Transcriptional Induction of Cholesterol 7alpha -Hydroxylase by Dexamethasone in L35 Hepatoma Cells Requires Sulfhydryl Reducing Agents*

(Received for publication, September 10, 1996, and in revised form, October 24, 1996)

John D. Trawick , Shui-Long Wang , David Bell and Roger A. Davis Dagger

From the Mammalian Cell and Molecular Biology Laboratory, Department of Biology and Molecular Biology Institute, San Diego State University, San Diego, California 92182-0057

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

It is known that hepatic levels of reduced glutathione correlate with the activity of the liver-specific enzyme cholesterol-7alpha -hydroxylase. We examined the possibility that sulfhydryl reducing agents activate transcription of cholesterol 7alpha -hydroxylase. Adding dithiothreitol (DTT, 1 mM) and dexamethasone to L35 hepatoma cells increased the content of 7alpha -hydroxylase mRNA 3-fold above the levels observed with dexamethasone alone. Without dexamethasone, DTT had no affect. The addition of reduced glutathione to L35 cells demonstrated a similar potentiation of expression dependent on dexamethasone. Nuclear run-on assays showed that in the presence of both dexamethasone and DTT, the transcription of the 7alpha -hydroxylase gene was clearly increased. In contrast, by itself, dexamethasone did not cause a detectable increase in the transcription of the 7alpha -hydroxylase gene. Dexamethasone and DTT did not affect the transcription of beta -actin, suggesting a selective induction of the 7alpha -hydroxylase gene. DTT reversed repression of 7alpha -hydroxylase expression by insulin but not the repression by phorbol ester. Our data show for the first time that the sulfhydryl redox potential of the hepatocyte (i.e. level of reduced glutathione) has a marked influence on the transcription and expression of the liver-specific gene 7alpha -hydroxylase.


INTRODUCTION

The conversion of cholesterol to bile acids is the major quantitative pathway through which cholesterol is removed from mammals (1, 2). The initial step controlling bile acid synthesis is catalyzed by cholesterol 7alpha -hydroxylase (EC 1.14.13.17). This cytochrome P-450 enzyme is expressed only in the liver (3). Hepatic expression of 7alpha -hydroxylase1 provides this organ with the unique ability to take up cholesterol ester-rich lipoproteins from the plasma and to excrete cholesterol into bile in the form of bile acids and cholesterol (1, 2, 4). This liver-specific cholesterol excretory pathway may help to maintain cholesterol homeostasis. Recent studies of mice having a targeted deletion of a functional 7alpha -hydroxylase gene show that they have marked disruption of hepatic function, lipid metabolism, and premature death (5, 6). Many of these pathological effects could be relieved by supplementation with bile acids and fat-soluble vitamins, providing evidence for the essential function of bile acid production (5). Thus, it is clear that 7alpha -hydroxylase plays an essential role both in regulating cholesterol homeostasis and in digestion and absorption of lipids including fat-soluble nutrients.

Expression of 7alpha -hydroxylase varies extensively in response to diet (7, 8), hormones (9-11), diurnal variation, and the enterohepatic circulation (1, 2). Recent studies indicate that the expression of 7alpha -hydroxylase is regulated mainly through changes in gene transcription (7, 11-15).

We have used a unique line of rat hepatoma cells (L35 cells) to examine the molecular mechanisms regulating 7alpha -hydroxylase (9, 16). These cells show the unique ability to express 7alpha -hydroxylase in cultured cells at levels similar to those observed in vivo (9). Expression of 7alpha -hydroxylase by L35 cells is sensitive to dexamethasone (induced) and insulin (repressed)(16) in a manner similar to that which occurs in vivo. In this study, we examine the mechanism responsible for these changes. The results show that by itself, dexamethasone induces 7alpha -hydroxylase mRNA expression mainly by a posttranscriptional mechanism (no detectable change in transcription). However, when presented in combination with the sulfhydryl reducing agent dithiothreitol (DTT), transcription of 7alpha -hydroxylase was increased, resulting in an increased mRNA expression that was greater than that observed with dexamethasone alone. Hepatic levels of reduced sulfhydryl reagents may have a significant influence on 7alpha -hydroxylase expression.


