(Received for publication, November 14, 1994; and in revised form, January 19, 1995)
From the
The key metabolite of vitamin D,
1
,25-dihydroxyvitamin D
(1,25-D
), induces
rapid cellular responses that constitute a so-called
``non-genomic'' response. This effect is distinguished from
its ``classic'' genomic role in calcium homeostasis involving
the nuclear 1,25-D
receptor. Evidence is presented that
protein kinase C (PKC) is directly activated by 1,25-D
at
physiological concentrations (EC
= 16 ± 1
nM). The effect was demonstrable with single PKC-
,
-
, and -
isoform preparations, assayed in a system containing
only purified enzyme, substrate, co-factors, and lipid vesicles, from
which it is inferred that a direct interaction with the enzyme is
involved. The finding that calcium-independent isoform PKC-
was
also activated by 1,25-D
shows that the calcium binding C2
domain is not required. The level of 1,25-D
-induced
activation, paired with either diacylglycerol or
4
-12-O-tetradecanoylphorbol-13-acetate, was greater than
that achievable by any individual activator alone, each at a saturating
concentration, a result that implies two distinct activator sites on
the PKC molecule. Phosphatidylethanolamine present in the lipid
vesicles potentiated
4
-12-O-tetradecanoylphorbol-13-acetate- and
diacylglycerol-induced PKC activities, whereas
1,25-D
-induced activity decreased, consistent with
1,25-D
-activated PKC possessing a distinct conformation.
The results suggest that PKC is a ``membrane-bound receptor''
for 1,25-D
and that it could be important in the control of
non-genomic cellular responses to the hormone.
The active metabolite of vitamin D, 1,25-dihydroxyvitamin
D
(1,25-D
), (
)a steroid type
hormone, is known for its regulatory role in calcium homeostasis. The
hormone, derived either from 7-dehydroxycholesterol by the action of
ultraviolet light on the skin or from the diet, facilitates the
absorption of calcium from the intestine, its mobilization from bone,
and its resorption in the kidney (DeLuca et al., 1990;
Burgos-Trinidad et al., 1990; Studzinski et al.,
1993). Apart from its role in calcium uptake, 1,25-D
is
widely acknowledged to be an important regulator of cell growth and
differentiation (Nemere et al., 1993; Darwish and DeLuca,
1993; Lowe et al., 1992).
Within target cells, it binds to
the vitamin D receptor in the nucleus, which regulates gene expression
by interacting with transcription factors. However, recently, it has
become clear that many cellular responses to the hormone are too rapid
to be mediated by gene expression controlled by the vitamin D receptor.
For example, 1,25-D rapidly stimulates the turnover of
phosphoinositides and phosphatidylcholines in a wide range of cell
types, leading to increases in the levels of inositol triphosphate and
diacylglycerol (DAG) (de Boland et al., 1994; Morelli et
al., 1993; Civitelli et al., 1990; Bourdeau et
al., 1990; Wali et al., 1990). These rapid effects,
typical of a membrane receptor-type response, have led to the
recognition of the importance of the non-genomic role for 1,25-D
in cell regulation.
PKC occupies a central position in signal
transduction and controls diverse cellular processes, including growth
and differentiation (Nishizuka, 1992; Stabel and Parker, 1991; Bell et al., 1992; Hug and Sarre, 1993). The release of calcium
from intracellular stores, induced by inositol triphosphate, along with
an increase in extracellular calcium influx promoted by 1,25-D (Morelli et al., 1993) triggers a phospholipid-dependent
translocation of calcium-dependent PKC isoforms to the membrane. DAG
liberated by phosphoinositide hydrolysis then activates the
membrane-bound PKC by inducing a conformational change in the enzyme. A
number of studies implicate an involvement of PKC in the non-genomic
actions of 1,25-D
(for example, see Boland et
al.(1991), Simboli-Campbell et al.(1994), de Boland and
Norman(1990), Simpson et al.(1989) Lissoos et
al.(1993), van Leeuwen et al.(1992)). Evidence for this
comes from a similarity in the effects of PKC activators (e.g. phorbol esters) to those of 1,25-D
on certain cellular
responses and from the reversibility of these responses by PKC
inhibitors (Simpson et al., 1989; Khare et al., 1993;
de Boland and Norman, 1990; van Leeuwen et al., 1992).
