Effects of all-trans retinoic acid on UVB-irradiated human skin substitute

Retinoids are frequently used for treatment of photodamaged skin. We wished to find out whether photodamage could be attenuated by applying all-trans retinoic acid (RA) during repetitive irradiation. For this purpose, we used human cutaneous cells and tissue: pure monolayer cultures containing either keratinocytes or fibroblasts, and human skin substitute (SS) containing both cell types. All cultures were exposed to 8 mJ/cm² of UVB and were immediately treated with RA (0, 1.5, or 3 μM). The irradiation and RA treatment protocol was repeated until the cells of the nonirradiated culture had reached confluence. In the irradiated SS, RA preserved the structure (epidermal stratification and differentiation) and ultrastructure (well-organized intermediate filaments and desmosomes) in a state comparable to that observed in nonirradiated SS. As well RA maintained secretion of basement membrane components (laminin and type-IV collagen). Following irradiation, cutaneous cells also displayed more proliferative capacity when SS was treated. In the irradiated monolayer cultures, RA maintained the proliferative capacity of fibroblasts and decreased their differentiation whereas the opposite effect was seen on keratinocytes. In conclusion, RA clearly helps protect human skin against photodamage induced by repeated exposure to UVB. J. Cell. Physiol. 181:14–23, 1999. © 1999 Wiley-Liss, Inc.     

The molecular cloning and expression of two CRABP cDNAs from human skin

Retinoic acid (RA) is known to have a profound effect on the growth and differentiation of human epidermal cells in vivo and in vitro. One of the proteins thought to be involved in mediating the action of RA is the cellular retinoic acid-binding protein (CRABP). We have used PCR technology to generate cDNAs for two distinct CRABPs from human skin and skin-derived cells. One is highly homologous to the CRABP I cDNAs previously cloned from bovine and murine sources. The second shares extensive deduced amino acid homology with CRABP II, a protein recently described in newborn rat and embryonic chick. Although both mRNAs can be detected in neonatal foreskin, CRABP II mRNA is the predominant one in this tissue, as well as in cultured newborn fibroblasts and keratinocytes. Northern blot analysis showed CRABP II mRNA level was only slightly reduced by addition of 10(-6) or 10(-5) M RA to cultures of neonatal foreskin-derived fibroblasts, as was the CRABP I mRNA level in cultured human gut epithelial cells. In contrast, expression of CRABP II mRNA by cultured neonatal keratinocytes was strongly downregulated by RA. We conclude that CRABP II is the predominant CRABP in human skin, at least in the newborn period, and that it is differentially regulated in fibroblasts versus keratinocytes. Our data are consistent with a role for CRABP in regulating the amount of RA delivered to the nucleus.     

A Ca(2+)-dependent protein kinase with characteristics of protein kinase C in leaves and mesophyll cell protoplasts from Digitaria sanguinalis: possible involvement in the C(4)-phosphoenolpyruvate carboxylase phosphorylation cascade

We have shown previously that fibroblasts derived from fat or dermal tissue differ in their functional properties, such as proliferation rate and contractile properties. To study these differences further, two-dimensional electrophoresis (2D PAGE) was performed on proteins isolated from cultured subcutaneous fat and dermal fibroblasts. The 2D gels were screened for proteins that were differentially expressed in all donors (n = 5). Five protein spots were subjected to further analysis by mass spectrometry. Two proteins could be identified: brain acid soluble protein 1 (BASP1) and cellular retinoic acid binding protein-II (CRABP-II). CRABP-II is of interest in terms of re-epithelialisation and was clearly expressed in dermal fibroblasts but not in fat fibroblasts. Real time PCR was performed to confirm the 2D data on CRABP-II. The CRABP-II mRNA level was significantly increased in dermal tissue and cultured dermal fibroblasts compared to fat tissue and cultured fat-derived fibroblasts, respectively. The mode of action of CRABP-II in skin is to mediate retinoic acid activity. Retinoic acid is known to inhibit migration and to stimulate differentiation of keratinocytes. The expression of CRABP-II by dermal fibroblasts implicates a role for these fibroblasts in wound re-epithelialisation, in contrast to subcutaneous fat-derived fibroblasts.     

