(Received for publication, August 31, 1994; and in revised form, November 18, 1994)
From the
Recent studies have demonstrated that 1,25-dihydroxyvitamin
D (D3) can activate Raf kinase and induce Egr expression in
cultured rat hepatic Ito cells (Lissoos, T. W., Beno, D. W. A., and
Davis, B. H.(1993) J. Biol. Chem. 268, 25132-25138).
Since Raf is an upstream activator of mitogen-activated protein kinase
(MAPK), the current study evaluated the ability of D3 to activate MAPK.
D3-activated MAPK and induced its cytoplasmic to perinuclear
translocation in Ito cells. MAPK activation was found to be protein
kinase C-dependent, which was analogous to previous studies of D3 and
Raf activation. To further explore the D3 cascade, a series of
transient transfections were performed using dominant negative raf and
MAPK mutant plasmids which effectively block Ras-induced Raf and MAPK
activity, respectively. D3 induced a marked increase in the expression
of a chloramphenicol acetyltransferase reporter gene linked to the Egr
promoter (egr-CAT). When the dominant negative Raf plasmid was
co-transfected, there was no significant reduction in egr-CAT. In
contrast, when the dominant negative MAPK plasmid was co-transfected,
egr-CAT induction was completely abolished. These results suggest that
1) D3 stimulates MAPK via a protein kinase C-dependent pathway, 2)
D3-induced Egr expression can occur via a pathway independent of
Rasinduced Raf, and 3) D3 absolutely requires MAPK activity for Egr
expression.
The major active metabolite of vitamin D, 1,25-dihydroxyvitamin
D (D3), (
)is known to promote cell proliferation
in several cell types(1, 2, 3) . D3 is a
member of the steroid superfamily, and until recently, this
secosteroid's actions were largely attributed to its nuclear receptor(4, 5) . However, recent studies have
identified several acute non-genomic effects of D3 that occur within
minutes of exposure(6, 7, 8) . The
non-genomic cytoplasmic components of the D3 cascade may participate in
D3's mitogenic actions and potentially serve as a model for other
non-tyrosine kinase receptor-linked agonists.
D3 acutely induces calcium mobilization, inositol triphosphate turnover, and protein kinase C (PKC) translocation(6, 7, 8) . To further characterize the D3 cascade, we have recently studied D3's pre-genomic effects in rat hepatic Ito cells. This mesenchymal cell type proliferates in part due to its de novo expression of the platelet-derived growth factor (PDGF) surface receptor during liver injury and fibrosis(9, 10) . PDGF-induced mitogenesis was markedly enhanced when Ito cells were exposed to D3 and PDGF simultaneously (11) . It was shown that D3 acutely stimulated Raf kinase and this required PKC(11) . Raf is a key regulatory cytoplasmic serine/threonine kinase stimulated by PDGF and numerous other mitogens(12, 13) . The known upstream activators of raf kinase include Ras and PKC(12, 14, 15, 16) . D3's requirement for PKC is in contrast to PDGF which has previously been shown to activate Raf by PKC-independent pathways(12, 17) . The D3/PDGF synergistic effect may be due at least, in part, to Raf activation by PKC-dependent and -independent pathways.
D3 induces proto-oncogene expression in several cell types and in Ito cells, we have found that D3 induces the expression of the nuclear proto-oncogene, Egr(11) . Constitutively activated Raf also leads to proto-oncogene expression(12) . D3's induction of Egr could therefore be attributed to its activation of Raf(12) . The downstream pathway which potentially links raf to Egr expression is complex. Recent work suggests that mitogen-activated protein kinase (MAPK) represents a key link in transmitting the Raf-generated signal to the nucleus in many cell types (14, 18, 19, 20, 21, 22) . MAPKs are a family of serine/threonine kinases that are regulated by threonine and tyrosine phosphorylation. Studies have shown that Raf is one of several MAPK kinase kinases(23, 24, 25, 26, 27, 28) .
In the current study, we characterized the post-Raf transduction cascade that follows D3 treatment. We found that D3 activates MAPK and induces its perinuclear translocation. The kinetics of its activation resemble MAPK activation kinetics by other mitogens(14) . D3-stimulated MAPK activation required PKC and was therefore similar to the previous studies with D3-stimulated raf activation. To determine whether D3 required Ras-induced Raf activation and/or MAPK for nuclear signaling, we performed a series of transfection experiments utilizing dominant negative plasmids which abolish either Ras-induced Raf or MAPK. We found that D3 required MAPK to activate Egr. In contrast, D3 could activate Egr in the absence of Ras-induced Raf. Our results suggest that vitamin D induces nuclear signaling by a PKC- and MAPK-dependent pathway which may be independent of Ras-induced Raf.
