THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 21, Issue of July 25, PP. 12424-12433,1SS0 Printed in U.S. A.

Vitamin Bs Influences Glucocorticoid Receptor-dependent Gene Expression*

(Received for publication, October 10, 1989)

Victoria E. Allgood, Frances E. Powell-Oliver, and John A. Cidlowski$j From the Department of Physiology and the $Cancer Cell Biology Program, University of North Carolina, Chapel Hill, North Carolina 27599-7545

We have examined the influence of intracellular vi- tamin Bs concentration on glucocorticoid receptor function in HeLa Sa cells transfected with a glucocor- ticoid-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid. CAT activity is induced from this plasmid specifically by glucocorticoid hormones in a glucocorticoid receptor-dependent manner. The in- tracellular concentration of pyridoxal phosphate, the physiologically active form of the vitamin, was ele- vated by supplementation of the culture medium with the synthesis precursor pyridoxine and lowered by exposure to the pyridoxal phosphate synthesis inhibi- tor 4-deoxypyridoxine. Analysis of glucocorticoid re- sponsiveness revealed that elevated concentrations of intracellular pyridoxal phosphate suppressed the amount of glucocorticoid-induced CAT activity whereas moderate deficiency enhanced the level of glucocorticoid receptor-mediated gene expression. In contrast, modulation of the intracellular pyridoxal phosphate concentration had no effect on either basal CAT activity derived from cells not stimulated with dexamethasone or on CAT activity derived from two glucocorticoid-insensitive reporter plasmids. The mod- ulatory effects of pyridoxal phosphate concentration occur without changes in glucocorticoid receptor mRNA levels, glucocorticoid receptor protein concen- tration, or the steroid binding capacity of the receptor. These observations demonstrate that vitamin Be selec- tively influences glucocorticoid receptor-dependent gene expression through a novel mechanism that does not involve alterations in glucocorticoid receptor con- centration or ligand binding capacity.

Vitamin Bs is an essential water-soluble vitamin required for normal growth and development (1). The physiologically active form of the vitamin, pyridoxal5’-phosphate (pyridoxal phosphate), is derived from inactive dietary precursors and functions as a cofactor in numerous enzymatic reactions of amino acid metabolism (2). Pyridoxal phosphate is also crucial to the regulation of neural function through its involvement as a cofactor in the biosynthesis of many neurotransmitters, including dopamine, norepinephrine, histamine, serotonin, and y-aminobutyric acid (3-6). In addition to these well

* This work was supported by National Institutes of Health Grant DK 32459 and by the Institute of Nutrition, University of North Carolina. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be sent: 460 Medical Sciences Research Bldg., CB 7545, University of North Carolina, Chapel Hill, NC 27599-7545.

documented roles, there is increasing evidence to suggest that vitamin Bs may also function in the regulation of steroid hormone action.

Steroid hormones are known to modulate the expression of specific target genes (7-9). Each hormone exerts its effects through interaction with its specific cognate receptor protein, resulting in the formation of a high affinity steroid-receptor complex. Through the poorly understood process of activa- tion, the steroid-receptor complex localizes to the nucleus and acquires the capacity to associate with specific DNA se- quences, termed hormone regulatory or responsive elements, ultimately resulting in the modulation of target gene expres- sion. The precise mechanism(s) through which steroid-recep- tor complex interaction with hormone regulatory or respon- sive elements leads to alterations in the expression of specific genes has not been elucidated and is currently the subject of intense investigation in many laboratories.

There have been a number of studies with both tissue and cell homogenates which demonstrate that pyridoxal phos- phate influences several biochemical properties of steroid hormone receptors, including their molecular conformation, polyanion binding, surface charge, and susceptibility to exo- geneous proteolysis (10-15). In addition, pyridoxal phosphate affects both the subcellular localization and the DNA binding capacity of steroid receptors. In animals, translocation of receptors from the cytoplasmic to the nuclear compartment of cells appears to be increased under conditions of vitamin Bs deficiency (16, 17) and decreased under the opposing condition of elevated vitamin concentrations (18). Steroid receptors also display decreased affinity for DNA following treatment with pyridoxal phosphate in vitro (19). Although the physiological significance of these alterations in the phys- ical properties of steroid hormone receptors is not completely understood at present, such observations do suggest that vitamin Bs, possibly by influencing nuclear localization and/ or DNA binding capacity of receptors, may serve as a modu- lator of steroid hormone action.

Several laboratories have attempted to establish a definitive correlation between vitamin Bs and steroid hormone action. DiSorbo and Litwack (20) have investigated the effect of vitamin Bs on the induction of tyrosine aminotransferase by glucocorticoids and have reported that the amount of hor- mone-induced tyrosine aminotransferase activity is slightly increased after restriction of the vitamin and is decreased following exposure to pharmacological doses of the vitamin. Interpretation of these data, however, is complicated by the fact that tyrosine aminotransferase is itself a vitamin Bg- dependent enzyme (21), and these studies relied on the detec- tion of enzymatic activity. Similarly, the work of Majumder et al. (22) has shown that increased concentrations of vitamin Bs inhibit glucocorticoid-induced casein messenger RNA ac- cumulation in mouse mammary gland. Finally, work in our

12424

Vitamin Bs and Glucocorticoid Hormone Action 12425

own laboratory has demonstrated that the induction of alka- line phosphatase by glucocorticoids in HeLa Ss cells is im- paired under conditions of vitamin Bg excess (23). However, since each of these endogenous marker genes cited is subject to regulation by multiple factors in addition to glucocorticoid hormone (22, 24-26), it has not been possible to determine unequivocally whether vitamin Bs directly or indirectly affects glucocorticoid hormone action.

In this manuscript, we describe studies that were designed to evaluate the influence of vitamin Bs on steroid hormone- regulated gene expression. We have utilized a system for analyzing gene expression which is positively regulated by glucocorticoid hormones in a glucocorticoid receptor-depend- ent manner and demonstrate that alteration of the intracel- lular vitamin Bg concentration has profound effects on glu- cocorticoid receptor-mediated gene expression. These findings provide direct evidence in support of a role for vitamin Bs in the mechanism(s) of glucocorticoid hormone action in uiuo.

EXPERIMENTAL PROCEDURES

Materials-Joklik’s minimal essential medium (JMEM)’ with and without pyridoxine was obtained from GIBCO. Dulbecco’s modified essential medium was from Hazleton Research Products (Denver, PA). Calf serum and fetal calf serum were from HyClone Laboratories (Logan, UT). Acetyl coenzyme A was obtained from Pharmacia LKB Biotechnology Inc. Pyridoxine, 4-deoxypyridoxine, HEPES, gluta- mine, Tris, glycine, EDTA, cu-thioglycerol, and sodium molybdate were from Sigma. Dexamethasone, cortisol, progesterone, Sa-dihy- droxytestosterone, and 17B-estradiol were obtained from Steraloids (W&on, NH). [i4C]Chloramphenicol (40-60 mCi/mmol), [3H]dexa- methasone mesvlate (48.9 Ci/mmol). and EN13H1ANCE were ob- tained from Du Pant-New England’ Nuclear. ku’ 486 was kindly provided by Dr. Deraedt at the Centre de Recherches Roussel-UCLAF (Romainville, France). Centricon 30 microconcentrators were pur- chased from Amicon Corp. (Danvers, MA), nitrocellulose was from Schleicher & Schuell, andoligo(dT)-cellulose was from Collaborative Research (Lexineton, MA). Aaarose, acrvlamide. bisacrvlamide. SDS. TEMED, ammonium persulfate, RNA sjze markers, and high molec: ular weight prestained protein standards were from Bethesda Re- search Laboratories. Other chemicals were obtained from Fisher.