MATERIALS AND METHODS

All reagents used for biochemical techniques were purchased from Sigma, VWR, or Fisher. Enzymes for restriction or labeling of cDNA probes were purchased from New England Biolabs or Boehringer Mannheim. Cell culture medium was obtained from Life Technologies, Inc./BRL, and serum was from Gemini. DTT and glutathione (obtained from Sigma) were stored as powdered forms (at -20 °C, desiccated without exposure to light). Immediately prior to use, each agent was dissolved in culture medium to a final concentration of 1 mM. This concentration of sulfhydryl reductant was chosen based on previous studies by our laboratory and others showing that it affects the sulfhydryl redox state of the cell as shown by changes in gene expression, the secretion of specific proteins, and the expression of 7alpha -hydroxylase without causing toxicity (see "Discussion"). The cDNA probes used for hybridizations have been described (3, 9, 16).

Cell Culture Lines and Conditions

L35 rat hepatoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 8% serum, as described (9, 16). Prior to each experiment, the medium was changed to DMEM containing 0.5% fetal bovine serum and 1% Nutridoma HU for 3 days. The medium was then changed to DMEM lacking serum but containing the indicated concentrations of additives (as indicated in the figure legends). When used, dexamethasone, dissolved in ethanol, was added to DMEM to a final concentration of 0.1 mM (9, 16). Control cells received ethanol only.

RNA Isolation and Quantitation

Cells were harvested, at the times indicated in the figure legends, by removing the culture medium and adding guanidinium isothiocyanate (18), with modifications (16). Poly(A)-containing RNA was obtained using the miniscale oligo(dT) cellulose (Collaborative Biotech type 3) method as described (16).

RNA (3-10 µg of poly(A) RNA) was loaded onto 0.8% agarose, 3% formaldehyde gels (19) and subjected to electrophoresis. The gels were blotted onto Zetaprobe GT (Bio-Rad) nylon membranes and hybridized with nick translated cDNA probes in the conditions described for Zetaprobe by Bio-Rad. After hybridization and washing, Northern blots were exposed to storage phosphor screens of a Molecular Dynamics PhosphorImager, Kodak Biomax MS film, or to DuPont Reflection Autoradiography film.

Transcription Run-On Assays of Nuclei

Transcription run-on assays were performed with modifications of the procedures outlined by Ausubel et al. (17). L35 cells, cultured as described above, were harvested by scraping the cells from the plate and pelleting the cells in a Beckman GPR centrifuge at 1000 rpm using a swinging bucket rotor for 5 min at 4 °C. The cell pellet was washed with cold, sterile PBS, and 2.5 ml of hypotonic lysis buffer (10 mM Tris, pH 7.4, 1 mM KCl, and 3.0 mM MgCl2) were added and then brought up to 5.0 ml of hypotonic lysis buffer with 0.3% Nonidet P-40. Then the tubes were incubated on ice for 5 min. The resulting nuclei were pelleted at 500 rpm for 5 min at 4 °C. They were washed in 5 ml of hypotonic lysis buffer with 0.3% Nonidet P-40 and pelleted for 5 min at 500 rpm; then the supernatant was removed. The wash was repeated using hypotonic lysis buffer without Nonidet P-40. The nuclei were flash frozen in liquid nitrogen and stored at -70 °C in 100 µl of nuclear storage buffer (40% glycerol, 50 mM Tris, 5 mM MgCl2, and 0.1 mM EDTA, pH 8.3).