Figure 1:
Vitamin D stimulates renaturable MBP
kinases in Ito cells. Ito cells were made quiescent by culture in media
containing 0.4% fetal calf serum for 24 h. Cells were incubated for 10
min with vehicle alone(-) or with 1,25-dihydroxyvitamin D (Vitamin D) (10
M) or
platelet-derived growth factor (PDGF) (20 ng/ml) and then
scraped in lysis buffer. After lysing, comparable aliquots of cell
protein were mixed with four
Laemmli solubilization buffer,
heated (95 °C
4 min), and separated by SDS-PAGE in an 8%
resolving gel containing MBP. Kinase activity was determined as
described by Kameshita and Fujisawa(33) . Molecular weight
markers are shown on the left. The arrows on the right identify the two kinases which are stimulated by both
vitamin D and PDGF.
Several
mitogen-activated renaturable protein kinases capable of
phosphorylating MBP have been identified(20, 21) .
These kinases are members of the extracellular signal regulated kinase
or ERK family(20, 21) . In an effort to identify the
D3 activated renaturable kinase, we probed Ito cell lysate Western
blots with a pan-ERK monoclonal antibody capable of identifying several
different ERK species, including ERK 1 (MAPK) and ERK 2
(MAPK
). As shown in Fig. 2(lanes 1 and 2), Ito cells express predominantly MAPK
. The
same molecular weight species was obtained after immunoprecipitating
Ito cell lysates with a MAPK
-specific polyclonal
antibody (Fig. 2, lanes 3 and 4).
Figure 2:
MAPK expression in Ito cells. Ito cell
lysates were either (lanes 1 and 2) separately by
SDS-PAGE in a 10% resolving gel or following immunoprecipitation with a
polyclonal MAPK antiserum (lanes 3 and 4). Following Western blotting with a MAPK monoclonal antibody
(ER-16), a single molecular mass species (
44 kDa) is
visualized.
Using the
monoclonal antibody which identified a single 44-kDa MAPK species, we
localized the MAPK protein in unstimulated Ito cells, as
well as cells treated with either vitamin D or fetal calf serum for 40
min. As shown in Fig. 3A, MAPK
is
present diffusely in the cytoplasm prior to stimulation. In contrast,
following either D3 or serum stimulation, MAPK
translocates to a perinuclear domain (Fig. 3, B and C). This redistribution of the kinase is consistent
with recent studies that examined MAPK (ERK 1 and ERK 2) activation in
other cell types(44) . Staining with comparable amounts of
non-immune mouse IgG gave only background staining (Fig. 3D).
Figure 3:
MAPK localization in Ito cells. Quiescent
Ito cells were either fixed directly in acetone/methanol (50/50) (A) or following treatment with vitamin D (B) or 20% fetal calf serum (C) for 40 min. The
fixed cells were stained with the monoclonal MAPK antiserum (ER 16)
followed by a biotinylated secondary antibody. Antigen localization was
carried out with the avidin-biotin-peroxidase complex followed by
hydrogen peroxide with diaminobenzidine for color formation. Note that
the unstimulated cells (A) contain a diffuse cytoplasmic
distribution of the MAPK
protein (small straight
arrows) in contrast to the perinuclear distribution of the protein
following vitamin D
(B) or serum (C)
stimulation. The curved arrows in B and C demonstrate that the protein has predominantly shifted from the
cytoplasm toward the nucleus. Note the lightly stained fibrillar
cytoplasmic pattern in contrast to the densely stained perinuclear
pattern in B and C. The variations in cell size are
characteristic of the stellate-shaped Ito cells in subconfluent
culture. Parallel quiescent cells were fixed and stained with
comparable amounts of non-immune mouse IgG (D) and showed only
background staining, although the cellular stellate appearance and
variability in size are still apparent. Hoffman optics; final
magnification:
150.