Recombinant Plusmids-Plasmid pGMCS, which contains the chloramphenicol acetyltransferase (CAT) gene fused downstream of the mouse mammary tumor virus promoter and glucocorticoid regu- latory element and upstream of the murine sarcoma virus glucocor- ticoid regulatory element was provided by Drs. Roger Miesfeld and Keith Yamamoto (University of California, San Francisco, CA). Plasmid pBLCAT2, obtained from Drs. Bruno Luckow and Gunther Schutz (Institute for Tumor and Cell Biology, Heidelburg, FRG), contains the thymidine kinase promoter from herpes simplex virus fused to the CAT gene. Plasmid pRSVCAT, which contains the Rous sarcoma virus promoter fused to the CAT gene, was obtained from Dr. Frank Rutter (University of California, San Francisco, CA).

Cell Culture-HeLa SI cells were grown as monolayer cultures in a 5% CO, humidified atmosphere at 37 ‘C in JMEM supplemented with 2 mM glutamine and 3% serum (1:l mixture of heat-inactivated fetal calf serum and calf serum). Where indicated, cells were propa- gated in JMEM deficient in pyridoxine or in medium supplemented with either pyridoxine or I-deoxypyridoxine. Harvesting of cells was accomplished by removing the medium and incubating with versene (2.7 mM KCl, 1.5 mM KHZPO,, 137 mM NaCl, 0.5 mM EDTA, 8 mM Na,HPO,) for 10 min followed by centrifugation at 1500 x g for 5 min in an IEC clinical centrifuge (International Equipment Company, Needham Heights, MA). Cell pellets were resuspended in medium without glutamine or serum and renlated or counted for exnerimental use. Cells were counted in 0.85% saline using a Zf Coulter Counter (Coulter Electronics, Hialeah. FL). Pvridoxine and I-deoxwvridox- ine were dissolved in water and diluted from stock solutio”ns”of 100 and 250 mM, respectively. Dexamethasone was dissolved in water and the concentration determined spectrophotometrically; other steroids

’ The abbreviations used are: JMEM, Joklik’s minimal essential medium; CAT, chloramphenicol acetvltransferase: SDS. sodium do- decyl sulfate; TEMED, k,N,N’,iV’-tetramethylenediamine; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid.

and RU 486 were dissolved in ethanol at a concentration of 200 pM. Cell Transfections-Twenty-four hours prior to transfection, cells

were plated at 2 X lo6 cells/l50-cm* tissue culture flask in Dulbecco’s modified essential medium supplemented with 100 units/ml penicil- lin, 100 pg/ml streptomycin, 2 mM glutamine, and 3% serum. Four hours before transfection, medium was replaced with fresh medium. Plasmid DNA (20 rg/flask) was prepared as a calcium phosphate precipitate (27) and incubated with cells for 4 h followed by a 30-s shock with 15% glycerol (28). Cells were then incubated for 16 h in fresh JMEM. At this point, the medium was removed and replaced with control medium or medium containing steroid or vitamin Bg analog as indicated in the appropriate figure legends.

Determination of Cytosolic Pyridoxal Phosphate Concentratian- The cytosolic pyridoxal phosphate concentration was determined essentially as described by Wada and Snell (29) with slight modifi- cations. Cells were lysed by homogenization, and a high speed super- natant was prepared. Cytosolic protein concentration was determined by the method of Lowry et al. (30), and aliquots containing equal amounts of protein were precipitated with 10% trichloroacetic acid, the resulting supernatant was reacted with phenylhydrazine in sul- furic acid as described (29). The phenylhydrazine calorimetric reac- tion was conducted at 0 “C to allow detection exclusively of pyridoxal phosphate and not of other derivatives of vitamin Bg.

Determination of CAT Activity-CAT assays were performed es- sentially as described by Gorman et al. (31): Briefly, a lysate was nrepared from 1 X lo6 cells, inactivated bv heating at 68 “C for 6 min. and adjusted to 156 mM Tris, 1 mM acetyl coenzime A, and 0.1 gCi of [i4C]chloramphenicol in a final volume of 150 ~1. The CAT reaction was then allowed to proceed for 24 h at 37 “C. Samples were applied to a thin layer chromatography plate and chromatographed in chlo- roform:methanol (95:5). After autoradiography, the amount of CAT activity was quantitated by removing the silica gel from the surface of the thin layer chromatography plate and quantitating [i4C]chlor- amphenicol and acetylated derivatives by scintillation counting in a Beckman LS 3801 scintillation counter (counting efficiency was greater than 75%).

Whole Cell Affinity Labeling of Glucocorticoid Receptor-HeLa cells were suspended in unsupplemented JMEM (5-10 x lo7 cells/ml) and incubated at 0 “C! with 40 nM [3H]dexamethasone mesylate minus or plus a l,OOO-fold excess of unlabeled dexamethasone. After 2 h of incubation at 0 “C, cells were centrifuged at 1,500 X g and the cell pellets resuspended in 10 mM Tris, 1 mM EDTA, pH 8.3, and homogenized at 0 “C by three 10-s bursts with a Tekmar Ultra Turrax homogenizer (Tekmar, Cincinnati, OH). The homogenates were cen- trifuged at 100,000 x g for 1 h at 0 “C. The high speed supernatant (cytosol) was treated with the pellet from an equal volume of dextran- coated charcoal (1% activated charcoal, 0.1% dextran in 1.5 mM MgCl,) to remove free steroid, concentrated 3-5-fold in volume with a Centricon 30 microconcentrator, mixed with an equal volume of 2 X sample buffer (20% (w/v) glycerol, 4.6% (w/v) SDS, 0.125 M Tris, pH 6.8), heated at 100 “C for 2.5 min. and kent at -70 “C until electrophoresis. Samples were electrophoresed by the method of Fair- banks et al. (32) on SDS-polyacrylamide gels (7.5% polyacrylamide) and electrophoretically transferred to nitrocellulose (33). The nitro- cellulose blots were either treated with EN[3H]ANCE and fluoro- graphed to determine steroid labeling or used for Western blot analy- sis.