The nuclear run-on transcription buffer contained 30% glycerol, 2.5 mM DTT, 1.0 mM MgCl2, 70 mM KCl, 0.25 mM each of GTP, CTP, ATP, and 100 mCi of [32P]UTP (3000 Ci/mmol). The transcription reaction was run for 15 min at 26 °C and stopped by adding EDTA, tRNA, and guanidinium isothiocyanate-RNA isolation mix. After the 32P-labeled nuclear RNA was isolated and precipitated, incorporation was estimated by binding to DE81 paper (Whatman), and equal counts of each reaction hybridized to nylon-bound unlabeled cDNAs for 7alpha -hydroxylase and beta -actin. Hybridization was performed at 65 °C in 0.015 M Tris, 0.7 M NaCl, 0.015 M EDTA, 1 × Denhardt's, 2% (w/v) Na4P2O7, 0.2% SDS, and tRNA to a final concentration of 1 mg/ml, pH 7.5. After washing, the blots were exposed to DuPont Reflection Autoradiography film.


RESULTS

DTT Potentiates the Induction of 7alpha -Hydroxylase mRNA by Dexamethasone but Has No Effect by Itself

When incubated with L35 cells for 48 h together with DTT (1 mM), dexamethasone increased the expression of 7alpha -hydroxylase to levels that were 2-3-fold greater than the levels obtained by incubating with dexamethasone alone (Fig. 1). In contrast, DTT by itself had no effect on the expression of 7alpha -hydroxylase mRNA. The increased abundance of 7alpha -hydroxylase mRNA caused by the combination of dexamethasone and DTT was specific, as demonstrated by no significant effect on the expression of beta -actin (Fig. 1). Furthermore, the effect of DTT was not associated with any sign of toxicity. Protein synthesis, cell viability, and growth were unaffected (data not shown). These data suggest that the sulfhydryl reducing agent, DTT, potentiates the induction of 7alpha -hydroxylase caused by dexamethasone.


Fig. 1. DTT potentiates the induction of 7alpha -hydroxylase by dexamethasone. L35 cells were incubated for 48 h in serum-free DMEM (control), dexamethasone (Dex, 100 µM), DTT (1.0 mM), and dexamethasone and DTT (Dex & DTT). After 48 h, the RNA was harvested and poly(A)-containing RNA was Northern blotted and hybridized with probes to 7alpha -hydroxylase and beta -actin. The multiple forms of 7alpha -hydroxylase mRNA expressed by L35 cells were estimated to be 3.6, 2.9, 2.4, and 1.7 kilobases. These forms are similar to those previously reported in L35 cells (9, 16) and in rat liver (3).
[View Larger Version of this Image (53K GIF file)]


Induction of 7alpha -Hydroxylase Expression by Dexamethasone Is Also Potentiated by Reduced Glutathione

Results similar to those obtained using DTT were obtained using the natural sulfhydryl reducing agent glutathione (Fig. 2). In the presence, but not in the absence, of dexamethasone, reduced glutathione (1 mM) induced the expression of 7alpha -hydroxylase mRNA up to 4-fold greater than the level obtained with dexamethasone alone. In the absence of dexamethasone, reduced glutathione did not induce 7alpha -hydroxylase mRNA above that observed with culture medium alone (Fig. 2). Furthermore, the increased abundance of 7alpha -hydroxylase mRNA caused by glutathione was specific, as demonstrated by no significant effect on the expression of beta -actin


Fig. 2. Reduced glutathione (GSH) causes the same effect as DTT on dexamethasone stimulation of 7alpha -hydroxylase gene expression. Reduced glutathione (GSH) was added with and without dexamethasone (Dex) to serum-free DMEM. L35 cells were incubated with these media for 48 h. RNA was harvested, blotted, and hybridized as described for Fig. 1.
[View Larger Version of this Image (66K GIF file)]


