Do 1,25-dihydroxyvitamin D3 receptors depend on vitamin K?

NUTRITION RESEARCH, vol. 9, pp. 725-733, 1989 0271-5317/89 $3.00 + .00 Printed in the USA. Copyright (c) 1989 Maxwell Pergamon Macmillan plc. A l l rights reserved.

DO 1,25-DIHYDROXYVITAMIN D3 RECEPTORS DEPEND ON VITAMIN K? I . N. Sergeev, Ph.D. and V. B. Spirichev, D.Sc. I n s t i t u t e of N u t r i t i o n , Academy of Medical Sciences of the USSR, Moscow 109240, USSR

ABSTRACT Vitamin K deficiency in rats caused a rise of in vivo occupied 1,25(OH)2D3 receptor level in chromatin of the intestinal mucosa and a marked (2-2.5-fold) increase of intestinal cytosolic 1,25(OH)2D3-receptor complex binding with heterologous DNA, whereas 1,25(OH)2D3 binding with receptors did not change. Preincubation of renal and intestinal cytosol of K-avitsminotic rats with microsomal vitamin K-dependent gamma-carboxylating system reduced sharply 1,25(OH)2D3-receptor complex binding with DNA. In rats treated with vitamin K antagonist along with a low Ca diet, no dramatic decrease of occupied 1,25(OH)2D3 receptors occurred after the animals were maintained on a high Ca diet. No such effect was observed in vitamin K-replete rats. In v i t r o Ca2+ sharply diminished 1,25(OH)2D3-receptor complex binding with DNA only in vitamin K-replete rats (EDso = 2.5 9 10-6 M). The data demonstrate vitamin K-dependent, Ca-sensitive q u a l i t a t i v e modifiest i o n of 1,25(OH)2D3 receptor decreasing i t s a f f i n i t y to DNA. KEY WORDS:

Vitamin K I, 1,25(OH)2D 3 receptor, DNA.

INTRODUCTION Modern knowledge of the vitamin D-endocrine system suggests that the hormonally active metabolite, 1,25(OH)2D3, produced in kidneys, mediates its functions, in particular, in intestinal calcium absorption, by interaction with an intracellular receptor (1, 2). The receptor structure and activity regulation have not been closely studied. Recent work has shown that

725

726

I.N. SERGEEVand V.B. SPIRICHEV

1,25(OH)2D 3 and other D 3 metabolites (3, 4) as well as glucocorticoids and retinoids (5, 6) modulate 1,25(OH)2D 3 receptor concentration. Pyridoxal 5'-phosphate reduces the interaction of 1,25(OH)2D3 receptor with its nuclear binding components (7) and dietary phosphate deprivation alters its affinity to ligand (8). Qualitative modification of the receptor in the form of phosphorylation is probably needed to activate the interaction of the 1,25(OH)2D3-receptor complex with ehromatin (9). A mechanism for 1,25(OH)2D 3 receptor inactivation has not been described yet. When studying calcium metabolism and the vitamin D-endocrine system in vitamin K-depleted rats, we found unexpectedly an increase in the 1,25(OH)2D 3 receptor level occupied in vivo and a significant (2-2.5-fold) increase in the 1,25(OH)2D30-receptor complex binding with heterologous DNA in vitro. The preincubation of renal and intestinal cytosol of K-avitaminotic rats with microsomal vitamin K-dependent gamma-carboxylating system reduced the ability of the 1,25(OH)2D3-receptor complex to bind with DNA up to normal level. The findings reported suggest a vitamin K-dependent mechanism, which regulates 1,25(OH)2D 3 receptor properties. The mechanism whereby vitamin K down-regulates the binding of 1,25(OH)2D 3 receptor with DNA may include vitamin K-dependent gamma-carboxylation of the Glu-containing region of the receptor, enabling it to bind Ca 2+. This in turn can decrease the affinity of receptor to DNA or initiate its inactivation.