Equilibrium Steroid Hormone Binding Analysis-HeLa cells were suspended in 10 mM Tris, 1 mM EDTA, 12 mM a-thioglycerol, 20 mM sodium molybdate, pH 7.5, at 4 “C and homogenized as described above. The homogenates were centrifuged at 100,000 x g for 1 h at 0 “C and protein concentrations of the supernatants determined by the method of Lowry et al. (30). Aliquots of each cytosol containing equal amounts of protein were removed to prechilled tubes containing [3H]dexamethasone at concentrations ranging from 1 to 600 nM. After incubation at 4 “C for 2 h, an aliquot was removed for quanti- tation of total steroid concentration and the remainder incubated with an equal volume of dextran-coated charcoal (1% activated char- coal, 0.1% dextran in 1.5 mM MgCl,) for 5 min at 4 ‘C to absorb unliganded steroid. The charcoal was pelleted at 4 “C by centrifuga- tion at 11,000 rpm, and steroid remaining in the supernatant was quantitated. Data were analyzed by the method of Scatchard (34) to determine the dissociation constant and total number of dexameth- asone binding sites.

Western Blot Analysis-Following electrophoretic transfer of [3H] dexamethasone mesylate-labeled cytosols, nitrocellulose blots were washed for 4 h at room temperature in 5% nonfat dry milk in Tris- buffered saline (200 mM NaCl, 50 mM Tris, pH 7.4). Blots were then

12426 Vitamin B6 and Glucocorticoid Hormone Action

incubated overnight at 4 “C with a 1:lOO dilution of serum containing anti-glucocorticoid receptor antibody in nonfat dry milk buffer. Sub- sequently. blots were washed in freshly prepared milk buffer at room temperature and then incubated for 2 h at room temperature with a peroxidase-conjugated secondary antibody (goat anti-rabbit, Cappel, West Chester, PA). The peroxidase color reaction was developed by incubation of the blot in 2 mg/ml 4-chloro-1-naphthol, 0.003% H,O, in Tris-buffered saline; the calorimetric reaction was stopped by immersion of the blot in H,O.

Northern Riot Analysis-Polyadenylated mRNA was isolated from HeLa cells as described by Badley et al. (35). RNA was denatured with glyoxal and dimethyl sulfoxide (36), electrophoresed through 1% agarose gels. and then transferred to Biotrans nylon membrane (ICN, Irvine, CA). After transfer, the membrane was baked, prehybridized, and hybridized under the conditions of Wahl et al. (37). The gluco- corticoid receptor and actin mRNA probes were constructed by in- sertion of the complete human glucocorticoid receptor cDNA or chicken @-actin cDNA. respectively, into a Riboprobe in uitro tran- scription vector (Promega Biotec, Madison, WI) and a cRNA probe synthesized by incorporation of [‘“P]UTP under conditions recom- mended by the manufacturer; probes were routinely labeled to a specific activitv of 0.021 pCi/pg. After hybridization, the blots were washed according to Wahl et al. (37) and processed for autoradiog- raphy. The autoradiographs were densitometrically scanned in order to determine mRNA levels qualitatively.

RESULTS

CAT Activity Is Induced Specifically by the Glucocorticoid Class of Steroid Hormones in HeLa Cells--Since the endoge- nous genes employed in previous evaluations (20, 22, 23) of the potential relationship between vitamin Bs and steroid hormone action are subject to regulation by multiple factors, the selective effects of vitamin B, on hormone-induced gene expression have yet to be determined unambiguously. To address this issue, we have introduced a glucocorticoid-re- sponsive CAT reporter plasmid, pGMCS, into HeLa & cells, a stable cell culture line that has high affinity saturable receptors for glucocorticoid hormones but lacks receptors for other steroid hormones (38,39). These cells, when transiently transfected with the plasmid pGMCS, respond to administra- tion of the synthetic glucocorticoid dexamethasone with an increase in CAT activity. In the absence of dexamethasone only 0.8% of precursor [‘“Clchloramphenicol is converted to its acetylated forms, reflected as the two upper spots in the representative autoradiograph in Fig. 1A; however, addition of 100 nM dexamethasone for 8 h results in 15.6% conversion, an approximate 19-fold increase over control. Cortisol, the naturally occurring glucocorticoid, is also effective although less potent (3.6% conversion). This difference probably re- flects the higher affinity of the glucocorticoid receptor for dexamethasone as well as the inability of dexamethasone to be bound by serum corticosteroid-binding globulin. Half-max- imal induction of CAT activity is achieved at a dexamethasone concentration of 50 nM (data not shown); this concentration is consistent with that reported for induction of mouse mam- mary tumor virus RNA (40, 41) and for regulation of enzy- matic activity encoded by another glucocorticoid-responsive gene, alkaline phosphatase, in HeLa cells (26).

Progestins and androgens have also been reported to regu- late the expression of reporter genes that are under the transcriptional control of DNA sequences derived from the mouse mammary tumor virus genome (40, 42), but, as the data in Fig. 1A demonstrate, estrogen, progesterone, and androgen fail to induce CAT activity above control levels in this system. The ineffectiveness of these other steroid hor- mones most likely reflects the absence of their cognate recep- tor proteins in HeLa cells. Thus, induction of CAT activity in these transfected cells occurs selectively in response to glucocorticoid hormone treatment.

To determine if the glucocorticoid receptor is required for

A d)r, ..,

0,

CON DEX CORT Ep PROG DHT

CON DEX RU RU+DEX

FIG. 1. Specificity of induction of CAT activity in trans- fected HeLa cells. HeLa cells were transfected with the glucocorti- coid-responsive CAT reporter plasmid pGMCS as described under “Experimental Procedures.” A, steroid specificity. Sixteen hours after transfection with pGMCS, cells were treated for 8 h with no hormone (CON) or the indicated hormone (dexamethasone (DEX), cortisol (CORn, 17p-estradiol (EJ, progesterone (PRO@, or 5cu-dihydrotes- tosterone (DHT)) at a concentration of 100 nM prior to analysis of CAT activity. The experiment shown is representative of observations made in two independent transfections. B, effect of a glucocorticoid receptor antagonist. Sixteen hours after transfection with pGMCS, cells were treated for 8 h with no hormone, 100 nM dexamethasone, 1 FM RU 486 (RU), or simultaneously with 1 j.tM RU 486 and 100 nM dexamethasone prior to CAT activity analysis. The experiment shown is representative of observations made in two independent transfec- tion experiments.

induction of CAT activity in transfected HeLa cells, we ex- amined the effect of the glucocorticoid receptor antagonist RU 486 on this response. This competitive antagonist has been shown to inhibit the glucocorticoid-mediated induction of tyrosine aminotransferase and tryptophan oxygenase (43) as well as the glucocorticoid-stimulated transcription of mouse mammary tumor virus RNA (44). As shown in Fig. lB, treatment of transfected cells with dexamethasone resulted in the characteristic appearance of CAT activity whereas treatment with the receptor antagonist failed to induce CAT activity. Further, with simultaneous exposure to dexametha- sone and RU 486, the receptor antagonist completely pre- vented induction by the agonist, indicating that the induction of CAT activity by dexamethasone is mediated through the glucocorticoid receptor.

