Purification and characterization of cholesterol sulfotransferase from rat skin.

Previous work has demonstrated that the activity of the enzyme cholesterol sulfotransferase is rapidly and dramatically increased upon squamous differentiation of a variety of epithelial cells in culture, including epidermal keratinocytes. As a step toward understanding the molecular mechanisms underlying this differentiation-related change, we now report the partial purification and characterization of this enzyme activity from rat skin. Supernatant solutions from rat skin homogenates were subjected to a series of column chromatography steps including anion exchange, gel filtration, chromatofocusing and hydrophobic interaction chromatography. The purification procedure resulted in cholesterol sulfotransferase activity purified 2,700-fold with a 11% recovery. The most purified preparation yielded a major Coomassie blue-stained band on denaturing polyacrylamide gel electrophoresis of an apparent molecular weight (MW) of 40,000 Da. Photoaffinity labeling with the donor substrate, 3'-phosphoadenosine-5'-phospho-[35S]-sulfate resulted in a single radiolabeled protein band on denaturing polyacrylamide gel electrophoresis, again of apparent MW 40,000 Da, strongly suggesting that the major Coomassie blue-stained band in the most purified preparation is the cholesterol sulfotransferase protein. Among 3beta-hydroxysteroids with a A5 double bond that were tested, each served as a substrate, while androgens, estrogens, corticosteroids, p-nitrophenol and DOPA did not serve as substrates. Apparent Michaelis constants for the 3beta-hydroxysteroid substrates ranged from 0.6 to 8 microM.

Correlation of carotenoid production, decreased membrane fluidity, and resistance to oleic acid killing in Staphylococcus aureus 18Z.

Staphylococcus aureus is susceptible to killing by host-derived fatty acids. Studies were performed to test for a correlation between carotenoid production by S. aureus and protection against oleic acid. Oleic acid killing of cells grown in carotenoid expression medium was determined as the dosage of oleic acid in 2 M NaCl-2 mM EDTA that would kill 20% of the cells in 60 min at 37 degrees C (i.e., the 20% lethal dose). Compared with the wild-type strain (18Z), a carotenoid-deficient mutant strain (18Z-76) and strain 18Z grown in a medium that suppressed carotenoid production both showed increased sensitivity to oleic acid. Spontaneous revertants of strain 18Z-76 that regained the ability to produce carotenoids were as resistant to oleic acid as the wild-type strain. Oleic acid was shown by fluorescence polarization to decrease polarization values. Lower polarization values indicate a more-fluid membrane. To determine whether protection against oleic acid killing might depend on carotenoid stabilization of membranes, fluorescence polarization values were determined for strains showing different levels of carotenoid production. An indirect correlation was found between membrane fluidity and carotenoid production. We were able to conclude that there is a direct correlation between carotenoid production (i.e., cell pigmentation), cell membrane stability, and resistance to oleic acid-induced cell killing.

Inhibition of growth and squamous-cell differentiation markers in cultured human head and neck squamous carcinoma cells by beta-all-trans retinoic acid.

