(Received for publication, December 1, 1994; and in revised form, March 21, 1995)
From the
Deprivation of vitamin A (retinol) leads to reduced potential of B cell proliferation and nearly complete block of T cell activation in vitro. Retinol, which is thought to function as a pro-hormone, is enzymatically converted into intracellular messenger molecules. Thus, 14-hydroxy-retro-retinol (14-HRR) is an intracellular messenger molecule linked to activation and growth regulation of lymphocytes; whereas, anhydroretinol, another natural retro-retinoid, is an antagonist of 14-HRR effects. In this article, we describe the isolation, structure determination, synthesis, and biological properties of a new intracellular retinol derivative, 13,14-dihydroxy-retinol (DHR), which also supports the viability of retinol-deprived lymphocytes. DHR is found in numerous cell lines representing a large cross-section of tissues and animals from insects to mammals. In T lymphocytes the production of DHR and 14-HRR is up-regulated by phorbol ester. DHR is converted to 14-HRR by mild acid treatment, but not by cells; therefore DHR is not a biosynthetic intermediate in the conversion of retinol to 14-HRR. DHR is a distinct end point of retinol metabolism. Although it is linked to cell proliferation, its biological role remains to be determined.
When B or T lymphocytes encounter an antigen, they execute, when
this event is appropriately amplified by co-receptor interactions, a
preordained program of proliferation and differentiation. The receptor
stimulation triggers a complex network of signals needed to induce
changes in the cell biochemistry and architecture, and to provide
commands for the requisite genomic changes. While this signal network
is highly organized and includes many interwoven circuits, one mode of
signal transduction is carried out by small diffusable messenger
molecules, which comprise polar substances (i.e. inositol
phosphates, cyclic nucleotides), and small lipophilic molecules
(steroids, vitamin D, thyroid hormone, and retinoic acid). The latter
class of compounds bind to and activate specific receptors which belong
to the superfamily of steroid receptors, inducing transcriptional
activation(1, 2, 3, 4) . Thus,
all-trans-retinoic acid 1 (Fig.1) and its 9-cis
isomer bind to and activate the retinoic acid nuclear receptors,
whereas, at higher concentrations, 9-cis-RA ()activates also
the retinoid X nuclear receptors(5, 6) . In addition,
4-oxo-RA, considered so far to be an elimination catabolite product of
RA, has been reported to be a potent ligand of the retinoic acid
nuclear receptors.
Figure 1: Retinoid structures.
On the other hand, retinol 2 itself, or metabolites distinct from the retinoic acid series, are essential for the regulation of spermatogenesis (8, 9) and the immune system(10, 11, 12) . In particular, 14-hydroxy-4,14-retro-retinol (14-HRR) 3, a new retinol metabolite, and the first bioactive retro-retinoid reported, is a potent intracellular mediator of B cell proliferation and T cell activation and growth(13, 14) . In contrast, anhydroretinol (AR) 4, another naturally occurring retro-retinoid(15, 16) , long known as the inactive retinoid (17, 18) , acts as a reversible inhibitor of retinol and 14-HRR effects (19, 20, 21) . 14-HRR and AR have been identified in various mammalian and insect cell lines(19, 20, 21, 22) ; they constitute the first naturally occurring agonist/antagonist pair of small lipophilic messenger molecules described.
In addition to these retro-retinoids, we have identified in lymphoblastoid 5/2
cells grown in the presence of [H]retinol,
another retinol metabolite P1, more polar than 14-HRR; we have now
characterized it as 13,14-dihydroxy-retinol (DHR) 5. DHR is also
active in the B cell proliferation and the T cell activation assays.
Because of its structure and its shared biological properties with
14-HRR, P1 was thought first to be the precursor of 14-HRR. However, P1
is not converted to 14-HRR by the cells; this leads to the conclusion
that both retinoids are independent end points of retinol metabolism.
To evaluate the effects of retinoids on T cell activation,
multiwell plates were coated with anti-CD3 antibody at 10 µg/well
in phosphate-buffered saline overnight, and washed. Retinoids were
added to washed thymocytes of 4-6-week-old BALB/c mice, which
were suspended in ITLB medium at 2 10
cells/ml.