Mouse conceptuses have a limited capacity to elevate the mRNA level of cellular retinoid binding proteins in response to teratogenic doses of retinoic acid.

In these studies, we wished to determine the effect of teratogenic doses of retinoic acid on the expression of cellular retinoic acid binding protein I (CRABP-I) mRNA, cellular retinoic acid binding protein II (CRABP-II) mRNA, cellular retinol binding protein I (CRBP-I) mRNA, and cellular retinol binding protein II (CRBP-II) mRNA in mouse conceptuses. Levels of CRABP-II mRNA and CRBP-I mRNA were modestly elevated (2.5-fold and 1.5-fold, respectively) in 9-day gestation conceptuses following treatment of dams with 100 mg/kg b.w. of retinoic acid. These levels were elevated by 6 hr following treatment and remained elevated until 48 and 24 hr, respectively. Two other retinoids, etretinate and retinoyl beta-glucuronide, also moderately elevated CRABP-II mRNA and CRBP-I mRNA levels in conceptuses. In contrast, the levels of CRABP-I mRNA in the conceptuses remained unaffected by treatment with any of these three retinoids. These results demonstrate that conceptuses have a limited capacity to elevate the cellular retinoid binding proteins mRNA levels and presumably the synthesis of their respective proteins in response to high, teratogenic doses of retinoic acid. As a result, an excess of free retinoic acid becomes available to the nuclear retinoic acid receptors, which may lead to inappropriate gene expression and eventual maldevelopment.     

Ultraviolet irradiation of human skin causes functional vitamin A deficiency, preventable by all-trans retinoic acid pre-treatment

We report here that ultraviolet irradiation substantially reduced the mRNA and protein of the two major nuclear retinoid receptors, RAR-gamma and RXR-alpha, in human skin in vivo. Pre-treatment with retinoic acid mitigated this loss of nuclear retinoid receptors. Ultraviolet irradiation caused a near-total loss of retinoic acid induction of two RAR/RXR target genes, cellular retinoic acid binding protein-II and RA 4-hydroxylase, but did not affect 1,25-dihydroxyvitamin D3 induction of the vitamin D receptor/RXR-regulated gene vitamin D 24-hydroxylase. In effect, ultraviolet irradiation causes a functional vitamin A deficiency that may have deleterious effects on skin function, contributing to skin photo-aging and carcinogenesis.     

Low synthesis of retinoic acid due to impaired cytochrome P450 1a1 expression in mouse xeroderma pigmentosum fibroblasts

New tumor formation was suppressed by retinoic acid (RA) administration in xeroderma pigmentosum (XP) patients who have a defect in nuclear excision repair. However, the inhibition is not due to enhanced removal of UV-damaged DNA. These results prompted us to investigate whether or not RA metabolism is abnormal in XP fibroblasts and what the underlying mechanism is. Compared with wild type fibroblasts, low activities of RA synthesis were determined on HPLC in mouse fibroblasts lacking XP group A (XPA) gene and UV-induced XPA deficient cancer cells. Moreover, we observed an impaired expression of cytochrome P450 1a1 in XPA deficient fibroblasts by RT-PCR and a decreased expression of retinoic acid receptor gamma in XPA deficient cancer cells by Western blotting. Finally, pre-treatment of RA isoforms significantly protected the XPA deficient fibroblasts from UV-induced death. These results suggest that decreased structure activity of RA synthesis, resulting from impaired mRNA expression of cytochrome P450 1a1 may, at least together with UV irradiation, involve in skin carcinogenesis in XP patients.     