Vitamin A and some of its metabolites such as beta-all-trans retinoic acid (RA) have been implicated in the regulation of differentiation of normal and malignant epithelial cells in vivo and in vitro. In the present study the effects of RA on the growth and differentiation of 7 cell lines derived from human head and neck squamous-cell carcinomas (HNSCCs) were examined. RA (greater than 0.01 microM) inhibited the proliferation in monolayer culture of 6 of 7 HNSCC cell lines. One cell line (UMSCC-35) was very sensitive, 5 (UMSCC-10A, -19, -30, -22B and HNSCC 1483) were moderately sensitive, and 1 (HNSCC 183) was insensitive. Three of the cell lines (UMSCC-22B, -30, and HNSCC 1483) were capable of forming colonies in semisolid medium--a capability that was suppressed by RA. The HNSCC cell lines expressed various levels of the squamous-cell differentiation markers type I (particulate, epidermal) transglutaminase (TGase) and cholesterol sulfate (CS). RA treatment (I microM, 6 days) decreased TGase activity by more than 50% in 3 (UMSCC-10A, -22B and 1483) of the 7 cell lines, and the effect on UMSCC-22B was dose-dependent. Type II TGase (soluble, tissue type) activity was detected in 3 cell lines, and after RA treatment its activity increased in HNSCC 1483 and 183 cells and decreased in UMSCC-19. Following RA treatment, CS levels decreased by 20, 25, 70, 76, 89 and 91% in cell lines UMSCC-30, -10A, 183, UMSCC-35, -22B, and HNSCC 1483, respectively. The suppression by RA of CS accumulation in the 1483 cells was dose-dependent. Cholesterol sulfotransferase activity, which is responsible for CS synthesis, was suppressed by 40-97% after RA treatment of UMSCC-19, -22B, and HNSCC 1483. Our results demonstrate that RA inhibits the growth and decreases the level of 2 squamous differentiation markers in HNSCC cells.

Action of phorbol esters, bryostatins, and retinoic acid on cholesterol sulfate synthesis: relation to the multistep process of differentiation in human epidermal keratinocytes.

This study examines the action of phorbol 12-myristate 13-acetate (PMA) on the synthesis of cholesterol sulfate in cultured normal and transformed human epidermal keratinocytes and assesses the antagonistic effects by retinoids and bryostatins on PMA action in relation to the multistep program of squamous differentiation. Treatment of normal human epidermal keratinocytes (NHEK) with PMA induces terminal cell division (irreversible growth-arrest) and causes a time- and dose-dependent increase in the incorporation of Na2(35)SO4 into cholesterol sulfate, a marker for squamous cell differentiation. This stimulation in sulfate incorporation appears specific for cholesterol sulfate and is due to increased levels of cholesterol sulfotransferase activity. The increase in cholesterol sulfate accumulation parallels the increase in transglutaminase type I, another marker for squamous differentiation. Several transformed NHEK cell lines do not exhibit increased levels of cholesterol sulfate and transglutaminase type I activity after PMA treatment, indicating that they acquired defects in the regulation of squamous differentiation. Bryostatins 1 and 2, and several diacylglycerol analogues neither inhibit cell proliferation nor increase cholesterol sulfate synthesis or transglutaminase activity, indicating that these agents do not induce terminal differentiation. In contrast, the bryostatins block the increase in cholesterol sulfate and transglutaminase activity as well as the commitment to terminal cell division by PMA. Bryostatin 1 inhibits the commitment to terminal cell division and the accumulation of cholesterol sulfate significantly even when added 8 h after PMA administration. Retinoids inhibit cholesterol sulfate accumulation and the increase in transglutaminase activity by PMA but do not affect the commitment to terminal cell division. In summary, phorbol esters induce in NHEK cells a program of squamous differentiation. This process of differentiation consists of the commitment to terminal cell division and expression of a squamous phenotype. Expression of this phenotype is accompanied by an accumulation of cholesterol sulfate and increased cholesterol sulfotransferase activity. Bryostatins 1 and 2 and retinoic acid affect this differentiation process at different stages.

Effects of bryostatins and retinoic acid on phorbol ester- and diacylglycerol-induced squamous differentiation in human tracheobronchial epithelial cells.

Previous studies have shown that normal human tracheobronchial epithelial (HBE) cells undergo squamous differentiation upon treatment with phorbol 12-myristate 13-acetate (PMA). In this study, we report that induction of this differentiation program is accompanied by an increase in the accumulation of cholesterol sulfate and in transglutaminase type I activity, two markers of squamous differentiation. Several carcinoma cell lines did not exhibit an increase in these differentiation markers after PMA-treatment and appear to have acquired a defect in the mechanism that triggers differentiation. The diacylglycerol analogue, didecanoylglycerol (diC10), was also able to induce squamous differentiation. Bryostatin 1, another activator of protein kinase C, did not induce terminal cell division or increase cholesterol sulfate accumulation or transglutaminase type I activity. Bryostatin 1 not only failed to inhibit cell proliferation and to induce differentiation but antagonized the PMA- and diC10-induced commitment to terminal differentiation. The bryostatin blocked both the PMA-induced terminal cell division as well as the expression of the two differentiation markers. Retinoids were found not to affect the PMA-induced commitment to terminal cell division but did inhibit the expression of the differentiated phenotype. Our results indicate that the bryostatins and retinoids affect the multistep process of squamous differentiation in tracheobronchial epithelial cells at two different stages.