Cell cultures were pulsed on day 4 for 4 h with
[
H]thymidine (0.8 µCi/well).
To evaluate
the production of DHR by normal T cells, pooled lymphocytes from
spleens and lymph nodes of BALB/c mice were purged of adherent cells by
culture in untreated Petri dishes for 1 h. Unattached cells were
transferred to Petri dishes coated with anti-IgM antibody (10
µg/ml) at a rate of 10 cells/10-cm dish, and the plates
were kept on ice for 3 h. 35% of input cells were recovered as
unattached T cells and enriched to 85% purity as shown by
immunofluorescence staining with anti-Thy-1.2 antibody. Aliquots of 4
10
cells in 1 ml of serum-free ITLB medium were
pulsed with 10 µl of HPLC purified [
H]retinol
and placed in a tissue culture incubator. At the onset of culture, half
of the aliquots were stimulated with phorbol myristate acetate (PMA) at
100 ng/ml. Stimulated and unstimulated cultures were each harvested at
times 0, 1, 2, and 4 h, and the cells extracted by the method of
McClean(24) . The extracts were analyzed by reversed phase HPLC
(analytical C
column, Vydac, Hesperia, CA) using the
elution gradient described in Fig.3. The approximate amounts of
DHR and 14-HRR formed were estimated by the integration of the
respective radioactive peaks and the known specific radioactivity of
the input retinol, assuming that dilution by endogenous retinoids did
not occur.
Figure 3:
High-pressure liquid chromatography of
retinoids from lymphoblastoid 5/2 cells. Analytical C reversed-phase column; water/methanol/chloroform gradient; flow
rate 1 ml/min; photodiode array detection. Disintegrations per min were
determined with an on-line scintillation counter. The 5/2 cells (1
10
cells in 20 ml of RPMI, 10% fetal calf serum
medium) were incubated with 40 µCi of
[
H]retinol; specific activity 49.3 Ci/mmol. After
16 h, retinoids were extracted from the washed cell pellet according to
the procedure of McClean et al.(24) . Unlabeled
synthetic retinoids (arrows) were used as reference standards:
1) 13,14-dihydroxy-retinol; 2) 14-hydroxy-4,14-retro-retinol;
3) retinol; 4) anhydroretinol; 5) retinyl
esters.
Cell growth curves of HL-60 cells were obtained by taking
aliquots from cultures in 12-well culture dishes (Costar, Cambridge,
MA) and counting the cells in a Neubauer chamber. Cell numbers were
kept between 1 and 5 10
cells/ml. Viability was
assessed by trypan blue exclusion. Assays were done in duplicate. For
detection of differentiation, HL-60 cells were tested for their ability
to reduce nitro blue tetrazolium (Sigma). During a 30-min stimulation
with 10
M PMA (Sigma), cells were provided
with 0.61 mM nitro blue tetrazolium and analyzed for staining
under the microscope(25, 26) . The percentage of
differentiated cells was determined in relation to the total number of
viable trypan blue negative cells. Cells of positive controls were
treated for 3 days with 10
M retinoic acid.
For quantitative assays of DHR and 14-HRR biosynthesis,
cells (40 10
) in ITLB medium were labeled with 10
µCi of [
H]retinol and cultured for the
indicated time periods. Cells were recovered by centrifugation in the
cold and extracted as described above(24) , using 1 ml of
extraction buffer. The recovered butanol extract was analyzed by HPLC
as described in the legend to Fig.3. The eluate was monitored
by an on-line scintillation counter (Radiomatic, Tampa, Fl) with
quench correction.
To check whether DHR was a biosynthetic precursor
of 14-HRR, 11,12-[H]DHR was isolated from
extracts of murine spleen cells stimulated with PMA in the presence of
11,12-[
H]retinol, and purified to homogeneity by
reversed phase HPLC. Lymphoblastoid 5/2 cells were cultured for 4 h in
serum-free medium in the presence of 6.4
10
M [
H]DHR, and 10
M unlabeled retinol. The cell extract was then analyzed
by reversed phase HPLC as described in the legend to Fig.8.