Modulation of Intracellular Pyridoxal Phosphate Concentra- tion-In those cells and tissues in which it has been measured, the concentration of pyridoxal phosphate, as well as its syn- thesis and metabolism, have been observed to differ substan- tially (45-50). In addition, there is wide variation in the effect on intracellular pyridoxal phosphate concentration arising from exogenous supplementation or withdrawal of the biolog- ical precursor pyridoxine (16, 18, 46, 48, 51). Thus, mainte-

Vitamin Bs and Glucocorticoid Hormone Action 12427

nance of intracellular pyridoxal phosphate concentration ap- pears to be both cell and tissue specific. These data prompted us to measure directly the concentration of pyridoxal phos- phate in HeLa cells and to determine if this concentration could be altered experimentally either by addition or removal of precursor from the culture medium or by pyridoxal phos- phate synthesis inhibition. To prevent synthesis of pyridoxal phosphate, we employed the compound 4-deoxypyridoxine. This analog acts specifically to inhibit pyridoxal kinase (52), a key enzyme in the biosynthesis of pyridoxal phosphate, and thereby prevents formation of the active form of the vitamin from inactive precursors or metabolites. Cells were cultured in either control medium, commercially available medium synthesized without pyridoxine and generally considered to be vitamin Bs deficient, or medium supplemented with either 1 mM pyridoxine or 5 mM 4-deoxypyridoxine for 48 h. At the end of this period, the intracellular pyridoxal phosphate con- centration was determined. Neither cell viability nor growth rate is significantly altered by culture under any of these conditions (data not shown). As summarized in Table I, HeLa cells grown in control medium have a concentration of 0.175 f 0.013 rmol of pyridoxal phosphate per mg of cellular pro- tein. Supplementation of growth medium with 1 mM pyridox- ine for 48 h produces an approximate 250% increase in the intracellular pyridoxal phosphate concentration, to 0.46 f 0.073 pmol/mg. Surprisingly, culture in pyridoxine-deficient medium does not produce a significant reduction in the intra- cellular concentration of pyridoxal phosphate. This finding suggests that HeLa cells are capable of efficiently recycling vitamin precursors and metabolites to prevent deficiency and thus prompted us to employ the pyridoxal phosphate synthe- sis inhibitor 4-deoxypyridoxine in order to achieve a state of vitamin Bs deficiency. When added to the culture medium at a concentration of 5 mM for 48 h, 4-deoxypyridoxine produces a decrease in the intracellular pyridoxal phosphate concentra- tion to 0.124 + 0.028 pmol/mg, approximately 70% of the control level.

Based on these data, it would appear that the concentration of pyridoxal phosphate in HeLa cells is tightly regulated by inherent homeostatic mechanisms. The intracellular concen- tration appears to be more easily elevated than lowered. However, significant changes in the intracellular concentra- tion of pyridoxal phosphate can be achieved by exposure to high extracellular concentrations of precursor or by exposure to the pyridoxal kinase inhibitor 4-deoxypyridoxine.

Effect of Intracellular Pyridoxal Phosphate Concentration on Glucocorticoid Receptor-mediated Gene Expression-Hav- ing established conditions under which the intracellular pyr- idoxal phosphate concentration in HeLa cells can be altered,

TABLE I Effect of modification of cell growth medium on intracellular

pyridonal phosphate concentration Cells were cultured in control medium, medium deficient in pyri-

doxine (-B6), medium supplemented with 1 mM pyridoxine (Pyr), or medium supplemented with 5 mM 4-deoxypyridoxine (4-Deoxy). After 48 h of growth under these conditions, cell were harvested, and intracellular pyridoxal phosphate was determined as described under “Experimental Procedures.” The data shown represent the average of triplicate determinations from four independent experiments.

Culture Intracellular pyridoxal medium phosphate

Nmol/mg cytosolic protein

Control 0.175 f 0.013 -J% 0.185 + 0.015 Pyr 0.461 & 0.073 4-Deoxy 0.124 _t 0.028

we sought to determine the effects of these modifications on glucocorticoid receptor-mediated gene expression. After transfection with the glucocorticoid-responsive reporter plas- mid pGMCS, HeLa cells were grown for 48 h in the media described above to alter intracellular pyridoxal phosphate levels and then treated with dexamethasone. The effects of altered intracellular pyridoxal phosphate concentration on the glucocorticoid receptor-mediated induction of CAT activ- ity are summarized graphically in Fig. 2. CAT activity meas- ured in dexamethasone-stimulated cells grown in unaltered medium is assigned a value of 1.0, and the amount of CAT activity derived from dexamethasone-stimulated cells grown under alternate media conditions is expressed as a percentage of the value obtained in unaltered medium; these data repre- sent the mean CAT activity + S.E. from three independent transfection experiments. In the absence of dexamethasone, there was only minimal CAT activity (see Fig. 1) that was not influenced by the pyridoxal phosphate concentration (not shown); thus, CAT activity derived from unstimulated cells is not presented. Treatment with 100 nM dexamethasone for 8 h in control medium produced the characteristic induction of CAT activity. In contrast, supplementation of the medium with 1 mM pyridoxine, which elevates the intracellular pyri- doxal phosphate concentration, suppressed the amount of dexamethasone-induced CAT activity to approximately 50% of that obtained in control medium. Following removal of pyridoxine from the culture medium, which did not signifi- cantly alter the intracellular pyridoxal phosphate concentra- tion, only minimal effects on the dexamethasone induction of CAT activity were observed. However, when the intracellular pyridoxal phosphate concentration was decreased by treat- ment with 4-deoxypyridoxine, an enhancement in the amount of glucocorticoid-induced CAT activity was observed, after 48 h in the presence of 5 mM 4-deoxypyridoxine, the amount of hormone-induced CAT activity was increased to approxi- mately 250% of that observed with the same dexamethasone treatment in control medium. These alterations in pyridoxal

FIG. 2. Effect of alterations in intracellular pyridoxal phos- phate concentration on glucocorticoid receptor-induced CAT activity in transfected HeLa cells. Sixteen hours after transfec- tion with pGMCS, cells were introduced into the following media in order to alter intracellular pyridoxal phosphate levels: unaltered Joklik’s minimal essential medium, the normal growth medium (CON); medium prepared without pyridoxine (-Be); medium supple- mented with 1 mM pyridoxine (PYR); or medium supplemented with 5 mM 4-deoxypyridoxine (4-DEOXY). After culture for 48 h under these conditions, cells were stimulated with 100 nM dexamethasone or no hormone for 8 h and the amount of CAT activity determined by quantitation of W by scintillation counting after autoradiography as described under “Experimental Procedures.” The values shown represent the mean f S.E. from three independent transfection experiments. The average CAT activity obtained from dexametha- sone-stimulated cells grown in unaltered medium is assigned a value of 1.0, and the average CAT activity derived from dexamethasone- stimulated cells grown under alternate media conditions is expressed as a percentage of this value. CAT activity is only minimal in cells not stimulated with dexamethasone and not influenced by the pyri- doxal phosphate concentration; thus, only data obtained in dexa- methasone-stimulated cells are presented.