DTT Potentiates Dexamethasone Induction of 7alpha -Hydroxylase

In cells cultured in the absence of either dexamethasone or DTT, the transcription of 7alpha -hydroxylase was barely detectable, whereas the transcription of beta -actin was clearly evident (Fig. 3). Adding DTT by itself did not induce 7alpha -hydroxylase expression. Although dexamethasone by itself caused dramatic increases in the expression of 7alpha -hydroxylase mRNA (Fig. 1), in three separate experiments performed on three separate occasions, its rate of transcription was similar to that of control cells. In marked contrast, adding DTT with dexamethasone increased the rate of transcription of 7alpha -hydroxylase 4.5-fold above the level of control cells and cells treated with only dexamethasone (Fig. 3). It may be important to point out that in a fourth experiment of treating L35 cells with dexamethasone alone, the transcription of 7alpha -hydroxylase was slightly greater than control cells. However, this rate of transcription was still less than 25% of cells treated with dexamethasone and DTT (data not shown). Neither dexamethasone nor DTT (separately or together) affected the transcription of beta -actin.


Fig. 3. In the presence of dexamethasone, DTT induces transcription of the 7alpha -hydroxylase gene in L35 cells. L35 cells were treated as described in the legend to Fig. 1. After 48 h, nuclei were isolated and transcribing RNA labeled with the addition of a [32P]UTP. The labeled RNA was hybridized to blots containing excess cDNA fragments of 7alpha -hydroxylase and beta -actin.
[View Larger Version of this Image (46K GIF file)]


DTT Blocks the Insulin Repression of 7alpha -Hydroxylase in L35 Cells but Does Not Affect Repression by Phorbol Ester in L35 Cells

Previous studies show that once induced by dexamethasone, L35 cells maintain an expression of 7alpha -hydroxylase in the absence of dexamethasone for at least 48 h, the longest time period examined (16). This apparently stable expression of 7alpha -hydroxylase can be reversed by either insulin or phorbol esters (16). Studies by others show that insulin (20) and phorbol esters (21) both repress 7alpha -hydroxylase by blocking transcription. In the absence of DTT, insulin repressed the expression of 7alpha -hydroxylase but had no effect on the expression of beta -actin (Fig. 4). However, in the presence of DTT, insulin had no significant effect on the expression of 7alpha -hydroxylase or beta -actin (Fig. 4). Moreover, either in the presence or absence of insulin, DTT in combination with dexamethasone caused the same degree of induction of 7alpha -hydroxylase (Fig. 4). DTT did not affect the ability of insulin to increase protein synthesis (as determined by the incorporation of [35S]methionine into trichloroacetic acid-precipitable protein (data not shown)). Similar data were obtained using reduced glutathione (1 mM)(data not shown).


Fig. 4. DTT blocks the insulin repression of 7alpha -hydroxylase mRNA. L35 cells were plated and grown and then switched to serum-free DMEM, and the indicated reagents were added: dexamethasone (Dex, 100 µM), DTT (1.0 mM), or insulin (INS, 0.1 µg/ml). After 48 h, cells were harvested, and poly(A)-containing RNA was purified and subjected to gel electrophoresis and Northern blotting.
[View Larger Version of this Image (71K GIF file)]


In contrast to the blocking insulin repression of 7alpha -hydroxylase, DTT had no effect on the repression caused by phorbol esters (Fig. 5). Both in the absence and presence of DTT, phorbol esters repressed the expression of 7alpha -hydroxylase (Fig. 5).


Fig. 5. DTT does not block the inhibition of 7alpha -hydroxylase mRNA by phorbol 12-myristate 13-acetate. L35 cells were cultured and induced with dexamethasone (Dex). After 48 h, phorbol 12-myristate 13-acetate (PMA, 1.0 nM) and DTT (1.0 mM) were added for 3 h, after which poly(A) RNA was purified and subjected to Northern analysis. A similar experiment in which DTT was added with dexamethasone for 48 h prior to phorbol 12-myristate 13-acetate addition gave similar results (data not shown).
[View Larger Version of this Image (64K GIF file)]



