holo-RBP2 for TTR. We observed that the high amounts of free holo-RBP2 and holo-RBP in sera of CRF patients were low in normal serum, suggesting that these forms are cleared by the kidney in normal conditions. The possible role of free holo-RBPs is discussed in the context of retinol recycling.

Characterization of two post-translationally processed forms of human serum retinol-binding protein: altered ratios in chronic renal failure.

Retinol-binding protein (RBP) is the specific blood carrier for the transport of retinol (vitamin A) to target tissues. As the kidney is involved in RBP metabolism, the analysis of RBP species in the serum of patients with chronic renal failure (CRF) was used as a model to study possible RBP alterations. SDS-PAGE-immunoblotting analysis of normal and CRF sera shows a doublet of RBP bands (band A and band B) near 21 kDa. Mass spectrometric analysis of purified RBPs from CRF and normal sera revealed the presence not only of full-length RBP (183 residues, migrating in band A) but also two forms of RBP differing from the native form by the loss of C-terminal Leu (i.e., RBP1 (residues 1-182), migrating in band A also) and the loss of C-terminal Leu-Leu (i.e., RBP2 (residues 1-181), migrating in band B). Interestingly, RBP2 was considerably increased in the serum of CRF, whereas it was low in normal sera. In healthy retinol target-tissues and in cultured HepG2 cells, RBP2 levels were significantly and variably present compared to RBP and RBP1. We propose that these post-translationally modified forms of RBP occur in cells and that after their release into the blood circulation RBP2 is cleared by the kidney in healthy individuals but accumulates in the serum of CRF patients. RBP2 may have an important physiological role in retinol transport and/or recycling.

Topical retinaldehyde on human skin: biologic effects and tolerance.

The present study was designed to explore if *etinaldehyde, a natural metabolite of vitamin A, has any biologic activity and is tolerated by human skin. Biologic activity was shown by the induction of cellular retinoic acid-binding protein type 2 (CRABP 2) mRNA and protein; the rank order for CRABP-2 increase was retinoic acid > retinaldehyde > 9 cis retinoic acid > retinol > beta carotene. In volunteers treated 1-3 months with 0.5, 0.1, and 0.05% retinaldehyde, there was a dose-dependent and significant increase in epidermal thickness, keratin 14 immunoreactivity, and Ki67-positive cells. The area of distribution of involucrin, transglutaminase, and filaggrin immunoreactivity was also increased in a dose-dependent manner, and keratin 4 immunoreactivity was induced in the upper epidermis. In pilot clinical tolerance studies, 229 patients received topical retinaldehyde at different concentrations; the 1% preparation was tolerated by up to 70% of the treated subjects; tolerance of the 0.5% preparation was slightly better, whereas both 0.1 and 0.05% preparations applied on facial skin were well tolerated and allowed prolonged use (up to 3 years) in patients with inflammatory dermatoses. These findings indicate that topical retinaldehyde has biologic activity and is well tolerated on human skin.

Topical glucocorticosteroids modulate the expression of CRABP I and II in human skin differently.

Epidermal cells express two retinotic acid-binding proteins (CRABP I and II). Because CRABP II protein is strongly induced by topical retinoic acid, the respective roles of the two proteins in the pharmacological activity and toxicity of topical retinoids deserve particular attention. Since topical steroids diminish the irritation induced by retinoic acid (RA), whereas retinoic acid may counteract the atrophogenic effects of steroids, the possible interplay of both compounds in the expression of CRABP I and II appeared worth studying. We have analyzed the effects of topical application of triamcinolone acetonide (TA) on the retinoic acid-induced altered expression of CRABP I and II in normal human skin, at the protein and mRNA levels. We found that CRABP II protein and mRNA were strongly increased upon retinoic acid application: this induction was significantly inhibited by concomitant application of triamcinolone acetonide; a more potent steroid, difluocortolone valerate, was also found to diminish normal endogenous expression of CRABP II. In contrast, CRABP I protein was decreased by topical retinoic acid, and the down modulating effect of retinoic acid was counteracted by triamcinolone acetonide.

Effect of retinoids on follicular cells.