MATERIALS AND METHODS

Hale Wistar rats, approximately b O g BW were used in our experiments. In the first experiment the rats were fed a vitamin D-free diet containing 0.62% Ca, 0.93% P for 4 weeks (lO). The animals were subdivided into 8 groups of various vitamin D and K supply (see Table 1). The vitamin K (menadione sodium bisulfite) content in the diet was 200 mg/kg; vitamin D 3 was administered per os in a dose of 1.O pg per rat every other day. In the second experiment the vitamin K antagonist, 3-alpha-(4'-nitrophenyl)-beta-acetylethyl-4-hydroxycoumatin, was injected ip in a dose of 30 mg/kg BW for 5 days into the rats maintained on vitamin D-replete diet. In the third experiment the rats were fed a vitamin D-replete low Ca diet (0.03% Ca, 0.7% P) for 17 days and then for 5 days a high Ca diet (2.4% Ca, 0.7% P). The vitamin K antagonist was administered to half of these rats at a dose of 15 mg/kg BW for the last 12 days of the experiment. Endogenously (in vivo) occupied and unoccupied receptors for 1,25(OH)2D 3 in crude chromatin fraction of the intestinal mucosa were quantified by the slightly modified method of Hunziker et al. (ll, 12). Briefly, to determine the concentration of unoccupied receptors, aliquots (100 pl, 0.3 mg protein) of chromatin suspension in TED buffer (i0 mM Tris-HC1, 1.5 mM EDTA, 1.0 mM dithiothreitol /DTT/, 0.3 mM phenylmethylsulfonylfluorid 7.4) were incubated for 17 h at 0-4~ with 1,25-dihydroxy/26,27methyl- H/cholecalciferol (specific activity 180 Ci/mmol, Amersham, UK) in the absence (total binding) or presence (nonspecific binding) of a 250-fold

/PMSF/,3PH

D3 RECEPTORS AND VITAMIN K

727

excess of unlabeled 1,25(OH)2D 3 (s kind gift from Dr. M. Uskokovic, Hoffman-LaRoche, USA). 3H-1,25(OH)2D3 saturating concentration (1.8 nM) was used for routine assay of chromatin 1,25(OH)2D 3 receptors level. To measure the occupied 1,25(OH)2D 3 receptors, the unoccupied binding sites were blocked by preincubation of chromatin fraction at 0-4~ with 200 pM Na-ptosyl-L-phenylalanine chloromethylketone following quantitation of occupied receptors by exchange incubation with 3H-1,25(OH)2D 3 at 37~ for 30 min (ll, 13). To determine the unoccupied and occupied 1,25(OH)2D3 receptor concentrations and binding kinetics in intestinal cytosol, total receptors were extracted with high-salt buffer (TED containing 0.3 H of KC1). Aliquots (I00 pl, 0.2 mg of protein) of cytosol, obtained by high-speed centrifugation, were incubated for 17 h at 0-4~ with increasing (0.055-1.8 nM) or saturating (i.0 nM) 3H-1,25(OH)2D 3 concentrations. The hormone bound to the cytosolic or chromatin receptors was separated from the free ligand by the hydroxylapatite (HAP) assay Ill). The washed HAP pellets were extracted with a mixture of chloroform-methanol (1:2, v/v) (14). DNA-cellulose (5-6 mg DNA/g) was prepared using salmon DNA by the method of Alberts and Herrick (15). The DNA-cellulose suspension in TED buffer (50~, w/v) was added to labeled cytosolic receptor preparations to give a final concentration of KCI of 0.15 M and incubated for 45 min at 0-4~ DNA-cellulose pellet was twice washed with TED buffer containing 0.SW Triton X-lO0. The effect of vitamin K-dependent gamma-carboxylation on 1,25(OH)2D 3 receptor properties was evaluated in rats treated with vitamin K-antagonist. For this purpose, intestinal mucosa and kidney homogenates in TD-KC1 buffer (lO mM Tris-HC1, lO mM DTT, 0.3 mM PMSF, 0.3 M KCI, pH 7.4) were centrifuged at 15,000 g, 4~ for 20 min; Triton X-lO0 was added to the supernatant to give a final concentration of 2% (v/v). Soluble microsomes were prepared by supernatant centrifugation at 105,000 g, 4~ for 1 h. Cytosol aliquots (1 ml, I0 mg of protein) were incubated for 3 h at 0-4~ in the presence of complete carboxylating system (0.I mM vitamin K1, 2 mM NADH, 1 mM NaHCO 3) (16, 17). All cytosol preparations were adjusted with TED-0.3 M KC1 buffer to approximately 1 mg/ml of protein prior binding assay. 1,25(OH)2D 3 receptor concentrations and their binding with DNA-cellulose were measured as described above, but an excess of non-labeled 25-OHD 3 (50 nM) was added to the kidney samples 1 h before the HAP treatment in order to circumvent unusually high nonspecific binding due to contamination with vitamin D-binding protein. To assess in vitro Ca 2+ effect (5"i0 -7 5"10 -4 M) on 1,25(OH)2D3-receptor complex binding with DNA-=cellulose and HAP, renal cytosol was prepared with TD-KC1 buffer. The same cytosol was used to quantify 45Ca binding with HAP and DNA-cellulose.

