Cholesterol Sulfotransferase Research Articles

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.

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).

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.

Cholesterol Sulfotransferase of Newborn Mouse Epidermis

Recent studies of the epidermis in patients with recessive X-linked ichthyosis indicate that cholesterol sulfate is an important endogenous substrate for steroid sulfatase in the stratum corneum. We report here that cholesterol sulfotransferase, which converts cholesterol to cholesterol sulfate, is present in the lower living epidermis. Epidermal cytosol also sulfates phenols and some steroids, and such reactions may be important in defending against compounds absorbed percutaneously and in modulating the pharmacologic activity of topical medicaments. Sodium salicylate and sodium citrate inhibit the sulfotransferase activity noncompetitively, and inhibition of cholesterol sulfate formation may be important in the desquamative action of these topical "keratolytics."

Sulfation of cholesterol in rat kidney and liver

Supernatant fractions obtained by high speed centrifugation of rat kidney and liver homogenates incorporated sulfate- 35S into an endogenous lipid. The radioactive product was identified as cholesterol sulfate- 35S by thin-layer chromatography and by co-crystallization with authentic cholesterol sulfate. More sulfate- 35S was incorporated into the lipid fraction when non-radioactive cholesterol was added to the incubation mixture. When tritium labeled cholesterol was added to the supernatant fraction from kidney, tritium labeled cholesterol sulfate was formed. Following parenteral administration of sulfate- 35S, cholesterol sulfate- 35S was identified as the predominant radioactive lipid in the kidney and liver. These studies demonstrate the presence of cholesterol sulfotransferase activity in rat kidney and liver.