1,25-D also induces an increase in the level of PKC
expression, independent from increases in DAG levels (Obeid et
al., 1990); however, this would again be too slow to explain the
relatively fast cellular responses to the hormone. The rapid nature of
the non-genomic responses, along with the differing specificities for
structural derivatives of 1,25-D
, compared with those
involving the vitamin D receptor (Norman et al., 1993), has
led to a proposal that there may be a cell membrane-bound receptor(s)
for 1,25-D
(Lieberherr et al., 1989; Nemere et
al., 1993). A recent report has provided evidence for the
existence of such a receptor (Nemere et al., 1994); however,
molecular details of the membrane-bound receptor or receptors, and the
mechanism by which the signal provided by 1,25-D
binding to
this receptor might be transduced, remains to be resolved.
The
similar effects of 1,25-D on PKC activation in vivo to those of DAG and phorbol esters suggested to us the possibility
of a direct activation of PKC and that the enzyme itself might act as a
membrane-associated 1,25-D
receptor. To test this
hypothesis, we compared the effects of 1,25-D
with those of
DAG and the phorbol ester
4
-12-O-tetradecanoylphorbol-13-acetate (TPA), on the
activity of PKC using a cell-free assay system with purified PKC. The
results reveal that PKC is directly and potently activated by
1,25-D
at physiological concentrations in a manner similar
to that by DAG.
The hypothesis that PKC might be directly activated by
1,25-D, and therefore constitute a membrane-associated
receptor for the hormone, was first tested by comparing its effects on
PKC activity with that of DAG and TPA. Using an in vitro assay
system, in which phosphatidylserine, calcium, and substrate
concentrations corresponded to those yielding maximal stimulation,
calcium-dependent PKC-I (rat brain,
,
,
isoform
mixture) was activated by 1,25-D
in a dose-dependent
manner, as shown in Fig. 1. The concentration required for
half-maximal activation (EC
) was 16 ± 1
nM, close to that for TPA (calculated from the data of Fig. 1and Fig. 3, respectively), indicating that the
compound is a potent activator of PKC. Fig. 2shows that PKC
activation by the metabolic precursor, vitamin D
, which
lacks the 1- and 25-hydroxyl moieties, was negligible compared with an
equimolar concentration of 1,25-D
(150 nM), as was
activation by the same concentration of vitamin D
, which
also differs in the steroid side chain structure. Therefore, 1- and/or
25-hydroxylation appear to be required for PKC recognition.
Figure 1:
Dose response curve for the activation
of PKC by 1,25-D. 1,25-D
was incorporated into
BPS/POPC LUV (1:4, molar) at the required concentration, and PKC-I
activity was determined as described under ``Experimental
Procedures.'' Data are the average of triplicate determinations
(±S.D.).
Figure 3:
The effect of a maximally activating
concentration of 1,25-D on the dose response curve for PKC
activation by TPA. 1,25-D
was incorporated into vesicles
consisting of BPS/POPC LUV (1:4, molar) at a concentration of 150
nM, and PKC-I activity was determined as described under
``Experimental Procedures.'' Filled and hollowcircles, with and without 1,25-D
,
respectively. Data are the average of triplicate determinations
(±S.D.).
Figure 2:
Effect of vitamin D-related
metabolites on the activity of PKC. Activity was measured in the
presence of equimolar concentrations (150 nM) of
1,25-D
, vitamin D
, vitamin D
, or
TPA at a concentration of 0.5 µM, each incorporated into
BPS/POPC LUV (1:4, molar), and PKC-I activity was determined as
described under ``Experimental Procedures.'' Data are the
average of triplicate determinations
(±S.D.).
The
possibility that the effect of 1,25-D on PKC activity was
mediated by an alteration in membrane bilayer physical properties, such
as membrane fluidity or lipid order, was discounted due to the
negligible effect that 1,25-D
had on the anisotropy of
1,6-diphenyl-1,3,5-hexatriene, used as a measure of membrane fluidity
(Slater et al., 1994a), even at the high level of 1 µM (results not shown).
Figure 4:
Activation of PKC-I, -, -
, and
-
preparation by 1,25-D
in combination with DAG or
TPA. Activities of PKC-I, -
, and -
, stimulated by TPA and
DAG, were measured in the presence (filledbars) and
absence (hollowbars) of 1,25-D
. A, PKC-I; B, PKC-
; C, PKC-
; D, PKC-
. Data are the average of triplicate determinations
(±S.D.). Other details are as described under
``Experimental Procedures.''