Figure 8:
Lymphoblastoid 5/2 cells do not convert
13,14-DHR to 14-HRR. Lymphoblastoid 5/2 cells were cultured for 4 h in
serum-free medium in the presence of [H]DHR and
10
M unlabeled retinol. The cell extract
was analyzed using the conditions described in the legend to Fig.2. The radioactive peaks were monitored by an on-line
liquid scintillation counter (dotted line); the solid line corresponds to the chromatogram monitored at 348 nm, the
max
of 14-HRR. The positions of DHR (1), 14-HRR (2), and
retinol (3) were determined in calibration
runs.
Figure 2:
Synthetic steps for
(13S,14R)-, and
(13R,14R)-13,14-DHR: (a) CHMgBr,
6, THF, reflux, followed by addition of 7 in THF, reflux, 84%; (b) flash chromatography SiO
, gradient
hexane/ethyl acetate 95/5 to 90/10; (c) pTsOH,
CH
OH, RT, 65%; (d) lithium aluminum hydride (LAH), ether, reflux, 80%.
The C-13 absolute configuration of synthetic DHR 10b and 11b was determined by the exciton chirality method (29) after lithium aluminum hydride reduction of the triple bond of the all-trans-14,15-acetonides 8b and 9b, respectively, and conversion of 13-OH to 13-p-methoxycinnamoyl ester(30) .
Figure 4:
A,
CD spectrum in CHOH; and B,
H NMR in
CD
CN of natural DHR:
1.06 (s, 6H,
1-(CH
)
), 1.22, 1.23 (2 s, 3H, 13-Me), 1.46 (m,
2H, 2-CH
), 1.61 (m, 2H, 3-CH
), 1.68 (s, 3H,
5-CH
), 1.91 (s, 3H, 9-CH
), 2.02 (t, J = 8 Hz, 2H, 4-CH
), 3.32-3.62 (m, 3H, 14-H
and 15-H
), 5.79, 5.81 (2 d, J = 16 Hz, 1H,
12-H), 6.07 (d, J = 16 Hz, 1H, 8-H), 6.08 (d, J = 12 Hz, 1H, 10-H), 6.17 (d, J = 16 Hz, 1H,
7-H), 6.64 (dd, J = 12 Hz, 16 Hz, 1H,
11-H).
The high resolution EI/MS ()gave an
observed value of 320.2354 (calculated for
C
H
O
= 320.2351),
indicating that P1 has two hydrogen atoms and two oxygen atoms (which
could be accounted for by two hydroxyl groups) more than retinol,
C
H
O.
The H NMR spectrum (Fig.4B) displays four olefinic protons which
establish P1 as a 13,14-DHR; thus P1 has two asymmetric centers at C-13
and C-14. The two singlets at 1.22 and 1.23 ppm (Fig.4B) (
)are characteristic of a
C(OH)CH
group and correspond to 13-Me: P1 very likely
exists as a mixture of diastereomers. That P1 exists as a mixture of
diastereomers is supported by two doublets at 5.79 and 5.81 ppm (12-H; J 16) integrating for 1H. This was also confirmed by
synthesis. Namely, the
H NMR spectra of minor natural P1
(
13-Me 1.23 ppm), and major natural P1 (
13-Me 1.22 ppm) are
identical to those of the synthetic diastereomers
all-trans-DHR 10b and 11b. The absolute
configurations at C-13 and C-14, however, remain to be established.
Therefore, we synthesized one set of the optically active
diastereomers: (13S,14R)- and
(13R,14R)-DHR.
Fig.5A shows the CD spectra of (13S,14R)- and (13R,14R)-all-trans-DHR 10b (first positive CD) and 11b (first negative CD), respectively, and their absorption spectra. The (13S,14R)- and (13R,14R)-9-cis isomers exhibit opposite CD signs to that of their corresponding all-trans diastereomer (curves not shown). This inversion of the CD signs has been observed also with mono-cis carotenoids as compared to the all-trans isomer of the same carotenoid(31) .