DISCUSSION

Our results show that DTT affects the ability of dexamethasone and insulin to alter the expression of 7alpha -hydroxylase. In contrast, DTT by itself has no affect on 7alpha -hydroxylase expression. Furthermore, DTT did not affect the ability of phorbol esters to repress 7alpha -hydroxylase. Additional data demonstrated that the effect of DTT could be recapitulated with the endogenous sulfhydryl reducing agent glutathione (22). Together, our data indicate that in the unique hepatoma cell line (L35 cells), the relative concentration of sulfhydryl reducing agents selectively affects the ability of hormones to alter the expression of 7alpha -hydroxylase mRNA. Previous studies show that the changes in 7alpha -hydroxylase mRNA levels in L35 cells correspond to parallel changes in enzyme activity (9). In addition, the changes in 7alpha -hydroxylase mRNA levels in L35 cells also correspond to parallel changes in protein levels, determined by Western blot analysis (data not shown). The question remains as to whether our results obtained in L35 cells apply to other liver cell systems or to the in vivo situation. Although our studies are the first to show that sulfhydryl reductants affect 7alpha -hydroxylase gene transcription, there are several studies that show that sulfhydryl reductants affect the expression of 7alpha -hydroxylase enzyme activity, mRNA levels, and rates of bile acid synthesis in vivo (rats)(23) and in human hepatoma cells (23-27).

It is well established that hepatic levels of the natural sulfhydryl reductant glutathione are an important indicator of liver function (22). Hepatic levels of glutathione change in response to liver injury, inflammation, and disease. Our results showing a significant role of sulfhydryl reductants in the expression of 7alpha -hydroxylase may explain the basis for decreased levels of expression in one or more of these altered states. The level of glutathione in liver (23) and in HepG2 cells is positively correlated with bile acid synthesis (23) as well as 7alpha -hydroxylase mRNA levels (24, 25). Induction of 7alpha -hydroxylase was caused by altering glutathione levels (23) and by increasing the level of L-cysteine (also a sulfhydryl donor)(26). Increased cysteine levels were related to induction of bile acid synthesis by taurine in HepG2 cells (27). Our data show that in L35 cells, sulfhydryl reducing agents by themselves cannot increase transcription of 7alpha -hydroxylase (Fig. 3). These cells require the addition of dexamethasone to uncover the transcriptional activation of the 7alpha -hydroxylase gene.

The redox state of the cell, or the addition of sulfhydryl reducing agents such as DTT, can affect several components of the transcription apparatus. The binding affinity of some transcription factors, including the glucocorticoid receptor (28), SP1 (29), and c-Myb (30) may be increased by the addition of DTT to cultured cells. Not only is the DNA binding affinity of thyroid transcription factor I increased by DTT addition (31) but so are its dimerization and activity in inducing gene expression in FRTL-5 cells (31). Moreover, in lung adenocarcinoma A549 cells, DTT can activate the transcription factor NF-kappa B (32). Interestingly, in these cells, DTT had no affect on SP1 (32). NF-kappa B also responds to reduced glutathione levels and a number of different thiols in T cells (33). These effects of reducing agents on gene expression are thought to be related to physiologic responses, in at least T cells (33) and the liver (29), via the relative levels and of intracellular reduced glutathione. DTT and the cytokine, tumor necrosis factor, were antagonistic to each other in the regulation of NF-kappa B activity in HeLa cells, with tumor necrosis factor activating and DTT inhibiting NF-kappa B (34). Regulation of NF-kappa B by sulfhydryl agents is mediated via I-kappa B phosphorylation, dissociation, and proteolysis (35-37).

The expression of 7alpha -hydroxylase has been shown to be negatively regulated by phorbol esters (16, 21) and various cytokines including tumor necrosis factor and interleukin-1 (38). Phorbol esters also decreased the level of intracellular thiols (35). Although based on these findings, it might be possible that phorbol esters repress 7alpha -hydroxylase via a mechanism associated with reduced intracellular sulfhydryl agents; however, our finding that DTT and glutathione could not reverse the phorbol ester repression of 7alpha -hydroxylase in L35 cells argues against this.