Figure 5:
Effect of PE on PKC activated by
1,25-D. Activation by 1,25-D
(150 nM),
TPA (0.5 µM), and DAG (4 mol % of the total phospholipid
concentration) was measured with (filledbars) and
without (hollowbars)
1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE). The vesicle
compositions were BPS/POPC LUV (1:4, molar) or BPS/POPC/POPE LUV
(1:2:2, molar), containing 150 nM 1,25-D
where
added. Data are the average of triplicate determinations
(±S.D.). Other details are as described under
``Experimental Procedures.''
Figure 6:
Effects of 1,25-D and DAG on
the calcium requirement for PKC activation. PKC-I activity was
determined as a function of calcium concentration using calcium-EGTA
buffers. The 1,25-D
concentration was 150 nM, and
DAG was 4 mol % of the total phospholipid concentration. Hollowcircles, basal; filledcircles,
1,25-D
; filledtriangles, DAG. Data are
the average of triplicate determinations (±S.D.). Other details
are described under ``Experimental
Procedures.''
The principal result of this study is the finding that PKC is
potently and directly activated by 1,25-D. This suggests
that PKC serves as a membrane-bound receptor for the hormone, providing
a rapid mechanism for transducing a 1,25-D
-initiated
signal, additional to the well known activation of the enzyme initiated
by hormone receptor-activated phospholipase C-induced phosphoinositide
breakdown. The assay system used contained only PKC, phospholipid
vesicles, substrate, and necessary cofactors so that a direct
interaction of 1,25-D
involving some specific site on the
enzyme itself is indicated. This is supported by the finding that the
metabolic precursor vitamin D
and also vitamin
D
, which lack the 1- and 25-hydroxyl moieties of
1,25-D
, were without significant effect on the enzyme
activity, indicating a degree of structural specificity.
The finding
that the level of activation achieved by 1,25-D, in
combination with either a maximally stimulating concentration of TPA or
DAG, was greater than that achievable by any activator alone suggests
1,25-D
is not simply competing with the other activators at
a single activator binding site. A similar increased activity was also
obtained in the presence of DAG added together with TPA (Slater et
al., 1994b). A trivial explanation that the individual isoforms
comprising the PKC-I preparation (each had differing affinities for
each activator) is ruled out since the effect was demonstrable with
single PKC-
, -
, and -
isozymes.
Previous studies
indicate that PKC has two activator binding sites, one on each of the
two C1 domain-zinc finger regions, which act as high and low affinity
phorbol ester binding sites (Burns and Bell, 1991; Kazanietz et
al., 1992). The nuclear 1,25-D receptor also contains
the zinc finger motif; therefore, it is possible that 1,25-D
binds at the region of the two C1-zinc finger domains contained
within PKC in a similar manner. The results of the present study are
inconsistent with 1,25-D
, TPA, and DAG activators binding
with equal affinities at each site, since equal competition by paired
activators for binding and activation at each site would not have led
to a greater activity than that obtainable with a single activator.
Thus, the activator pairs, 1,25-D
and TPA or 1,25-D
and DAG, most likely bind with reversed affinities to the two
sites, as previously proposed for TPA and DAG (Slater et al.,
1994b). Thus, for TPA and DAG added together, for example, TPA would
bind and/or activate more strongly to the so-called high affinity
phorbol ester site while DAG would bind more strongly and/or activate
more strongly at the second low affinity phorbol ester site.
An
alternative explanation is that occupation of only one of the two
activator binding sites is responsible for activation, while the other
``effector'' site acts to promote or amplify activation. This
is consistent with the observation that addition of a second activator increases the level of activation beyond the overall
level of stimulation achievable by the first activator alone. With this model, a single activator alone would promote its own
stimulation by interacting at both sites, or it could promote the
effect of a second activator, provided the second activator bound more
strongly to the effector site. 1,25-D both activates PKC
and augments the level of TPA and DAG stimulation, suggesting that
1,25-D
can also act both as an effector and promoter. This
model implies an order of binding affinities for activator pairs
opposite for the two sites. While binding studies should answer this
question, this is not trivial. Usually in the study of the binding of
phorbol esters, the short chain phorbol-12,13-dibutyrate is used since
it has low nonspecific binding to the membrane. However, this would not
be adequate to test the above model since phorbol-12,13-dibutyrate has
a very different activating potency from TPA. Further, TPA and
1,25-D
both bind nonspecifically to the membrane so that
separation of bound from free activator is experimentally difficult.
The idea of a promoter site raises the intriguing and testable
hypothesis that there may be a class of compounds that act solely as
promoters while alone being unable to activate the enzyme.
