Stanley S. Shapiro

Characterization of the action of retinoids on mouse fibroblast cell lines.

Abstract Retinoic acid affects 3T6 and 3T3 cells by inhibiting growth, causing a morphological change and increasing cell-to-substratum adhesiveness. Retinoic acid does not exert such effects on virus-transformed 3T3SV cells. Retinoic acid treatment of 3T6 cells causes a concentration-dependent increase in generation time and a reduction in saturation density. Analysis of cell surface proteins shows that a high molecular weight band of 230 000 D, corresponding to the position of the LETS glycoprotein, is more intensely labeled by iodination of cells treated with retinoic acid compared to control cells. Retinoic acid substantially stimulates the incorporation of 35SO4 into cell-associated glycosaminoglycans and causes a less dramatic increase in glycosaminoglycans excreted into the medium. The relationship between the increase in these cell surface components and the enhanced adhesiveness is discussed. A retinoic acid binding protein is detectable in the cytosol of 3T6 and 3T3 cells but not in 3T3SV cells, suggesting that the action of retinoids on these cells is mediated via this protein.

Effect of retinoic acid on chondrocyte glycosaminoglycan biosynthsis

Abstract The effect of retinoic acid on glycosaminoglycan biosynthesis was investigated in rat costal cartilage chondrocytes in vitro. At levels of 10−9 to 10−8 m retinoic acid, 35SO4 uptake into glycosaminoglycans was reduced 50%. At these low levels of retinoic acid there was no evidence of lysosomal enzyme release. The results are explained best in terms of modification of glycosaminoglycan synthesis, rather than accelerated degradation. Retinoic acid selectively modified the incorporation of 35SO4 or [14C]glucosamine into individual glycosaminoglycans fractions under the conditions studied. The relative incorporation of radiolabeled precursor into heparan sulfate (and/or) heparin increased three- to fourfold. The relative incorporation of radiolabeled precursor remained constant for chondroitin 6-sulfate, whereas incorporation into chondroitin 4-sulfate and chondroitin (and/or) hyaluronic acid decreased. Under the conditions studied, retinoic acid did not appear to be cytotoxic and did exhibit selective control over glycosaminoglycan biosynthesis. It is suggested that the decreased incorporation of 35SO4 into glycosaminoglycans at hypervitaminosis A levels of retinol may be accounted for by the presence of low levels of retinoic acid, a naturally occurring metabolite.

Retinoic acid-induced alterations of growth and morphology in an established epithelial line.

Abstract Retinoic acid induced alterations in cellular growth, morphology, and adhesion in a human intestinal epithelial line. Inhibition of growth was dependent on cell density, with the greatest inhibition of growth observed at low cell density. As cells approached confluence, the extent of inhibition was diminished. Cells grown in the presence of retinoic acid appeared elongated and showed enhanced detachability with trypsin. Control cells grew in tight clusters with maximum cell-cell contact. This is in contrast to retinoic acid-treated cells which did not grow in clusters. In addition, it was observed that retinoic acid inhibited glycosaminoglycan biosynthesis. These observations are uniquely different from those reported for non-epithelial cells.

Effect of Retinyl Acetate on Sulfated Glycosaminoglycan Biosynthesis in Dermal and Epidermal Cells in Vitro

The effects of retinyl acetate on the biosynthesis of sulfated glycosaminoglycans in dermal and epidermal cells isolated from newborn mice was investigated. Three compartments were analysed for [35S]-glycosaminoglycans; the culture medium, the cellular matrix, and the cells. The individual levels of chondroitin 4-sulfate, dermatan sulfate and heparin and/or heparan sulfate in these compartments as a function of retinyl acetate was also analysed. The addition of retinyl acetate resulted in a dose dependent increase 35SO4 incorporation in the cellular and matrix compartments of the dermis in vitro. At the optimum concentration of 1.8 X 10(-6) M, this increase was 50%. The levels of35SO4 incorporated into medium glycosaminoglycans were relatively constant. There were also changes in the levels of the individual sulfated glycosaminoglycans. The glycosaminoglycan profile was modified differently in each of the three compartments analysed. In the epidermal cells, retinyl acetate at an optimum concentration of 1.8 X 10(-6) M resulted in a dose dependent increase in 35SO4 incorporation in the cellular, matrix and medium compartments. There was no apparent change in the glycosaminoglycan profile, with heparin and/or heparan sulfate being the major sulfated glycosaminoglycan.

MODULATION OF GLYCOSAMINOGLYCAN BIOSYNTHESIS BY RETINOIDS

In conclusion, retinoids modulate phenotypic changes such as morphology, adhesion, and growth rate. These changes also result in specific alterations of glycosaminoglycans. We have observed increases in the degree of sulfation in fibroblast matrix heparan sulfate and, a change in the ratio of sulfamido to ester sulfate in matrix heparan sulfate in 407 cell surface. Matrix glycosaminoglycans have been functionally implicated in cellular interactions, and have a specific role in adhesion and growth rates. Our results are consistent with the proposed role for heparan sulfate. It is possible that some of the modulation in cellular behavior resulting from retinoid treatment may be mediated by cell surface and cellular changes in heparan sulfate and other glycosaminoglycans.