12428 Vitamin B6 and Glucocorticoid Hormone Action

phosphate concentration do not affect the retention of trans- fected plasmid (data not shown); thus, these results are not due to gene dosage effects.

The inhibitory effect of pyridoxine and the stimulatory effect of 4-deoxypyridoxine on the glucocorticoid receptor- mediated induction of CAT activity are concentration de- pendent, as demonstrated in Fig. 3. In these experiments, CAT activity obtained by treatment with 100 nM dexameth- asone in control medium is assigned a value of 1.0, and the effects on CAT activity induction resulting from 48-h culture under conditions of increasing concentrations of either pyri- doxine (Fig. 3A) or 4-deoxypyridoxine (Fig. 3B) are expressed as percentage of control; where error bars are indicated, the data represent the mean CAT activity -+ S.E. from at least two independent transfections. At the lowest pyridoxine con- centration tested (0.1 mM), an approximate 42% decrease in glucocorticoid-induced CAT activity was observed relative to control. Further increases in pyridoxine supplementation caused progressive reductions in the amount of glucocorticoid- induced CAT activity. Treatment with 3 mM pyridoxine in- hibited the glucocorticoid induction by 85%; however, concen- trations of pyridoxine of 3 mM and above also decreased the growth rate and altered the morphology of the cells. In con- trast, treatment with either 1 or 2 mM pyridoxine under these conditions had no effect on these parameters. Thus, we do not consider the results of experiments using concentrations of pyridoxine above 2 mM to represent physiological actions of the vitamin.

The enhancement in glucocorticoid-induced CAT activity as a function of 4-deoxypyridoxine concentration is demon- strated in Fig. 3B. Although culture in pyridoxine-deficient medium alone (0 mM 4-deoxypyridoxine) has no significant effect on the amount of glucocorticoid-induced CAT activity, addition of 2.5 mM 4-deoxypyridoxine produces a 25% in- crease in dexamethasone-induced CAT activity. Increasing the 4-deoxypyridoxine concentration to 5 mM resulted in a further enhancement of CAT activity to approximately twice that observed in unaltered media, with no effect on cell growth rate or morphology. Additional increases in the 4-deoxypyri- doxine concentration did not result in CAT activity signifi-

PYRIDOXINE (mM) 4-OEOXYPYRIOOXINE (mt.4)

FIG. 3. Effect of supplementation of culture medium with increasing concentrations of pyridoxine or I-deoxypyridox- ine on glucocorticoid receptor-induced CAT activity. Sixteen hours after transfection with pGMCS, HeLa cells were exposed to unaltered medium (CON), medium supplemented with increasing concentrations of pyridoxine (left panel), or medium supplemented with increasing concentrations of 4-deoxypyridoxine (right panel). After 48-h culture under these conditions, cells were stimulated with 100 nM dexamethasone for 8 h and then harvested, and CAT activity was determined as described under “Experimental Procedures.” CAT activity observed in dexamethasone-stimulated cells grown in unal- tered medium is assigned a value of 1.0; the amount of CAT activity observed in dexamethasone-stimulated cells grown under altered media conditions is expressed as percentage of this. Where error bars are indicated, the values are representative of the mean + SE. from at least two individual transfection experiments; the absence of an error bar indicates a measurement from a single transfection experi- ment.

cantly greater than that observed with 5 mM treatment. It is important to note that neither cell growth rate, viability, nor morphology is significantly affected by 48-h exposure to con- centrations of 4-deoxypyridoxine up to 10 mM or to concen- trations of pyridoxine up to 2 mM.

Influence of Vitamin Bs Levels on Glucocorticoid-insensitive CAT Gene Expression-The next series of experiments was designed to determine if the effects of altered pyridoxal phos- phate concentration represent specific effects on glucocorti- coid receptor-dependent gene expression or perhaps reflect nonspecific actions on other transcription factors involved in the induction of CAT activity. To this end, we examined the influence of vitamin Bs concentration on CAT activity from other transfected CAT reporter plasmids. For comparison with pGMCS, two other CAT reporter plasmids were also used for transfection, pBLCAT2 and pRSVCAT. These plas- mids contain viral promoter elements, derived from herpes simplex virus and Rous sarcoma ViNS, respectively, as de- scribed under “Experimental Procedures”; CAT expression derived from pBLCAT2 and pRSVCAT is constitutive and glucocorticoid insensitive (data not shown). The influence of intracellular pyridoxal phosphate concentration on CAT en- zymatic activity observed in HeLa cells transfected with either of these three plasmids is presented in Fig. 4, these data were analyzed and presented as described for Fig. 2 and represent the mean CAT activity f S.E. from two independent trans- fection experiments. In marked contrast to the effects on glucocorticoid-induced CAT activity derived from pGMCS, alterations in the pyridoxal phosphate concentration had no effect on CAT activity derived from either of the constitu- tively expressing plasmids pBLCAT2 or pRSVCAT. Simi- larly, in cells transfected with pGMCS, the low level of basal CAT activity (derived from cells not stimulated with dexa- methasone) is also completely unaffected by altered intracel- lular vitamin Bs concentrations (not shown). These observa- tions strongly suggest that vitamin Bs does not influence gene expression nonspecifically but acts in a selective manner to modulate glucocorticoid receptor-induced gene expression. These data also demonstrate that neither pyridoxine nor 4- deoxypyridoxine acts directly on the CAT enzyme itself to influence its enzymatic activity or metabolic half-life. To- gether, these observations provide compelling evidence that vitamin B6 does not alter the normal transcriptional and translational activities of the cells. When coupled with our observations on cell growth rate, viability, and morphology described earlier for Fig. 3, these data demonstrate that the influence of vitamin Bs concentration on receptor-mediated gene expression occurs under conditions in which the normal homeostatic and metabolic processes of the cells are not affected. We conclude, then, that neither the intracellular pyridoxal phosphate concentration nor the agents used in these experiments to alter it act to influence gene transcrip- tion in a nonspecific manner or to affect CAT mRNA stability, translation, or catalytic activity.

Effect of Pyridonal Phosphate Concentration on Glucocorti- coid Receptor mRNA Levels, Cellular Glucocorticoid Receptor Concentration, and Steroid Binding Capacity-Using cell lines expressing different levels of glucocorticoid receptors, Van- derbilt et al. (53) have demonstrated that the degree of hor- mone responsiveness is dependent upon the cellular concen- tration of hormone receptors. In light of this observation, we wished to determine if the effects of intracellular pyridoxal phosphate concentration on glucocorticoid receptor-induced CAT activity are reflective of changes in the cellular concen- tration of glucocorticoid receptor. For this analysis, four in- dependent approaches were taken to quantitate the steady-

Vitamin B, and Glucocorticoid Hormone Action 12429

FIG. 4. Effect of alterations in intracellular pyridoxal phosphate concentration on CAT activity derived from glucocorticoid-responsive and nonresponsive plasmids in transfected HeLa cells. Follow- ing transfection with either the glucocorticoid-responsive plasmid pGMCS or a glucocorticoid-insensitive plasmid, pBLCAT2 or pRSVCAT, HeLa cells were cultured in unaltered medium (CON), pyridoxine-deficient medium (-BJ, or medium supplemented with either 1 mM pyridoxine (PYR) or 5 mM 4-deoxypyridoxine (I-DEOXY) for 48 h. Cells were then stimulated with 100 nM dexamethasone for 8 h and assayed for CAT activity. The values shown are representative of the mean + SE. from two independent transfections, with CAT activity from cells grown in unaltered medium assigned a value of 1.0, and CAT activity from cells grown in other media expressed as a percentage of this value. The first four lanes on the left are from cells transfected with pGMCS; the center four lanes, from cells transfected with pBLCAT2; and the four lanes on the right, from cells transfected with pRSVCAT.

state levels of glucocorticoid receptor in HeLa cells with altered intracellular vitamin Bg concentrations.