Evidence
supporting the idea that the 1,25-D-activated PKC
conformation differs from the TPA or DAG-activated forms, comes from
the opposite effects of PE on 1,25-D
activation. A number
of studies suggest that PE promotes an optimal PKC-lipid bilayer
interaction, thereby amplifying the level of activation (Slater et
al., 1994a; Kaibuchi et al., 1981; Bazzi et al.,
1992; Orr and Newton, 1992). However, the effect is dependent on the
experimental conditions, and we have shown PE can have an apparent
opposite inhibitory effect (Slater et al., 1994a). This was
explained on the basis of the level of PKC activity being a biphasic
function of the PE level or of the physical effect of PE, which is to
induce a stress at the head group region (Slater et al.,
1994a). This is caused by small head group compared to acyl chain
volume of PE leading to a molecular cone shape, by contrast to the
cylindrical shape of phosphatidylcholine. Fitting cone-shaped PE into
an essentially planar bilayer surface induces surface stress since
optimal packing of PE molecules induces a concave surface
(Israelachvili et al., 1980; Kirk et al., 1984;
Gruner, 1985; Hui and Sen, 1989; Seddon, 1990). The other half of the
bilayer, tending to curve in the opposite direction, and other lipids
such as phosphatidylcholine etc., frustrates the expression of
this curvature stress and maintains the bilayer form, although the
system retains an elastic curvature stress energy. PKC insertion into
the bilayer, together with the known conformational change (Bazzi and
Nelsestuen, 1988; Brumfeld and Lester, 1990; Snoek et al.,
1988; Bosca and Moran, 1993; Slater et al., 1994a), is
energetically favorable since it acts to reduce the curvature stress.
Thus, there is a level of curvature stress that leads to adoption of an
optimal conformation of PKC yielding maximal activity (Slater et
al., 1994a). The finding that PE addition leads to amplified
activity when DAG or TPA stimulated but reduced activity with
1,25-D
suggests that there are distinct activator-dependent
PKC conformations, with different levels of curvature stress for
optimal interaction/activity.
Although 1,25-D shares
with DAG and TPA an ability to translocate PKC to the membrane (for
example, see Morelli et al.(1993), Simboli-Campbell et
al.(1992, 1994), Wali et al.(1990)) and shows a
calcium-dependent activation, as shown here (for calcium-dependent
isoforms), some differences remain. For instance, 1,25-D
promotes cell differentiation and is anti-proliferative, while
phorbol esters promote proliferation (Blumberg, 1988). Interestingly,
as with 1,25-D
, the potential anti-tumor agent bryostatin
also activates PKC while promoting differentiation rather than
proliferation (Wender et al., 1988; Kraft et al.,
1986), and we have recently found that, as with 1,25-D
,
bryostatin in combination with TPA also produces a greater level of
activity than that achievable with either activator alone. (
)The possibility that distinct active PKC conformers
induced by TPA and 1,25-D
could contribute to the different
effects these compounds have on differentiation and proliferation is
supported by the results of the present study and would be a worthwhile
area for further investigation.
The finding that 1,25-D both induces differentiation and decreases proliferation, in a
variety of cell lines, has led to much interest in its potential
therapeutic properties. However, it is limited by accompanying
deleterious effects on calcium metabolism at the required doses. Thus,
much effort has been directed toward separating the structural
specificities required for effects on calcium metabolism from the
anti-proliferative and differentiation effects of the hormone
(Studzinski et al., 1993), mostly involving manipulation of
the sterol side chain (Norman et al., 1993). The finding that
PKC activation by the metabolic precursor vitamin D
was
negligible, as was activation by the same concentration of vitamin
D
, indicates that 1- and/or 25-hydroxylation are required
for PKC recognition. Therefore, although further studies are required
to fully elucidate the structural specificity of PKC activation by
1,25-D
, comparison with the structures of other known PKC
activators and inhibitors may allow a more rational design of vitamin D
analogs, optimized for inducing PKC-mediated differentiation and
anti-proliferative effects rather than calcium homeostasis.
In
summary, we present evidence that 1,25-D directly activates
PKC at physiological concentrations. Thus, PKC acts as a membrane-bound
receptor for 1,25-D
and as such may be responsible for many
of the non-genomic cellular responses to the hormone. Recently, a
membrane-associated protein was isolated that also binds 1,25-D
(Nemere et al., 1994). Although this protein itself
could be PKC, it appears unlikely, and it may be that there are several
membrane proteins that can act as 1,25-D
receptors. The
present finding that PKC is directly activated by 1,25-D
reinforces the importance of this hormone in signal transduction
events.