Effect of substratum and retinoids upon the mucosecretory differentiation of airway epithelial cells in vitro.

The lining of the trachea consists of a pseudostratified, mucociliary epithelium that under a variety of conditions, such as vitamin A deficiency, toxic and mechanical injury, becomes a stratified squamous epithelium. Several in vitro cell culture models have been established to study the regulation of the mucosecretory phenotype. Such studies have indicated that the mucosecretory phenotype in tracheal epithelial cells can be modulated by substratum and the presence of retinoids. Cells grown on a collagen type I gel matrix in the absence of retinoids undergo stratification and squamous cell differentiation. Cells grown on a collagen gel matrix in the presence of retinoids express a mucosecretory phenotype. As in the normal tracheal epithelium, these cultures contain columnar, polarized cells that exhibit apical tight junctions and secretory granules. Biochemical analysis of radiolabeled glycoconjugates released into the medium indicate the synthesis of mucinlike glycoproteins. Retinoids appear to determine whether tracheal epithelial cells become committed to a pathway of squamous differentiation or to a mucosecretory pathway of differentiation. The collagen gel matrix appears not to determine the commitment of the pathway of differentiation but allows the expression of the secretory phenotype in retinoic acid-treated cultures. The mechanisms by which retinoids and substratum modulate differentiation in tracheal epithelial cells is still poorly understood. It is clear that differentiation into squamous or mucous cells requires the activation and suppression of different genes. In the case of retinoids, the alterations in gene activity may be mediated by the nuclear retinoic acid receptor. In summary, in tracheal epithelial cells the substratum and extracellular matrix in conjunction with hormonal factors such as retinoids determine the ultimate function of these cells.

Increased cholesterol sulfate and cholesterol sulfotransferase activity in relation to the multi-step process of differentiation in human epidermal keratinocytes.

In this study the synthesis of cholesterol sulfate is examined in relation to the process of squamous differentiation in normal human epidermal keratinocytes (NHEK) in culture. During the exponential growth phase, NHEK cells exhibit a relatively high colony-forming efficiency and appear undifferentiated on the basis of their morphology and expression of biochemical characteristics. At confluence, the cells undergo terminal differentiation that is characterized by the commitment to terminal cell division (reduction in colony-forming ability) and expression of the differentiated phenotype. An accumulation of cholesterol sulfate accompanies this program of differentiation. This accumulation of cholesterol sulfate parallels the increase in transglutaminase type I activity and the competence to form cross-linked envelopes, whereas it precedes the "spontaneous" formation of cross-linked envelopes. Increased cholesterol sulfotransferase activity appears to account for the increase in cholesterol sulfate. The cholesterol sulfate accumulation, as well as the increase in cholesterol sulfotransferase and transglutaminase activity, are inhibited by retinoids. However, the presence of retinoids does not prevent NHEK cells from undergoing terminal cell division at confluence. Two NHEK cell lines expressing SV40-large T antigen also undergo terminal differentiation at confluence and start to accumulate cholesterol sulfate. Two other, differentiation-defective cell lines do not exhibit an increase in cholesterol sulfate at confluence. These results show that epidermal keratinocytes in culture, like cells in the epidermis, accumulate cholesterol sulfate when undergoing squamous differentiation. This program appears to consist of a retinoid-insensitive step (commitment to terminal cell division) and a retinoid-sensitive step (expression of the squamous differentiated phenotype).