To examine first the levels of glucocorticoid receptor gene expression, we isolated polyadenylated RNA from HeLa cells after 48-h culture in pyridoxine-deficient medium, medium supplemented with 5 mM 4-deoxypyridoxine, or control me- dium (Fig. 5A, left three lanes). In a separate experiment, cells were harvested after comparable culture in medium supple- mented with 1 mM pyridoxine or control medium (Fig. 5A, right tlvo lanes). These mRNAs were electrophoresed and hybridized with a glucocorticoid receptor cRNA probe as described under “Experimental Procedures.” The autoradi- ographic signal corresponding to hybridization with the 7- kilobase human glucocorticoid receptor mRNA is indicated by the arrow in the upper portion of Fig. 5A. As can be seen in these two representative experiments, glucocorticoid recep- tor mRNA is present in each of the experimental groups, regardless of the intracellular pyridoxal phosphate concentra- tion. Although there is some variability in the level of human glucocorticoid receptor mRNA, when these blots are normal- ized by hybridization with actin (indicated in the lowerportion of Fig. 5A) and analyzed by densitometric scanning, there is no significant change in the amount of glucocorticoid receptor mRNA derived from transfected cells grown under altered media conditions (pyridoxine-deficient and 4-deoxypyridox- ine uersus control; pyridoxine uersus control). Similar analysis of human glucocorticoid receptor levels in three additional experiments, each normalized with actin as described above, confirms that neither moderate pyridoxal phosphate deli- ciency nor elevation has a significant effect on the steady- state level of glucocorticoid receptor mRNA. The major band just below the human glucocorticoid receptor signal represents cross-hybridization to 28 S ribosomal RNA; this signal is observed when the hybridization probe, either human gluco- corticoid receptor or actin, is prepared from the Riboprobe in vitro transcription vector and, therefore, most likely repre- sents nonspecific hybridization with vector sequences.

We next investigated the effect of alterations in pyridoxal phosphate concentration on the level of immunologically de- tectable glucocorticoid receptor protein in HeLa cells. For this analysis, we used an antibody directed against a glucocorticoid receptor-specific epitope in the amino terminus of the gluco-

corticoid receptor (54). Cytosols were prepared after 48-h culture in the media described above to alter intracellular pyridoxal phosphate concentrations. AIiquots of cytosol con- taining equal amounts of protein were examined by electro- phoresis and subsequent Western blot analysis. Interaction of the anti-glucocorticoid receptor antibody with the M, 94,000 glucocorticoid receptor protein is indicated by the arrow in Fig. 5B. The experiment shown here, which is representative of three, demonstrates that the amount of immunoreactive glucocorticoid receptor present in each of the cell groups is qualitatively the same regardless of the vitamin Bs concentration of the cells. This observation has been confirmed subsequently (data not shown) using an antibody that recognizes a different epitope of the glucocorticoid recep- tor (55). By analysis of three independent experiments using these two different antibodies, we conclude that the intracel- lular pyridoxal phosphate concentrations in these experi- ments do not have significant effects on the level of immu- nologically detectable glucocorticoid receptor protein present in HeLa cells. Together, the data in Fig. 5, A and B, suggest that neither the concentration of glucocorticoid receptor pro- tein nor its mRNA is affected by alteration in the intracellular pyridoxal phosphate concentration. Therefore, the effects of altered vitamin Bs concentration on glucocorticoid receptor- induced CAT activity cannot be attributed to changes in the concentration of glucocorticoid receptor protein or its mRNA.

Since previous studies have defined at least one pyridoxal phosphate binding site on the steroid binding domain (mero form) of the glucocorticoid receptor (56), we next wished to determine if the effects of pyridoxal phosphate concentration on glucocorticoid receptor-mediated gene transcription are reflective of changes in the steroid binding capacity of the glucocorticoid receptor. For this analysis, cells were collected after 48-h culture in pyridoxine-deficient medium, medium supplemented with 5 mM 4-deoxypyridoxine, or control (Fig. 5C, left six lanes). In a separate experiment, cells were col- lected after 48-h culture in medium supplemented with 1 mM

pyridoxine or control (Fig. 5C, right four lanes). Cells were labeled with [3H]dexamethasone mesylate in the absence and presence of excess radioinert dexamethasone, and cytosols were prepared and analyzed by polyacrylamide gel electropho- resis and subsequent fluorography. Although several cytosolic

12430 Vitamin B,; and Glucocorticoid Hormone Action

NORTHERN BLOl

proteins associate with the covalent affinity ligand [“H]dex- amethasone mesylate, only the M, 94,000 protein is saturably bound by excess radioinert dexamethasone. This band of

B WESTERN EL01

pB-- - - -

- _- - CI* -hGR

BSA-

0”

C AFFINITY LABEL

ca- .~ -.

DEX - + - + - + - + - + MEDIA. Con -86 “-Day CO” w

FIG. 5. Effect of altered intracellular pyridoxal phosphate concentration on glucocorticoid receptor mRNA and protein levels and whole cell affinity labeling capacity. A, Northern blot analysis. HeLa cells were cultured for 4X h in pvridoxine-def’icierlt medium f-H,:), medium supplemented with 5 mbt bdeoxypyridoxine (.l-lIc~.~,v), or control medium ((‘on) (Icft thrw lanes). In a separate experiment, cells were cultured in medium supplemented with 1 mM pyridoxine t&r) or control (r$hi irc,o Inncs). Polyadenylated RNA was isolated. electrophoresed through agarose gels, and transferred to nylon membranes as described under “Experimental Procedures.” Blots ivere then probed with a glucocorticoid receptor cRNA probe and subsequently aith an actin cRSA probe. The signal representing hybridization with the glucocorticoid receptor (II mRNA is indi- cated with the orrou in the upper portion of’ the f’imre: hybridization to actin is indicated 1)~ the nrrorc. in the lo~r~~r portion of the figure. The experiment shown is representative of’ three independent exper- iments. H. \\:estern blot analysis. After 48-h culture in control me- dium. pgridoxine-deficient medium. or medium supplemented with either 5 rnbl Gdeoxvpyridoxine or 1 mbt pyridoxine. cells were har- vested and labeled for 2 h with 40 IIM [ ‘Hldexamethasone mesylate. A high speed cytosol was then prepared, electrophoresed, and electro- phoretically transf’erred to nitrocellulose as described under “Exper- imental Procedures.” The membrane was incubated with an antibody directed against the glucocorticoid receptor followed by a peroxidase- conjugated secondary antibody. Immunoreactive glucocorticoid recep tar is indicated by the nrro~. Prestained molecular mass markers, electrophoresed in an ndjn~nt Inne, are: phosphorylase R (PR), 95.400 Da: bovine serum albumin (HSrl), 68,000 Da; and ovalhumin ( O V ) ,