Ascorbic acid-2-sulfate sulfhohydrolase activity of human arylsulfatase A.

Pure human arylsulfatase A (EC 3.1.6.1) was found to hydrolyze ascorbic acid 2-sulfate to ascorbic acid and inorganic sulfate at rates from 200 to 2000 mumol/mg per h depending on the method of assay. This rate was lower than that observed with the synthetic substrate 4-nitrocatechol sulfate, but higher than that seen with the physiological substrate cerebroside sulfate. Extracts of cultured fibroblasts from normal subjects were also shown to hydrolyze ascorbic acid 2-sulfate; extracts of fibroblasts from patients with metachromatic leukodystrophy, known to be deficient in arylsulfatase A, did not. Similarly, hydrolysis of ascorbic acid 2-sulfate was not observed when a partially purified preparation of human arylsulfatase B was tested under a variety of conditions. Thus, in the human, arylsulfatase A appears to be the major, if not the only, ascorbic acid-2-sulfate sulfohydrolase.

Sulfated mucopolysaccharides of midgestation embryonic and extraembryonic tissues of the mouse

Abstract Incorporation of [ 35 S]sulfate into sulfated mucopolysaccharides has been characterized in midgestation mouse embryo, yolk sac, trophoblast, and decidua. Enzymatic analysis indicated that chondroitin sulfates contained approximately half of the label in embryo, trophoblast, and decidua, but less than 20% in yolk sac. While the labeled chondroitin sulfate fraction of trophoblast and decidua was mainly chondroitin-4-sulfate, only embryo contained a significant proportion of labeled chondroitin-6-sulfate. The relative incorporation into embryo chondroitin-6-sulfate was also substantially higher than that observed in four adult soft tissues. Labeled dermatan sulfate was absent from the embryo and yolk sac, but small amounts might have been synthesized by the placenta. Nitrous acid degradation studies revealed that essentially all the chondroitinase resistant MPS was N -sulfated, i.e., heparan sulfate and/or heparin. Electrophoretic profiles indicate that the bulk of the N -sulfated material resembles heparan sulfate rather than heparin. Electrophoretic heterogeneity and slow migration rates relative to standard markers suggest that the majority of labeled chondroitin sulfates may be undersulfated. The different mucopolysaccharide patterns in the various tissues may reflect their specialized properties and functions.

RETINOID EFFECTS ON SEBOCYTE PROLIFERATION

The human sebocyte model offers several advantages over the current animal models. Foremost among these is the correlation of in vitro activity with clinical results, which was not true for arotinoids in the animal models. It is also possible to study several parameters (total cell number, [3H]thymidine uptake, protein and lipid composition/synthesis, hormone response, receptor regulation, etc.) in the same system. The proliferation of isolated sebocytes is inhibited by retinoids, such as isotretinoin and tretinoin, which are known to be clinically active in human acne. Sebocytes are not responsive to the arotinoid temarotene, which is active in the aforementioned animal models and against dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma but inactive clinically in acne. Additionally, this model is not responsive to etretinate, a compound known to be active in psoriasis but inactive in acne. The in vitro model is, therefore, more predicative of clinical efficacy than the animal models alone.

Mitochondrial inclusions in keratinocytes of hairless mouse skin exposed to UVB radiation

Mitochondrial inclusions were observed in keratinocytes during an ultrastructural investigation of the skin of hairless mice exposed to UVB radiation. Mice were irradiated 3 times a week for 5–6 months with sunlamps at individual doses seldom exceeding 0.06 J/cm2. Strips of dorsal skin were processed for electron microscopic examination; blocks were sectioned to include both epidermis and dermis. Mitochondrial inclusions were observed in keratinocytes of the basal, spinous and granular layers. They were spherical in shape and of moderate and homogeneous electron density. Mitochondria toward the upper regions of the epidermis were swollen and had fragmented cristae; mitochondria in the lower areas of the epidermis usually contained smaller and less dense inclusions and intact or partially disrupted cristae. Because mitochondria are essential in providing the energy for cellular function, keratinocyte mitochondrial damage induced by UVB radiation may have serious pathological consequences. Possible mechanisms involved in mitochondrial inclusion formation are suggested.

Ascorbate-2-sulfate levels in metachromatic leukodystrophy patients.

Metachromatic leukodystrophy (MLD), an autosomal recessive neurological disease associated with myelin abnormalities, is caused by the lack of arylsulfatase A activity. In vitro, ascorbate-2-sulfate is a substrate for arylsulfataseA. The purpose of this study is to determine if there are abnormally high levels of ascorbate-2-sulfate in the urine or blood leukocytes of MLD patients. Ascorbate-2-sulfate was identified in the urine of both MLD patients and normal controls. The results show that urinary ascorbate-2-sulfate levels (micrograms per milligram of creatinine) of the total subject population were correlated negatively with the age of the subjects (r = −0.78, p < 0.01). The urinary ascorbate-2-sulfate levels of the MLD patients did not differ significantly from those of age-matched controls. Furthermore, ascorbate-2-sulfate was not detectable in the leukocytes from MLD patients. These results do not support the hypothesis that there are abnormally high levels of ascorbate-2-sulfate in MLD patients.