Cholesterol sulfate accumulation in tumorigenic and nontumorigenic rat esophageal epithelial cells: evidence for defective differentiation control in tumorigenic cells.

In this study the regulation of squamous cell differentiation in several rat esophageal epithelial cell lines is examined. Nontumorigenic RE-149 cells undergo a program of squamous cell differentiation at confluence. This program of differentiation is influenced by the concentration of calcium in the medium and by the presence of retinoic acid. High calcium concentration stimulates terminal cell division, as indicated by a reduction in colony-forming efficiency, and increases the expression of the differentiated phenotype as indicated by an increase in cholesterol sulfate accumulation and cross-linked envelope formation. Retinoic acid inhibits squamous cell differentiation as both cholesterol sulfate accumulation and cross-linked envelope formation are reduced. Two tumorigenic cell lines, RE-B2 and RE-2BT, do not undergo squamous cell differentiation in vitro. High calcium concentration in the medium did not significantly reduce colony-forming efficiency or induce cross-linked envelope formation. High calcium concentration or retinoic acid had only a limited effect on the accumulation of cholesterol sulfate. RE-B2T cells exhibit high levels of cholesterol sulfate and cholesterol sulfotransferase activity. These levels appear no longer controlled by calcium or retinoic acid, indicating that the synthesis of cholesterol sulfate occurs in a constitutive manner. The altered responses of RE-2B and B2T cells to calcium and retinoic acid suggest that these malignant cells have acquired one or more defects in the control of differentiation.

Human bronchial epithelial cells synthesize cholesterol sulfate during squamous differentiation in vitro.

Epithelial cells of the airways can, under pathological conditions, undergo squamous metaplasia. The accumulation of cholesterol sulfate has recently been described as a new marker for squamous cell differentiation in rabbit tracheal epithelial cells. We now report that normal human bronchial epithelial cells in culture metabolically incorporated [35S]-sulfate and [3H]-mevalonate into material indistinguishable from cholesterol sulfate by the criteria of solubility in organic solvents, behavior on ion-exchange chromatography, susceptibility to solvolysis, and behavior on thin-layer chromatography before and after solvolysis. The accumulation of cholesterol [35S]-sulfate correlated well with squamous cell differentiation (as measured by cross-linked envelope formation), which occurred when the cells reached confluency. The increase in the level of cholesterol sulfate could be inhibited by the inclusion of retinoic acid in the cell-culture medium. The addition of phorbol-12-myristate-13-acetate or the presence of high Ca2+ concentration in the medium stimulated the accumulation of cholesterol sulfate. An increased activity of cholesterol sulfotransferase seems to account for the cholesterol sulfate accumulation. The original observation of cholesterol sulfate accumulation during squamous differentiation thus extends across species lines and strengthens the suggestion that the cholesterol sulfate may play an important role in this type of differentiation. Moreover, cholesterol sulfate provides a sensitive biochemical marker to study this pathway of differentiation of human bronchial epithelial cells.

Increase in cholesterol sulfotransferase activity during in vitro squamous differentiation of rabbit tracheal epithelial cells and its inhibition by retinoic acid.

It has previously been demonstrated that rabbit tracheal epithelial cells in primary culture undergo terminal differentiation at confluence to yield cornified cells much in analogy to epidermal keratinocytes and that one biochemical marker of this process seems to be the accumulation of cholesterol sulfate by the cells. The current work addresses the possible causes of this accumulation. Our studies show that the stimulation of cholesterol sulfate is paralleled by an increased activity of the biosynthetic enzyme cholesterol sulfotransferase. Squamous differentiated cells exhibited 20- to 30- fold higher levels of this enzyme activity than that in undifferentiated cells. As with other markers of squamous cell differentiation, the increase in cholesterol sulfotransferase can be prevented by the inclusion of retinoids in the cell culture medium. Inhibition of sulfotransferase levels can be observed at concentration of retinoic acid as low as 10(-11) M. The enzyme activity is optimal at pH 7 in buffers containing 0.2 M NaCl and 0.01% Triton X-100. Apparent Michaelis constants for the substrates 3'-phosphoadenosine-5'-phosphosulfate and cholesterol are 1 microM and 0.6 mM, respectively. Our results indicate that the increase in cholesterol sulfotransferase is the proximate cause for the accumulation of cholesterol sulfate in rabbit tracheal epithelial cells during squamous cell differentiation.