It has been demonstrated that topical application of all-trans retinoic acid and other retinoids can alter the hair-growth cycle in the C3H mouse model. The anagen phase is prolonged and the telogen phase is shortened. This effect is similar to the effect of minoxidil on the hair-cycle dynamics in this animal model. The levels of cellular retinoic acid binding protein measured by radioreceptor assay in whole skin of C3H mice were higher during anagen and lower during telogen. Topical application of certain retinoids caused elevated levels of cellular retinoic acid-binding protein (cRABP) in the whole skin homogenates during both phases of the cycle. Of the retinoids tested, those most effective in altering the levels of cRABP in the skin of the mice were also capable of significantly altering the hair-cycle dynamics. There appeared to be a relationship between the ability of retinoid to increase cRABP, increase 3H-thymidine incorporation, and alter the dynamics of the hair cycle. Only cRABP-II is detectable in human cultured dermal fibroblasts and dermal papilla cells. Dermal fibroblasts showed higher amounts of cRABP-II as compared to dermal papilla cells. The difference in cRABP-II expression might explain a distinct response to RA by these two cell populations. Whether the difference in expression of cRABP-II might be of physiologic importance remains to be determined. Treatment of human dermal papilla cells in culture with retinoic acid does not appear to affect proliferation, at least at the doses tested.

Characterization and expression of a novel human fatty acid-binding protein: the epidermal type (E-FABP).

Using PAGE--Autoradioblotting technique we have characterized an E--FABP in human epidermal cells that is distinct from liver-, heart-, intestine- and adipose tissue-FABPs. FABP radiobinding analysis was performed directly on protein extracts without prior partial purification. E-FABP has a Mr of approximately 15 kDa and binds oleic acid with high affinity but does not bind all-trans-, 13-cis- and 9-cis-retinoic acid nor all-trans-retinol. Expression levels of E-FABP were low in normal epidermis, higher in human cultured keratinocytes and still higher in psoriasis, a disease characterized by abnormal epidermal differentiation. These findings suggest that epidermal cells may have a distinct fatty acid metabolism compared to other tissues.

Expression of CRABP-I and -II in human epidermal cells. Alteration of relative protein amounts is linked to the state of differentiation.

The physiological role of cellular retinoic acid-binding proteins (CRABPs) may be to influence the intracellular level of free retinoic acid in the cell. In the present study two isoforms of CRABP, CRABP-I and CRABP-II were partially characterized in various human Malpighian epithelia and in human cultured keratinocytes expressing various patterns of differentiation. We have developed a new sensitive radiobinding assay using a PAGE/autoradioblotting technique which effectively separates CRABP-I and CRABP-II. This method allows the simultaneous quantification of these proteins. We show that CRABP-I and -II have similar M(r) values (15,000), but differ in their dissociation constant towards retinoic acid (Kd of 16.6 nM and 50 nM respectively), in pI (4.86 and 5.13) and in their relative mobilities (RF) on PAGE under nondenaturating conditions (RF values 0.65 and 0.44). In addition, we show that CRABP-II is the major isoform expressed in human keratinocytes, in vivo as in vitro. Furthermore, we demonstrate that CRABP-II is actually the CRABP previously studied in epidermal cells by a PAGE assay (Siegenthaler & Saurat (1987) Eur. J Biochem. 166, 209-214) and whose levels are dramatically increased by retinoic acid and its analogues in human epidermis. Keratinocytes, in the absence of full terminal differentiation, as well as hyperplasia, such as cultured human differentiating keratinocytes, psoriatic plaques, and non-keratinized oral mucosa, contained high levels of CRABP-II. CRABP-I was not detected in cultured keratinocytes, whereas normal skin (at full terminal differentiation) expressed CRABP-I and CRABP-II at a ratio of approx. 1:1.4. This value was approx. 1:17 in lesional psoriatic skin and 1:8 in oral mucosa. These observations suggest that CRABP-I and -II are regulated differently in human keratinocytes. The sharp increases in CRABP-II levels are associated with an alteration in the differentiation programme, as well as with cell response to retinoic acid overload, whereas CRABP-I might be a marker for terminal differentiation.

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.