An alternative mechanism that might account for reduced sulfhydryl reagents affecting the transcription of 7alpha -hydroxylase is through an indirect effect mediated by the secretory pathway. The activation of NF-kappa B occurs through proteolysis of the regulatory factor I-kappa B in the endoplasmic reticulum (35-37). DTT can alter the retention, degradation, and secretion of some proteins containing disulfide bridges by interfering with their ability to fold properly (39). Because NF-kappa B can be regulated by the level of unfolded proteins in the endoplasmic reticulum (34), it is possible that DTT induction of 7alpha -hydroxylase might be mediated through a signal derived from the secretory pathway. This signal could affect I-kappa B proteolysis and/or NF-kappa B itself or the content of other transcription factors or gene products. NF-kappa B does not always act positively on its target genes; at least one gene that NF-kappa B represses has been identified (40).

Our findings showing that sulfhydryl reducing agents have a marked influence on 7alpha -hydroxylase transcription and expression are analogous to those reported on the transcriptional regulation of CYP1A1 and CYP1A2 (41). In cultured hepatoma cells, all three hepatic cytochrome 450s (CYP7, CYP1A1, and CYP1A2) are repressed by insulin in a manner that is reversed by sulfhydryl reducing agents. CYP1A1 and CYP1A2 are also repressed by inflammation (41). Previous studies in C57BL/6 and BALB/c inbred mice show that CYP7 is repressed by feeding a high fat diet containing cholic acid (4). In marked contrast, feeding the same diet to inbred strain C3H does not cause a repression of CYP7 (4). Additional studies show that the high fat diet containing cholic acid causes a NF-kappa B-induced inflammatory response in C57BL/6 and BALB/c mice but not in C3H mice (42). This inflammatory response is associated with reduced hepatic glutathione content (42).

Our findings showing that sulfhydryl reducing agents block the ability of insulin to repress 7alpha -hydroxylase may help to explain an apparent enigma in regard to in vivo regulation. It is well established that the expression of 7alpha -hydroxylase in vivo is greatest soon after food consumption (1). Normally, this is also when insulin levels are greatest. Our findings would predict that in the context of normal hepatic function, the hepatic content of reduced glutathione is sufficient to block insulin repression of 7alpha -hydroxylase in vivo. An appreciation of our finding that sulfhydryl reducing agents have a marked influence on 7alpha -hydroxylase transcription may help to delineate the molecular events through which the transcription of this important gene product is altered in response to diet and physiologic state.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant HL52005 and also by a grant from the American Heart Association California affiliate. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 619-594-7936; Fax: 619-594-7937; E-Mail: rdavis{at}sunstroke.sdsu.edu.
1    The abbreviations used are: 7alpha -hydroxylase, cholesterol-7alpha -hydroxylase NADPH:oxygen oxido reductase; DTT, dithiothreitol; Dex, dexamethasone; DMEM, Dulbecco's modified Eagle's medium; NF-kappa B, nuclear factor-kappa B.

Acknowledgments

We gratefully acknowledge the technical support of Gina Moore and Karl Lewis. In addition, we thank Christian A. Drevon for reading the manuscript and giving helpful suggestions.