New benzoic acid derivatives with retinoid activity: lack of direct correlation between biological activity and binding to cellular retinoic acid binding protein.

In this paper the biological activity of several newly synthesized benzoic acid derivatives of the Am- and Ch- series, which are structurally different from retinoic acid and arotinoids, was examined. These compounds inhibit squamous cell differentiation of rabbit tracheal epithelial cells in vitro as indicated by the inhibition of transglutaminase Type I and cholesterol 3-sulfate levels. In contrast to the inhibition of differentiation in rabbit tracheal cells, these compounds induce differentiation of mouse embryonal carcinoma F9 and human promyelocytic leukemia HL60 cells. The Am- and Ch- series of compounds also affect several parameters of cell proliferation. These agents are very potent inhibitors of growth of melanoma S91 cells and inhibit the induction of ornithine decarboxylase activity by phorbol 12-myristate 13-acetate in 3T6 fibroblasts. These results show that the Am- and Ch- derivatives elicit in several cell systems the same cellular responses as retinoic acid. We propose, therefore, that they exhibit mechanism(s) of action similar to those of retinoids. Comparison of the biological response with the binding capacity to the cellular retinoic acid-binding protein shows a lack of a direct correlation.

Retinoic acid and substratum regulate the differentiation of rabbit tracheal epithelial cells into squamous and secretory phenotype. Morphological and biochemical characterization.

In this paper we show that the expression of the squamous differentiated phenotype and mucosecretory phenotype by cultured rabbit tracheal epithelial cells can be regulated by substratum and the presence of retinoic acid. Cells grown on a type I collagen gel matrix in the absence of retinoic acid stratify and undergo squamous differentiation as indicated by the appearance of squamous, cornified cells. Under these conditions cells are rich in desmosomes and heavy tonofilament bundles. These cells also express several biochemical markers for squamous differentiation such as high levels of type I transglutaminase and cholesterol sulfate. High levels of transglutaminase were also observed in areas of squamous metaplasia in tracheas of vitamin A-deficient hamsters. Treatment with retinoic acid not only blocked squamous differentiation as evidenced by the inhibition of the biochemical markers for squamous differentiation but induced the appearance of columnar, polarized cells many of which contained secretory granules. These granules stained positively with periodic acid thiocarbohydrazide and certain lectins indicating the presence of glycoconjugates. Analysis of radiolabeled glycoconjugates released into the medium indicated the synthesis of mucous glycoproteins. It appears that retinoic acid determines the pathway of differentiation whereas the collagen gel matrix is permissive for the expression of both phenotypes. The morphological and biochemical similarities between this in vitro cell system and the normal and metaplastic tracheal epithelium suggest that this rabbit tracheal epithelial cell system is a useful and relevant model to study the regulation of differentiation of the tracheobronchial epithelium.

Synthesis of mucous glycoproteins by rabbit tracheal cells in vitro. Modulation by substratum, retinoids and cyclic AMP.

One function of airway epithelium is the secretion of mucins, which comprise an important component of the mucous lining layer. We demonstrate that rabbit tracheal epithelial cells grown in primary culture incorporate [3H]glucosamine into material released into the medium which is characterized as mucin by the following criteria: high Mr, monosaccharide composition, ion-exchange behaviour different from that of glycosaminoglycans and oligosaccharides attached via N-acetylgalactosamine. The production of mucin by the cells requires growth on a substratum of collagen gel and is enhanced by retinoids in the extracellular medium. In the presence of retinoids, 8-bromo cyclic AMP and factors present in medium from 3T3 fibroblasts each further stimulate mucin production. These results indicate that an isolated epithelial-cell culture system, in the absence of nervous, mesenchymal or other tissue types, can be used to answer questions about the regulation of mucin production at the cellular level.