To verify the results obtained with the
kinase renaturation assay which utilized whole cell lysates, we
examined MAPK activation with an immunoprecipitation
assay. For these studies, we treated Ito cells with vitamin D and then
immunoprecipitated the MAPK
species using the previously
mentioned MAPK
-specific antibody. The immune complexes
were then incubated with MBP in a solution kinase assay and the
phosphorylated MBP was identified following separation by SDS-PAGE. As
shown in Fig. 4, D3 activated MAPK
within 4 min
of exposure. This stimulation persisted for at least 10 min
(preliminary studies found that D3 incubations for 1-2 min did
not induce significant ERK 1 activity). D3 consistently induced a
2.5-fold increase in ERK 1 activity and this degree of stimulation is
similar to many other mitogenic activators(19) . To investigate
the role of PKC as an upstream mediator of the D3 signal, we pretreated
the cells with PMA for 24 h which has previously been shown to
down-regulate PKC in Ito cells(35) . As shown in Fig. 5,
PMA pretreatment abolished D3's capacity to activate
MAPK
. In parallel experiments, acute PMA treatment
markedly stimulated MAPK
activity and PMA pretreatment
for 24 h abolished acute PMA activation of MAPK
. In
similar experiments (data not shown), pretreatment with the PKC
inhibitor, calphostin C, for 15 min also abolished D3's capacity
to activate MAPK
. These results together with our
earlier studies demonstrate that PKC mediates the activation of raf and
MAPK
by D3(11) .
Figure 4:
Vitamin D stimulates MAPK activity.
Quiescent Ito cells were lysed following treatment with ethanol vehicle
or vitamin D (10M) for 4 or 10 min as
indicated. MAPK
was immunoprecipitated from cell lysates
(600 µg of protein) as described under ``Materials and
Methods.'' The immune complexes were then washed and incubated
with MBP in kinase buffer (3 min, 30 °C). The reaction was stopped
in 4
solubilization buffer and heated (95 °C
4
min). After centrifugation, protein in the supernatant was separated by
SDS-PAGE on a 14% resolving gel. The gel was subsequently fixed, dried,
and exposed for autoradiography. The resulting radiolabeled MBP band is
indicated by the arrow on the right, and the
molecular weight markers are indicated on the left. This
audioradiogram is representative of four separate
experiments.
Figure 5:
Vitamin D-stimulated MAPK activity is
PKC-dependent. Ito cells were processed as described in the legend to Fig. 4. The cells were treated with vitamin D (10M) or PMA (100 nM) for 10 min as indicated. In
some groups, the cells were pretreated with PMA (1 µM) for
24 h. The radiolabeled MBP band is indicated by the arrow on
the right, and molecular weight markers are indicated on the left. These same findings were observed in two independent
experiments.
Recent work has shown that there are non-Raf pathways that also lead to MAPK activation(26, 27) . It was not clear whether D3 could utilize these Raf-independent pathways to activate MAPK. Moreover, a better characterization of the D3-induced activators of MAPK will provide useful clues for the mechanisms of action of D3.
We initially determined that overexpression of MAPK augmented egr expression over base line (Fig. 6). This induction was totally abolished when the cells were co-transfected with the dominant negative MAPK (DomNeg MAPK) plasmid. Co-transfection with the dominant negative raf (DomNeg Raf) plasmid caused a 50% reduction in the ability of MAPK to induce egr expression. These observations suggest that the overexpression of MAPK leads to egr induction, in part due to base-line Raf kinase activity(14, 19) . In addition, although MAPK is downstream of Raf, it may modulate Raf activity itself (i.e. an upstream effect of MAPK on raf function)(14) . In this regard, MAPK has been shown to phosphorylate raf kinase(14, 45) .
Figure 6:
MAPK-induced egr-CAT. Dominant negative
MAPK abolishes MAPK-induced egr-CAT, whereas dominant negative Raf
partially suppresses MAPK-induced egr-CAT. Ito cells were
co-transfected with an egr-CAT reporter plasmid (5 µg), a pMNC
empty vector plasmid(- -), and a pCMV-p41MAPK plasmid (5
µg) (MAPK). As indicated, some cells were co-transfected
with 15 µg of dominant negative MAPK or dominant negative Raf
plasmid instead of the pMNC empty vector plasmid: either
pCMV-p41(Ala54/Ala55)MAPK (15 µg) (DomNeg MAPK) or pMNC
301-1 (15 µg) (DomNeg Raf), respectively. All
transfections included pCMV--galactosidase plasmid (1 µg) to
permit normalization for transfection efficacy. The cells were
harvested 72 h following transfection after culture in media containing
0.4% fetal calf serum. The data are expressed as relative CAT activity
(as determined by the CAT diffusion assay) after normalization for
comparable protein content and
-galactosidase activity. Each bar represents the average of duplicate transfections ±
S.D. This is a representative experiment that was repeated three times
with similar results.