REFERENCES

  1. Myant, N. B., and Mitropoulos, K. A. (1977) J. Lipid Res. 18, 135-153 [Medline] [Order article via Infotrieve]
  2. Davis, R. A., Dueland, S., and Trawick, J. (1992) in Molecular Genetics of Coronary Heart Disease and Stroke (Lusis, A., Rutter, J., and Sparkes, R. S., eds), Vol. 14, pp. 208-227, Karger Press, New York
  3. Jelinek, D. F., Andersson, S., Slaughter, C. A., and Russell, D. W. (1990) J. Biol. Chem. 265, 8190-8197 [Abstract/Free Full Text]
  4. Dueland, S., Trawick, J. D., Nenseter, M. S., MacPhee, A. A., and Davis, R. A. (1992) J. Biol. Chem. 267, 22695-22698 [Abstract/Free Full Text]
  5. Ishibashi, S., Schwarz, M., Frykman, P. K., Herz, J., and Russell, D. W. (1996) J. Biol. Chem. 271, 18017-18023 [Abstract/Free Full Text]
  6. Schwarz, M., Lund, E. G., Setchell, K. D. R., Kayden, H. J., Zerwekh, J. E., Bjorkhem, I., Herz, J., and Russell, D. W. (1996) J. Biol. Chem. 271, 18024-18031 [Abstract/Free Full Text]
  7. Jones, M. P., Pandak, W. M., Heuman, D. M., Chiang, J., Hylemon, P. B., and Vlahcevic, Z. R. (1993) J. Lipid Res. 34, 885-892 [Abstract]
  8. Davis, R. A., Hyde, P. M., Kuan, J. C., Malone-McNeal, M., and Archambault-Schexnayder, J. (1983) J. Biol. Chem. 258, 3661-3667 [Free Full Text]
  9. Leighton, J. K., Dueland, S., Straka, M. S., Trawick, J., and Davis, R. A. (1991) Mol. Cell. Biol. 11, 2049-2056 [Medline] [Order article via Infotrieve]
  10. Princen, H. M. G., Huijsmans, C. M. G., Kuipers, F., Vonk, R. J., and Kempen, H. J. M. (1989) Biochem. J. 262, 341-348 [Medline] [Order article via Infotrieve]
  11. Hylemon, P. B., Gurley, E. C., Stravitz, R. T., Litz, J. S., Pandak, W. M., Chiang, J. Y. L., and Vlahcevic, Z. R. (1992) J. Biol. Chem. 267, 16866-16871 [Abstract/Free Full Text]
  12. Stravitz, R. T., Hylemon, P. B., Heuman, D. M., Hagey, L. R., Schteingart, C. D., Ton-Nu, H.-T., Hofmann, A. F., and Vlahcevic, Z. R. (1993) J. Biol. Chem. 268, 13987-13993 [Abstract/Free Full Text]
  13. Pandak, W. M., Vlahcevic, Z. R., Heuman, D. M., Redford, K. S., Chiang, J., and Hylemon, P. B. (1994) Hepatology 19, 941-947 [Medline] [Order article via Infotrieve]
  14. Pandak, W. M., Li, Y. C., Chiang, J. Y. L, Studer, E. J., Gurley, E. C., Heuman, D. M., Vlahcevic, Z. R., and Hylemon, P. B. (1991) J. Biol. Chem. 266, 3416-3421 [Abstract/Free Full Text]
  15. Waxman, D. J. (1992) J. Steroid Biochem. Mol. Biol. 43, 1055-1072 [CrossRef]
  16. Trawick, J. D., Lewis, K. D., Dueland, S., Moore, G. L., Simon, F. R., and Davis, R. A. (1996) J. Lipid Res. 37, 588-598 [Abstract]
  17. Current Protocols in Molecular Biology14.10.1.4.10.10John Wiley & SonsNew YorkAusubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) Current Protocols in Molecular Biology, Vol. 1, pp. 4.10.1.-4.10.10, John Wiley & Sons, New York
  18. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  19. Derman, E., Krauter, K., Walling, L., Weinberger, C., Ray, M., and Darnell, J. E. (1981) Cell 23, 731-739 [Medline] [Order article via Infotrieve]