Figure 5: A, CD spectra of synthetic (13S,14R)-, and (13R,14R)-all-trans-DHR 10b and 11b, respectively, and their corresponding absorption spectrum. B, CD spectrum of 14,15-acetonide of (13R,14R)-all-trans-DHR, its 13-p-methoxycinnamoyl ester showing a first positive Cotton effect characteristic of a 13R configuration, and their respective UV spectra.
Fig.5B (solid
line) shows that the CD spectrum of the 13,14-acetonide,
corresponding to (13R,14R)-all-trans-DHR 11b, also exhibits a first negative CD band. However, the
corresponding 13-p-methoxycinnamoyl-13,14-acetonide exhibits a
prominent split CD centered at 294 nm due to exciton coupling between
the polyene chain and the cinnamate chromophores, max = 292
and 311 nm, respectively(29) . The positive sign of the first
couplet (32) is in full agreement with the 13R absolute configuration in 11b. As expected, the
13-p-methoxycinnamoyl-13,14-acetonide, corresponding to the
(13S,14R)-diastereomer 10b, exhibits a split
CD with a first negative couplet (curve not shown) which is consistent
with the 13S absolute configuration in 10b.
Natural
DHR exists as a mixture of isomers, and we know from the NMR data that
at least two diastereomers are present in a 5/4 ratio. Indeed, the
major diastereomer ( 13-Me 1.22 ppm; Fig.4B) has
a
H NMR spectrum identical to that of
(13R,14R)-all-trans-DHR 11b (or its
antipode); whereas the minor P1 (
13-Me 1.23 ppm; Fig.4B), has the same
H NMR spectrum as
(13S,14R)-all-trans-DHR 10b (or its
antipode). Its CD spectrum (Fig.4A), however, does not
look like a simple summation of the CD curves of two diastereomers. In
addition, the optical activity of ``natural'' P1 varies with
the concentrations of retinol added to the cell cultures: under
unphysiological conditions (10
M retinol)
P1 was even racemic! Just like
14-hydroxy-retro-retinol(14) , optically active
natural P1 may result from the opening of optically active
13,14-epoxyretinol, which can be produced via stereospecific 13,14-ene
epoxidation of retinol bound to cellular retinol-binding protein
(1-3
10
M). Under
unphysiological conditions, most retinol within the cells is free or
nonspecifically bound to membranes and nonspecific cellular proteins,
leading to random epoxidation and ring opening. Thus, it is possible
that as in the case of 14-HRR(14) , the stereochemistry of P1
is dependent on subtle biosynthetic conditions and varies from case to
case (Fig.6). The minute amount of P1 extracted under
physiological conditions (i.e. cells cultured in medium
containing 10% serum) precluded the determination of its absolute
configuration.
Figure 6: Plausible routes leading to 14-HRR and DHR from the retinol precursor; the epoxide is a putative intermediate.
Figure 7:
Effect of DHR on T cell activation and
growth of promyelocytic HL-60 cells. A, comparative dose
responses of DHR, 14-HRR, and retinol in T cell activation. Thymic T
cells of BALB/c mice were activated with immobilized anti-CD3
antibody and graded doses of retinoids added at the onset of culture.
Proliferation was measured by incorporation of tritiated thymidine into
DNA on day 3. Means of triplicate measurements that were within 15% of
each other are plotted. Natural racemic DHR, a mixture of synthetic
9E/9Z diastereomeric DHR, and synthetic racemic 14-HRR were used. B, HL-60 cells from stock cultures in 10% fetal calf serum
were reseeded in serum- and retinol-free ITLB medium and natural
racemic DHR added daily to the final concentrations shown. Cell numbers
and viability were determined by trypan blue exclusion and microscopic
counting in cultures with different retinoid concentrations. The cell
density was kept between 3
10
and 2
10
cells/ml by providing additional culture medium
accordingly. Shown is the total cell number from days 4 through 12 of
cultures.
, 0.3 µM DHR;
, 0.1 µM
DHR;
, 0.03 µM DHR;
, 0.01 µM DHR;
, none.
Contrary to retinoic acid (10
M) and retinol (
2
10
M), the retro-retinoids 14-HRR and AR do not
induce differentiation of HL-60 cells to granulocytes(21) .
Similarly, DHR (2
10
or 1
10
M) does not induce HL-60 cell
differentiation as measured by the nitro blue tetrazolium reduction
assay.