RESULTS

Vitamin K deficiency did not affect the level of receptors occupied with 1,25(OH)2D3, but caused a moderate (non-significant due to the high spread in values) decrease of unoccupied receptor concentration leading to the increased percentage of occupied receptors. A similar pattern of the changes of unoccupied and occupied receptor levels was observed in the case of a combined vitamin D and K deficiency and also, when vitamin D-depleted rats were treated with that vitamin (Table 1).

728

I.N.

SERGEEV and V.B. SPIRICHEV TABLE i

C o n c e n t r a t i o n o f Unoccupied and I n V i v o Occupied 1,25(OH)2D 3 R e c e p t o r s i n Chromatin o f t h e I n t e s t i n a l Mucosa o f Rats w i t h V a r i o u s V i t a m i n D and K Supply

Treatment

i. 2. 3, 4. 5. 6. 7. 8.

+D+K +D-K -D+K -D-K -D+K, -D-K, -D+K, -D-K,

Note:

+Did +Did +D 5d +D 5d

Receptor Concentration Unoccupied Occupied fmol/mg prot

63.0 41.6 69.4 53.6 65.4 /;4.8 60.8 38.6

+ • • i • • i •

19.3 13.7 19.7 14.0 22.7 8.5 12.9 3.2

7.69 9.64 <1.0 <1.0 10.2 7.80 8.30 8.33

Per Cent Occupied

• 1.8 + 3.5

• i i •

10.9 18.8 <2.0 <2.0 13.5 14.8 12.0 17.4

3.4 2.1 1.2 0.i

+D and -D, rats replete and depleted of vitamin D. +K and -K, rats replete and depleted of vitamin K. ld and 5d, period of vitamin D 3 administration. The data are means • SE for three observations

Concentration, maximum binding capacity and affinity 1,25(OH)2D 3 receptors in vitamin K deficiency did not change.

of

cytosolic

A sharp rise (2-2.5-foid) of the 1,25(OH)2D3-receptor complex (holoreceptor) binding with DNA-cellulose was quite unexpected in vitamin K deficiency (Table 2). A similar effect was seen in combined deficiency of vitamin D and K and in the case of vitamin D 3 was administered to these rats. A significant effect of vitamin K deprivation on 1,25(OH)2D3-receptor complex binding with DNA gave rise to the probiem of finding the mechanism of that effect. Since the major biochemicaI function of vitamin K is to act as a cofactor in the posttranslational gamma-carboxylstion of some Ca-binding proteins (17, 1B), we tried to find a possible connection between this process and the observed changes of the properties of the 1,25(OH)2D 3 receptor. With that purpose in mind, we used 1,25(OH)2D ) receptors extracted with a high ionic strength buffer in the presence of Triton X-IO0 from the intestinal mucosa and kidney of rats treated with vitamin K antagonist. The cytosol preparations were incubated in vitro with the carboxylating system under conditions optimal for both gamma-carboxylation and following 1,25(OH)2D ) binding with receptor. In this second experiment, as in the first one, in rats receiving a vitamin K antagonist the binding of the cytosolic 1,25(OH)2D3-receptor complex both intestinal and renal with DNA-cellulose was 2-3 times as high as in controls (Table 3). The level of occupied receptors in kidney of rats treated with vitamin K antagonist was about twice as high as in the intact animals. Since occupied 1,25(OH)2D 3 receptors are located in nuclei, the data suggest that an increased binding of the 1,25(OH)2D3-receptor complex with DNA in vitamin K deficiency may occur not only in vitro but under physioiogical conditions (in vivo) as well.

D3 RECEPTORS AND VITAMIN K

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D3 RECEPTORS AND VITAMIN K

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The incubation of i n t e s t i n a l and renal cytosol of i n t a c t rats with a complete system for vitamin K-dependent gamma-earboxylation of endogenous substrates (vitamin K 1 + NADH + HCO~ + DTT) did not affect the parameters of 1,25(OH)2D3-receptor complex binding with DNA-cellulose. The incubation of intestinal and renal cytosol of rats, receiving vitamin K antagonist, with the same complete system leads to a decrease of 1,25(OH)2D3-receptor complex binding with DNA-cellulose to the level characteristic of intact animals. The incubation with vitamin K 1 in the absence of NADH and HC03 did not produce that effect (Table 3). Simultaneously, carboxylation of renal endogenous substrates of rats, treated with vitamin K antagonist, reduced cytosolic concentration and per cent of the in vivo occupied receptors for 1,25(OH)2D 3. No such effect was found in the intestinal cytosol.