4:3.000 Da. The experiment shown is representative of three inde- pendent experiments. c’. aff’inity labeling analysis. HeLa cells were cultured for 48 h in control medium, pyridoxine-deficient medium, or medium supplemented with 3 mu I-deoxypyridoxine (left six Innes). In a separate experiment. cells were cultured in control medium or medium supplemented with 1 mM pgridoxine for 48 h (right four [arzes). Cells were then collected and labeled for 2 h at 0 “C with 40 nhf [ ‘Hjdexamethasone mesylate in the absence (-) or presence (+) of300 go radioinert dexamethasone. Cytosols were prepared, electro- phoreeed through acrylamide gels. and analyzed by fluorography as described under “Experimental Procedures.” Saturable hinding, rep resentative of the 94.000-Da glucocorticoid receptor. is indicated by the nrrorc. and does not appear in the samples treated simultaneously with radiolabeled and radioinert steroid. Prestained molecular mass markers are as in H. including carbonic anhydrase ( C A ) , 29.000 Da. The figure shown is representative of three independent experiments.

saturable [“Hldexamethasone mesylate labeling, indicated by the arrow in Fig. 5C, comigrates with the protein giving rise to the immunoreactivity in Fig. 5B and therefore represents specific association of the glucocorticoid receptor with the affinity ligand. Results from the two different experiments shown in Fig. 5C demonstrate that culture of cells in pyridox- ine-deficient medium or medium supplemented with either 5 mM 4-deoxypyridoxine or 1 mM pyridoxine does not signifi- cantly alter the pattern of [“Hldexamethasone mesylate label- ing as compared with control. This observation has been confirmed through subsequent analysis of three identically conducted independent experiments. Thus, it appears that the intracellular pyridoxal phosphate concentration does not affect the in uiuo affinity labeling capacity of the glucocorti- coid receptor as determined by whole cell incubation with dexamethasone mesylate.

In an attempt to provide more quantitative information regarding the steroid binding capacity and affinity of the glucocorticoid receptor derived from cells of altered intracel- lular vitamin Bli concentrations, we have performed equilib- rium hormone binding studies using the glucocorticoid recep- tor agonist [“Hldexamethasone. For these studies, cytosols were prepared from HeLa cells cultured for 48 h in pyridoxine- deficient medium, medium supplemented with either 1 mM

pyridoxine or 5 mM 4-deoxypyridoxine, or control medium as described above. Aliquots of cytosol were incubated with [“HI dexamethasone, and hormone binding was measured as de- scribed under “Experimental Procedures.” The binding data were then analyzed by the method of Scatchard (34). The results from one experiment, which are representative of three independent measurements, are presented in Fig. 6. These data demonstrate that the glucocorticoid receptor from HeLa cells cultured in unaltered medium exhibits high affinity dexamethasone binding, with a dissociation constant of 4.5 nM observed; this value is consistent with the previously reported dissociation constant for dexamethasone binding by the glucocorticoid receptor from HeLa cells (26, 57). In addi-

FIN:. 6. Effect of altered intracellular pyridoxal phosphate concentration on equilibrium dexamethasone binding to glu- cocorticoid receptors from HeLa cells. Cytosolic extracts were prepared from HeI,a cells after 48-h culture in control medium, pyridoxine-deficient medium (GM?), or medium supplemented with either 5 mM 4-deoxypyridoxine (.l-Dfi:fJXY) or 1 mM pyridoxine (I’Yli). Aliquots of’ extracts were incubated with [“Hldexamethasone at concentrations ranging from 1 to 600 nM, the unliganded steroid absorbed with dextran-coated charcoal, and dexamethasone quanti- tated by scintillation counting. Binding data were analyzed hy the method of’ Scatchard (34) to permit determination of’ dissociation constant and numher of dexamethasone binding sites. The experi- ment shown is representative of’ three independent experiments. [GH]. glucocorticoid receptor concentration.

Vitamin Bg and Glucocorticoid Hormone Action 12431

tion, the dissociation constant does not appear to be signifi- cantly affected by alterations in the intracellular pyridoxal phosphate concentration, with dissociation constants ranging only from 4.4 to 5.1 nM in the data shown. Quantitative analysis of three individual experiments revealed dissociation constants (mean k S.E.) as follows: control, 6.1 f 2.7 nM; pyridoxine, 5.3 f 1.5 nM; pyridoxine-deficient medium, 6.5 +- 4.3 nM; and 4-deoxypyridoxine, 5.5 f 1.4 nM. Thus, possible alterations in the ligand binding affinity cannot account for the effects on glucocorticoid-induced CAT activity which arise from modulation of the intracellular pyridoxal phosphate concentration.

From the same binding data, we have also determined the total number of dexamethasone binding sites in cytosols pre- pared from HeLa cells. In cells cultured in unaltered medium, we measured 236 fmol binding sites/mg of cytosolic protein; this value does not appear to be significantly altered by changes in the intracellular pyridoxal phosphate concentra- tion (pyridoxine-deficient, pyridoxine, 4-deoxypyridoxine). Analysis of steroid binding in three independent experiments demonstrated the following receptor concentrations (mean f S.E.) control, 219 + 40 fmol/mg; pyridoxine, 220 f 11 fmol/ mg; pyridoxine-deficient, 232 + 47 fmol/mg; and 4-deoxypyr- idoxine, 195 f 26 fmol/mg. Thus, neither the dissociation constant nor the number of binding sites for dexamethasone is significantly affected by alterations in the intracellular concentration of pyridoxal phosphate.

Together, the data in Figs. 5 and 6 demonstrate that alter- ations in the intracellular pyridoxal phosphate concentration do not influence the amount of glucocorticoid receptor pro- tein, the steady-state level of glucocorticoid receptor gene transcription, or the capacity of the receptor protein to bind steroid. Thus, the modulation of glucocorticoid receptor-me- diated gene expression which is observed under these condi- tions of altered pyridoxal phosphate concentrations must occur by a mechanism(s) that does not involve changes in the level of receptor or its ligand binding ability.