Accumulation of cholesterol 3-sulfate during in vitro squamous differentiation of rabbit tracheal epithelial cells and its regulation by retinoids.

Rabbit tracheal epithelial (RbTE) cells in culture undergo terminal squamous differentiation characterized by enhanced transglutaminase activity, synthesis of specific keratins, and the formation of cross-linked envelopes. The expression of each of these markers of differentiation occurs spontaneously after the cells reach confluency, but this expression can be inhibited by the inclusion of retinoids in the extracellular medium. In the current work, we demonstrate that radioactive sulfate incorporation into the organic phase of a CHCl3/CH3OH (2:1) extract of RbTE cells increases 50- to 100-fold upon differentiation and that this accumulation can be completely blocked by the inclusion of retinoic acid in the culture medium. By the techniques of specific metabolic radiolabeling, thin layer chromatography, gas chromatography-mass spectrometry, and fast atom bombardment-mass spectrometry, the sulfated amphiphile was shown to be cholesterol 3-sulfate. Cholesterol sulfate accumulation begins 1 to 2 days after the RbTE cells reach the stationary phase of growth which is the same time that other differentiated functions begin to be expressed. The inhibition of accumulation by retinoic acid is concentration-dependent and half-maximal at 5 X 10(-11) M. The relative efficacy of a series of synthetic retinoids in inhibiting cholesterol sulfate accumulation correlated with their binding to the cellular retinoic acid-binding protein. These data taken together indicate that cholesterol sulfate is a marker of squamous differentiation in RbTE cells in culture. Possible biochemical mechanisms of the regulation of cholesterol sulfate levels during differentiation are discussed.

Biochemical characterization of mucous glycoproteins synthesized and secreted by hamster tracheal epithelial cells in primary culture.

Hamster tracheal epithelial cells growing on type I collagen gel synthesize and secrete high molecular weight glycoconjugates which elute in the void volume upon Sepharose CL-4B column chromatography. The presence of any proteoglycans in this void volume material was ruled out based on both enzymatic analysis and behavior on DEAE-ion exchange chromatography. Based on the incorporation of radioactive precursors, followed by strong acid hydrolysis or neuraminidase digestion, the material was shown to contain sialic acid, fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and sulfate. Complete susceptibility to papain digestion and reductive beta-elimination suggests that the material consists of O-linked glycoproteins. The identification of N-acetylgalactosaminitol in the beta-eliminated oligosaccharides confirms this notion. The molecular weight of the oligosaccharides following beta-elimination ranges from 4,000 to 15,000. We conclude that the high molecular weight glyconjugates produced by hamster tracheal epithelial cells in primary culture are mucous glycoproteins based on size, sensitivity to alkaline borohydride treatment, and monosaccharide composition. Further characterization of these mucous glycoproteins showed both size and charge microheterogeneity among molecules. Detailed structural analysis of oligosaccharides of these mucous glycoproteins is currently under way.

Glucose starvation alters lipid-linked oligosaccharide biosynthesis in Chinese hamster ovary cells.

The effect of glucose deprivation on the synthesis of lipid-linked oligosaccharides by Chinese hamster ovary cells has been studied. When these cells are placed in serum-free Dulbecco's minimal essential medium devoid of glucose, there is a rapid cessation of the synthesis of the usual Glc3Man9GlcNAc2 lipid-linked oligosaccharide and the accumulation of a smaller species with the composition Man9GlcNAc2. This latter compound is the glucosylated and transferred to protein where it is subsequently processed. These findings demonstrate that Chinese hamster ovary cells utilize an alternate glycosylation pathway when deprived of glucose.