A decrease of calcium content in the diet of rats from 0.6 to 0.03~ led to an increase of occupied 1,25(OH)2D 3 receptor level from 14-18~ to 46-52~. The administration of vitamin K antagonist to the rats did not markedly effect the level of occupied 1,25(OH)2D 3 receptors of rats fed low calcium diet (cot. 49 vs. 38~ - in the control), but abolished to a great extent a decrease of the percentage of occupied 1,25(OH)2D 3 receptors when calcium content in the diet was increased to 2.4~ (cot. 32 vs. 8~ - in the control). One hour exposure of in vitro occupied I t25(OH)2D 3 receptors, extracted with EDTA-free buffer, to Ca2+ (5"10-7 5"i0 -4 M) at 0-4~ dramatically decreased t h e i r binding with DNA-cellulose in vitamin K-supplied rats (ED50 = 2.5 -10-6 M), but p r a c t i c a l l y did not affect the binding in rats treated with vitamin K antagonist (ED50 over 5"10 -5 M). 1,25(OH)2D3-receptor complex adsorption on HAP remained unchanged in terms of the Ca 2+ concentration used (data not shown). But in vitamin K replete rats 45Ca binding by HAP with a simultaneous adsorbing 1,25(OH)2D3-receptor complex on it increased only at Ca 2+ over 5"10 -5 M. In vitamin K-deplete rats no such increase was found. 45Ca adsorption on DNA-cellulose was minimal and was not dependent on vitamin K (data not shown).

DISCUSSION These data demonstrate that vitamin K deficiency in rats caused s rise of in vivo occupied 1,25(OH)2D 3 receptor level in chromatin of the intestinal mucosa and a marked (2-2.5-fold) increase of intestinal cytosolic 1,25(OH)2D3-receptor complex binding with heterologous DNA. Preincubation of renal and intestinal cytosol of K-avitaminotic rats with microsomal vitamin K-dependent gamma-csrboxylating system reduced 1,25(OH)2D3-receptoz complex binding with DNA. In vitro Ca 2+ diminished 1,25(OH)2D3-receptor complex binding with DNA only in vitamin K-replete rats. These data suggest the possible role of vitsmin K-dependent gamma-carboxylation in modulation of 1,25(OH)2D3-receptor properties. A suitable explanation of gamma-carboxylation's possible role in 1,25(OH)2D 3 receptor modification may derive from the proposal that gammaglutamyl residues are present in the receptor protein. Since hormonal interaction of 1,25(OH)2D 3 with target ceils are accompanied by an increase in Ca 2+ intracellular concentration (19), it is possible that this ion may terminate hormone effect inactivating the 1,25(OH)2D 3 receptor by feedback regulation. Mechanism of such inactivation may include the interaction of Ca z+ with gamma-carboxyglutamyl residues in DNA-binding domain of the receptor that attenuates its affinity to DNA or triggers cleavage of the receptor, but, evidently, did not affect binding of 1,25(OH)2D 3 to the

732

I.N. SERGEEV and V.B. SPIRICHEV

receptor. No changes of maximum binding capacity and equilibrium dissociation of 1,25(OH)2D 3 receptor in various vitamin K supply of the rats speak in favour of this view. It should be noted that a significant influence of calcium on 1,25(OH)2D3-receptor complex binding with DNA in vivo and in vitro was found only in vitamin K-replete rats. These data may suggest that calcium sensitivity of 1,25(OH)2D ) receptor depends on vitamin K. We should also not rule out the possibility that vitamin K effect on 1,25(OH)2D 3 receptor involves any other modifying factor dependent on this vitamin. By analogy with well-known clotting factors, it may represent a serine proteinase. But the presence of PMSF, inhibitor of such activity, in the buffer for 1,25(OH)2D 5 receptor extraction enables us to discard this proposal. Our preliminary results also indicate that 1,25(OH)2D3 production in rat kidney in vitro does not significantly change in vitamin K deficiency. Evidently, occupied 1,25(OH)2D 3 receptors level hardly increases due to the simple elevation of circulating concentration of this hormone. Study with a highly purified 1,25(OH)2D 3 receptor is needed to further clarify the role of vitamin K-dependent posttranslational modification of 1,25(OH)2D ) receptor protein. Taking into account the role of Ca 2+ as the second messenger to external stimuli, it should be interesting to investigate in this context receptors of other steroid hormones and some intracellular Ca-binding proteins, which may be classified as Ca 2+ receptors. Thus, our data suggest for the first time that there is a mechanism of inactivation of the preformed 1,25(OH)2D 5 receptors involving vitamin K-dependent gamma-carboxylation of this protein.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the technical Sokolnikov and secretarial assistance of I. V. Urusova.

assistance

of

A.

A.

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G.

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Recent findings in understanding Medical Biol 1982; 60:16-2&. M.

Accepted for publication March 28, 1989.

Vitamin

K

and

1,25-

the biological

urogenital

tract.

Annu Rev Nutr 19Ba;