Effect of a medium with a low serum content on protein synthesis and secretion and RNA and DNA synthesis in a skin fibroblast culture from patients with rheumatoid arthritis and systemic scleroderma

Synthesis and secretion of protein, as well as synthesis of RNA and DNA by skin fibroblasts of patients with systemic sclerodermia (SSD) and rheumatoid arthritis (RA) upon prolonged culturing of fibroblasts in the medium with low (0.5-1%) serum content differ markedly in their direction and intensity from protein, RNA and DNA synthesis by skin fibroblasts of healthy donors (HD) and by fetal fibroblasts. It has been found that skin fibroblasts of patients with RA and SSD, as well as those of HD, secrete 75-80% of protein synthesized by fibroblasts de novo upon their culturing in DMEM medium with 1% human serum. Under the same conditions, on days 2-5 of culturing, RNA synthesis in the fibroblasts of patients with RA and SSD was increased 3-4-fold, while DNA synthesis was increased 2-3-fold. Collagenolytic and caseinolytic activity in the culture medium of skin fibroblasts from HD and patients with RA reached maximal levels on days 3-5. High protein secretion was observed in DMEM serum-free medium in the presence of vitamin mixture upon culturing skin fibroblasts of patients with SSD. The results obtained show that skin fibroblasts from HD differ in their functional activity from those of patients with rheumatic disorders. It might be suggested, therefore, that the mechanism of protein secretion plays an important role in the maintenance of constant intracellular protein levels in resting cells.     

Cellular retinoic acid-binding protein type 2 mRNA is overexpressed in human psoriatic skin as shown by in situ hybridization.

In situ hybridization with full length mouse cellular retinoic acid-binding protein type 1 and cellular retinoic acid-binding protein type 2 cDNA derived RNA probes showed overexpression of cellular retinoic acid-binding protein type 2 mRNA in lesional hyperplastic psoriatic skin whereas cellular retinoic acid-binding protein type 1 mRNA was undetectable. This suggests that the previously reported increase of cellular retinoic acid-binding protein in psoriatic epidermis corresponds to increased translation of cellular retinoic acid-binding protein type 2 gene. Cellular retinoic acid-binding protein types 1 and 2 mRNAs were not detectable in normal epidermis; however, type 2 message was detected in non-hyperplastic, non-lesional skin of psoriatic patients thus before altered epidermal differentiation and hyperplasia are morphologically detectable.     

Metabolism of glycoproteins and fibronectin in skin fibroblasts of patients with rheumatoid arthritis

The distribution in the cellular monolayer of the de novo synthetized pre-labeled glycoproteins and fibronectin upon culturing of fibroblasts in the medium with low serum content was analyzed. It was found that in rheumatoid arthritis (RA) the amount of total glycoproteins on the surface and within fibroblasts is higher and in the extracellular matrix is lower than in skin fibroblasts of healthy donors (HD). However, the amount of pre-labeled fibronectin on the surface of skin fibroblasts from patients with RA was considerably lower than in those from HD This finding as well as a rapid decrease in the amount of pre-labeled fibronectin in the extracellular matrix of RA fibroblasts is indicative of a more rapid metabolism of this protein in RA. In the skin fibroblasts from HD there was a practically uniform decrease in the amount of pre-labeled fibronectin in the cellular monolayer. The presence of caseinolytic activity in the culture medium even upon the first day of cell culturing in the serum-free medium, as well as the effect of various proteinase inhibitors on glycoprotein content in the cellular monolayer provide evidence that the rate of glycoprotein and fibronectin metabolism, especially in connective tissue cells of patients with RA, might possibly be determined not only by the level of their synthesis but also by the level of proteolytic activity in the connective tissue cells.     

Purification and partial characterization of a novel binding protein for retinoic acid from neonatal rat

This report describes the purification and partial characterization of a novel retinoic acid-binding protein (CRABP-II) from neonatal rat pups. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-cellulose, and high performance liquid chromatography (HPLC) on a DEAE 5PW column. Two retinoic acid-binding peaks were resolved at the DEAE-cellulose step, with CRABP-I in the major peak and CRABP-II in the minor peak. Apparent homogeneity was achieved for both binding proteins after the HPLC step. CRABP-II consists of a single polypeptide, migrating with an apparent Mr of 15,000 in sodium dodecyl sulfate-polyacrylamide gel. It has an isoelectric point of 5.0. The dissociation constant for CRABP-II of retinoic acid was estimated to be 65 nM by fluorescence titration. Amino-terminal sequence analysis showed that CRABP-II has a distinct sequence, while the CRABP-I sequence is exactly identical to that of the rat testis CRABP. Despite the extensive sequence homology between CRABP-I and CRABP-II, antibodies directed against CRABP-I did not cross-react with CRABP-II.