DISCUSSION

The intracellular concentration of vitamin Bs is known to influence two parameters of steroid receptors which are req- uisite for regulation of target gene expression, namely the subcellular localization of the receptor and its DNA binding capacity. Under conditions of vitamin Bs deficiency, it has been observed for estrogen and androgen receptors that both the uptake of receptors into target tissues and the localization of receptors to the nuclear compartment of cells are enhanced whereas the fraction of cytoplasmic receptors is diminished (16, 17). Under the contrasting conditions of vitamin Bs excess, both target tissue uptake and nuclear binding or localization of glucocorticoid, estrogen, androgen, and proges- terone receptors are decreased (11, 12, 18, 58, 59). Moreover, the in vitro DNA binding capacity of all steroid hormone receptors is decreased after treatment of receptor preparations with pyridoxal phosphate. These observations together sug- gest that vitamin Bs may act in viuo to modulate the ability of hormone receptors to bind to DNA, either indirectly by influencing nuclear localization or directly by influencing the DNA binding capacity through an unknown mechanism(s). Such alterations in the DNA binding capacity of receptors should be reflected as changes in the expression of hormone- regulated genes, and attempts have been made to establish such a link between vitamin Bs and steroid hormone function in humans and in whole animals. The early work of Symes et al. (16) demonstrated that the stimulation of rat prostate growth by androgen is enhanced in animals maintained on a

vitamin Bs-deficient diet. Similarly, the estrogen stimulation of rat uterine growth and peroxidase activity is enhanced under conditions of vitamin B6 deficiency (60) whereas the incidence of glucocorticoid-induced cleft palate in rats has been observed to decrease as a result of vitamin Be adminis- tration during gestation (61). Similar results (discussed in the Introduction) have also been demonstrated in cell and organ culture. Although these studies all suggest that responsiveness to steroid hormone is altered in response to changes in vitamin Bs status, they do not distinguish between direct and indirect effects of vitamin Bs on steroid hormone-dependent events. This prompted us to introduce a reporter gene with a defined promoter into a cell culture line. In this manner, the regula- tion of expression of the reporter gene can be controlled through judicious choice of promoter sequences, and the in- tracellular pyridoxal phosphate concentration can be manip- ulated by supplementation of precursor or antagonist to the culture medium, thereby comprising a model system in which to investigate the effects of pyridoxal phosphate concentra- tion on glucocorticoid receptor-mediated gene expression.

It has been documented clearly that by maintaining animals (rats, rabbits, and cats) on a defined diet supplemented with pyridoxine, it is possible to produce an elevation in the intra- cellular pyridoxal phosphate concentration, as measured in liver, kidney, brain, and plasma (49, 51, 62). In addition, humans receiving a dietary supplement of pyridoxine exhibit increased levels of plasma and urine pyridoxal phosphate (reviewed in Ref. 48). Both a rat liver cell line (20) and mouse mammary gland organ culture (22) respond to pyridoxine- supplemented medium with an increase in the intracellular levels of pyridoxal phosphate. Thus, the intracellular pyri- doxal phosphate concentration appears to be very tightly regulated, necessitating the administration of high doses of precursor to affect even modest increases in the intracellular concentration of the active form of the vitamin (reviewed in Ref. 48). The increase in pyridoxal phosphate concentration is, then, not proportional but is directly correlated with the pyridoxine supplement dose. In contrast, animals that have been maintained on a vitamin Be-deficient diet as well as a cell culture line maintained in a growth medium that is devoid of pyridoxine exhibit a decrease in intracellular pyridoxal phosphate concentration (20, 51, 63). However, the decrease does not occur rapidly, and even extended exposure to the deficient diet or culture medium does not deplete intracellular pyridoxal phosphate. This is due most likely to the recycling of precursors and metabolites of pyridoxal phosphate present in cells prior to exposure to the deficient diet or culture medium. Additionally, from data with a rat hepatoma cell line maintained in vitamin Bs-deficient media (47), Meisler and Thanassi have suggested the existence of at least one pathway for pyridoxal phosphate synthesis which is not observed under normal conditions. Thus, simple maintenance of animals or cultured cells on a vitamin Be-deficient diet or medium is not sufficient to produce a pyridoxal phosphate deficiency. In the present study, we elected to take an alternate approach to establish a pyridoxal phosphate deficiency by using an agent long known to act as a vitamin Bs antagonist, 4-deoxypyri- doxine. McCormick and Snell (52) have demonstrated that 4- deoxypyridoxine binds with high affinity and inactivates pyr- idoxal kinase; this enzyme is required for synthesis of pyri- doxal phosphate, and its inactivation prevents formation of pyridoxal phosphate from both precursors (pyridoxine, pyri- doxamine) and metabolites (pyridoxal), thereby creating a state of vitamin Bg deficiency. By direct measurement, we were able to determine that this approach does in fact produce small but reproducible alterations in the intracellular pyri-

12432 Vitamin Be and Glucocorticoid Hormone Action

doxal phosphate concentration of HeLa cells, as shown in Table I.

The effects of pyridoxal phosphate concentration on the glucocorticoid receptor-dependent induction of CAT gene expression (Fig. 2) and the lack of effect on glucocorticoid- insensitive gene expression (Fig. 4) imply that the ability of the glucocorticoid receptor to induce gene expression can be influenced by the intracellular vitamin B, concentration. Sup- port for this conclusion is derived from the observation that basal CAT activity, from cells not stimulated with dexameth- asone, is also not affected by modulations in the intracellular pyridoxal phosphate concentration (not shown). Taken to- gether, these data demonstrate that the vitamin Bs status of whole cells has profound effects on glucocorticoid receptor- mediated gene expression. From the data shown in Figs. 5 and 6, we can eliminate concomitant effects on glucocorticoid receptor concentration or its ligand binding capacity as pos- sible mechanisms through which alterations in the pyridoxal phosphate concentration influence glucocorticoid hormone action.

There is considerable evidence that strongly suggests a physical association of the glucocorticoid receptor with pyri- doxal phosphate in vitro; the isoelectric focusing pattern of the glucocorticoid receptor is shifted to more acidic forms after treatment with pyridoxal phosphate (64), and cleavage of the receptor by trypsin is prevented by prior treatment with pyridoxal phosphate (14). In addition, by Western blot analysis using an antibody directed against pyridoxal phos- phate (65), a physical association of the glucocorticoid recep- tor with pyridoxal phosphate in vitro has been detected.2 These observations are consistent with the hypothesis that pyridoxal phosphate interacts directly with the glucocorticoid receptor, and we speculate that this interaction is responsible for the observed effects of pyridoxal phosphate concentration on glucocorticoid receptor-mediated gene expression. How- ever, it should be noted that from the available data it is not possible to distinguish unequivocally between a direct inter- action of pyridoxal phosphate with the glucocorticoid receptor in viuo or with some other cellular protein or factor which, in turn, influences the transcriptional modulatory function of the receptor. Nonetheless, the data presented here demon- strate that vitamin Bs modulates the ability of the glucocor- ticoid receptor to induce gene expression within a cell and thereby establishes a direct biological correlation between vitamin Bs and glucocorticoid hormone action in vivo.

Acknowledgments-We are grateful to Deborah Bellingham for her critical evaluation of the manuscript and to Paul Wagner for assist- ance in preparing the figures.

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