  20. Twisk, J., Hoekman, M. F. M., Lehman, E. M., Meijer, P., Mager, W. H., and Princen, H. M. G. (1995) Hepatology 21, 501-510 [Medline] [Order article via Infotrieve]
  21. Stravitz, R. T., Vlahcevic, Z. R., Gurley, E. C., and Hylemon, P. B. (1995) J. Lipid Res. 36, 1359-1369 [Abstract]
  22. Meister, A., and Anderson, M. E. (1983) Annu. Rev. Biochem. 52, 711-760 [CrossRef][Medline] [Order article via Infotrieve]
  23. Hassan, A. S. (1988) Biochim. Biophys. Acta 963, 131-138 [Medline] [Order article via Infotrieve]
  24. Hassan, A. S., Bunick, D., and St. Denis, S. H. (1993) Biochem. Pharmacol. 46, 555-556 [Medline] [Order article via Infotrieve]
  25. Hassan, A. S., Bunick, D., Lund, L. A., and Bottje, W. G. (1992) Biochem. Pharmacol. 44, 1475-1477 [Medline] [Order article via Infotrieve]
  26. Hassan, A. S., Hackley, J. J., and Jeffery, E. H. (1984) Steroids 44, 373-380 [Medline] [Order article via Infotrieve]
  27. Stephan, Z. F., Lindsey, S., and Hayes, K. C. (1987) J. Biol. Chem. 262, 6069-6073 [Abstract/Free Full Text]
  28. Tienrungroj, W., Meshinchi, S., Sanchez, E. R., Pratt, S. E., Grippo, J. F., Holmgren, A., and Pratt, W. B. (1987) J. Biol. Chem. 262, 6992-7000 [Abstract/Free Full Text]
  29. Ammendola, R., Mesuraca, M., Russo, T., and Cimino, F. (1994) Eur. J. Biochem. 225, 483-489 [Abstract]
  30. Guehmann, S., Vorbrueggen, G., Kalkbrenner, F., and Moelling, K. (1992) Nucleic Acids Res. 20, 2279-2286 [Abstract]
  31. Arnone, M. I., Zannini, M., and Di Lauro, R. (1995) J. Biol. Chem. 270, 12048-12055 [Abstract/Free Full Text]
  32. Das, K. C., Lewis-Molock, Y., and White, C. W. (1995) Am. J. Physiol. 269, L588-L602 [Abstract/Free Full Text]
  33. Mihm, S., Galter, D., and Droge, W. (1995) FASEB J. 9, 246-252 [Abstract/Free Full Text]
  34. Pahl, H. L., and Baeuerle, P. A. (1995) EMBO J. 14, 2580-2588 [Abstract]
  35. Staal, F. J. T., Roedere, M., Herzenberg, L. A., and Herzenberg, L. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9943-9947 [Abstract]
  36. Shreck, R., Rieber, P., and Baeuerle, P. A. (1991) EMBO J. 10, 2247-2258 [Abstract]
  37. Roff, M., Thompson, J., Rodriguez, M. S., Jacquet, J.-M., Baleux, F., Arenzana-Seisdedos, F., and Hay, R. T. (1996) J. Biol. Chem. 271, 7844-7850 [Abstract/Free Full Text]
  38. Feingold, K. R., Spady, D. K., Pollock, A. S., Moser, A. H., and Grunfeld, C. (1996) J. Lipid Res. 37, 223-228 [Abstract]
  39. Lodish, H. F., and Kong, N. (1993) J. Biol. Chem. 268, 20598-20605 [Abstract/Free Full Text]
  40. Supakar, P. C., Jung, M. H., Song, C. S., Chatterjee, B., and Roy, A. K. (1995) J. Biol. Chem. 270, 837-842 [Abstract/Free Full Text]
  41. Barker, C. W., Fagan, J. B., and Pasco, D. S. (1994) J. Biol. Chem. 269, 3985-3990 [Abstract/Free Full Text]
  42. Berliner, J. A., Navab, M., Fogelman, A. M., Frank, J. S., Demer, L. L., Edwards, P. A., Watson, A. D., and Lusis, A. J. (1995) Circulation 91, 2488-2496 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.