We next addressed the role of Ras-induced raf or MAPK in mediating D3-stimulated egr gene expression. For these studies we compared the level of egr-CAT expression in cells stimulated by D3 alone with cells co-transfected with plasmids containing either the dominant negative Raf or dominant negative MAPK prior to D3 exposure. As shown in Fig. 7, the two dominant negative co-transfections had vastly different effects. The 7-fold induction of egr-CAT expression by D3 was not significantly reduced when the dominant negative Raf plasmid was co-transfected. In contrast, co-transfection with the dominant negative MAPK plasmid totally abolished D3's ability to induce egr-CAT expression. Each co-transfection used 15 µg of the dominant negative plasmid in a 3:1 ratio of dominant negative plasmid to Egr reporter plasmid.
Figure 7:
Vitamin D-induced egr-CAT: Raf versus MAPK dependence. Ito cells were treated as described in the legend
of Fig. 6. Cells were transfected with either pMNC empty vector
plasmid (- - and vitamin D groups) or with comparable
amounts of dominant negative raf (DomNeg Raf) or dominant
negative MAPK (DomNeg MAPK) plasmids, as indicated. 72 h
following transfection and culture in media containing 0.4% fetal calf
serum, the cells were exposed to vitamin D (D3) (10M) or ethanol as indicated (
6 h) prior to
harvesting. Data are expressed as relative CAT activity as described in
the legend to Fig. 6. Each bar represents the average
of duplicate transfections ± S.D. and is representative of three
independent experiments.
To further evaluate the failure of the dominant negative Raf plasmid to alter vitamin D-induced Egr expression, we performed a series of transfections using a higher relative concentration of the 301-1 dominant negative raf plasmid. As shown in Fig. 8A, when a 4:1 dominant negative Raf/egr-CAT ratio was used, vitamin D induced egr-CAT was still unaffected. All transfections contained comparable amounts of plasmid DNA and transfection efficacy was unaffected by these differing DNA concentrations. To assess the dominant negative Raf plasmid's capacity to alter Ito cell signal transduction in general, we performed similar transfections and used either IGF-I or serum as stimulants of egr-CAT. In other cell systems, IGF-I and serum have been shown to activate raf and MAPK and to require raf function for downstream signaling(12, 13, 14, 46) . In addition, IGF-I has previously been shown to be mitogenic for Ito cells and to potentiate the Ito cell's PDGF mitogenic response (47) . In this regard, IGF-I is analogous to our previous observations with vitamin D and PDGF(11) . As shown in Fig. 8B, we found that the dominant negative Raf plasmid completely abolished IGF-I induced Egr expression and caused a reduction in serum-induced Egr expression. These observations are consistent with the previously mentioned studies with IGF-I, serum, and Raf and demonstrate the relative importance of Ras-induced Raf in Ito cell signaling from non-vitamin D agonists(12, 13, 14, 46) .
Figure 8:
Dominant negative Raf alters IGF and
serum-induced egr-CAT expression but not vitamin D-induced egr-CAT
expression. Subconfluent Ito cells in 3.5-cm wells were
transfected with egr-CAT (0.25 µg/well) and empty vector pMNC
plasmid (1 µg/well) using the Lipofectamine method. In some
experiments, as indicated, the 301-1 dominant negative Raf plasmid (DomNeg Raf) was used in place of the pMNC plasmid. Parallel
transfections which included the CMV-
-galactosidase reporter
plasmid demonstrated equal transfection efficacy when either the pMNC
or 301-1 plasmid was used. Following transfection, the cells were
maintained in 0.4% serum containing media for the subsequent 48 h and
then exposed to either vitamin D (10
M) (A) or IGF-I (2 ng/ml) or fetal calf serum (20%) for 6 h (B). The cells were harvested, six wells were combined, and
following protein quantitation and heating at 65 °C
5 min,
CAT quantitation was performed. The data represent the average ±
S.D. of two separate experiments, each obtaining by pooling six
individual transfected wells.
In summary, the current studies demonstrate that D3 stimulates MAPK by a PKC-dependent pathway, analogous to the previous observation with D3-stimulated raf activation. Recent studies have identified other agonists that activate MAPK by PKC-dependent mechanisms(48) . These agonists utilize heterotrimeric GTP protein-coupled receptors and are unrelated to D3(48) . Although D3 can stimulate both Raf and MAPK, the dominant negative Raf/MAPK transfection experiments suggest that the nuclear signaling which is induced by D3 can proceed by a pathway that is independent of Ras-induced Raf. D3 absolutely requires MAPK activity to induce Egr. Future work is needed, however, to determine if MAPK is required for all D3-induced nuclear signaling.