The cell growth promoting effect of 14-HRR is reversibly antagonized by AR(19, 20) , suggesting that 14-HRR and AR bind to the same receptor. Surprisingly, assays carried out with the T cell line ERLD show clear evidence for reversible inhibition of AR cytotoxicity by 13,14-DHR (data not shown). Although structural differences suggest different receptors for 14-HRR and DHR, binding to the same receptor could be also rationalized by the fact that DHR, just like retinol bound to cellular retinol-binding protein(33, 34) , can adopt a 6-s-trans conformation leading to ring/chain coplanarity, which in 14-HRR is imposed by the retro structure.
The functional similarity
of DHR and 14-HRR and their common existence in cells suggest that DHR
could be the biosynthetic intermediate in the pathway to 14-HRR.
Indeed, DHR is easily converted to 14-HRR upon mild acid treatment.
However, studies with high doses (10M) of
synthetic DHR, or [
H]DHR (obtained
biosynthetically from [
H]retinol) did not lead to
detectable amounts of DHR in 5/2 lymphoblastoid cells. Indeed, the
chromatogram of extract from lymphoblastoid 5/2 cells cultured in the
presence of [
H]DHR and unlabeled retinol (Fig.8) does not exhibit radioactivity above background at 19.5
min, the retention time of 14-HRR. However, a small amount of 14-HRR is
synthesized from unlabeled retinol (see absorption spectrum in Fig.8, inset), indicating that the 14-HRR biosynthetic
pathway was active at the time of the experiment. The estimated limit
of detection is about 1-5% of the input radioactivity, and thus a
small amount of 14-HRR could have gone undetected. However, because
steady-state concentrations of 14-HRR and DHR in cells approximate a
1/1 ratio, we would have expected conversion of half the input DHR to
14-HRR. Thus, DHR is not a metabolic intermediate in the biosynthesis
of 14-HRR from retinol, but rather a distinct end point of retinol
metabolism alongside 14-HRR. In other experiments carried out with HeLa
and Jurkat T cells, synthetic unlabeled DHR was not metabolized to
14-HRR (data not shown).
The production of 14-HRR and DHR is not
limited to transformed cell lines. They are produced in normal T cells
where their synthesis is up-regulated by phorbol ester, as shown in Fig.9. Moreover, a limited trace labeling study with
[H]retinol shows a radioactive peak coeluting
with DHR in myeloid cells and tumor cell lines derived from lung,
colon, prostate, breast, and nervous tissues. The DHR peak is also
present in tissue culture cells of various insects (Aedes, Manduca, Drosophila). In fact, virtually all dividing
cells seem to produce simultaneously DHR and 14-HRR. DHR is also found
in amphibians. Thus, extraction of 7 ml of untreated Xenopus laevis oocytes, followed by HPLC purification using water/methanol and
water/acetonitrile gradients, successively, led to 4
10
M DHR and 2
10
M 14-HRR. These data suggest a broad evolutionary
conservation of retinol metabolic pathways, ranging from invertebrates,
to amphibians, to mammals.
Figure 9:
The
production of DHR and 14-HRR in T lymphocytes is up-regulated by
phorbol ester. Purified T cells of BALB/c mice were cultured in
serum-free medium in the presence of [H]retinol. A, control cultures produced both DHR and 14-HRR, reaching
steady state levels 60 min after the initiation of cultures. B, however, PMA treated cultures led to 2-4 times more
DHR and 14-HRR.
Our data support the hypothesis that metabolism of retinol leads to several molecules with different biological functions involving either control of cell growth or cell differentiation. The latter is exemplified by the acid derivatives of retinol, i.e. all-trans-RA, 9-cis-RA, and all-trans-4-oxo-RA, which induce differentiation accompanied by growth arrest via activation of nuclear receptors. On the other hand, 14-HRR and DHR serve as growth-promoting factors for retinol-dependent cells, but do not induce differentiation. AR is a functional antagonist of 14-HRR and DHR with growth-inhibiting activity. Whether 14-HRR, DHR, and AR function through nuclear, cytoplasmic, or membrane receptor is under investigation.