©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Kinase C and Mitogen-activated Protein Kinase Are Required for 1,25-Dihydroxyvitamin D-stimulated Egr Induction (*)

(Received for publication, August 31, 1994; and in revised form, November 18, 1994)

David W. A. Beno Lynda M. Brady Marc Bissonnette Bernard H. Davis (§)

From the Gastroenterology Section, Department of Medicine, University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Recent studies have demonstrated that 1,25-dihydroxyvitamin D(3) (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.


INTRODUCTION

The major active metabolite of vitamin D, 1,25-dihydroxyvitamin D(3) (D3), (^1)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.


MATERIALS AND METHODS

Chemicals

D3 (Steroids Ltd., Chicago, IL) used in the experiments was stored as a stock solution in 100% ethanol and was freshly diluted in tissue culture media prior to use. Control cultures treated with equivalent volumes of ethanol vehicle (final concentration < 0.1%) were indistinguishable from untreated cells. PDGF-BB and insulin-like growth factor-I (IGF) were obtained from Life Technologies, Inc. Phorbol 12-myristate 13-acetate (PMA) and calphostin C (Sigma) were freshly prepared prior to use. Myelin basic protein (MBP) (Sigma) was aliquoted (1 mg/ml) after solubilization in water and stored at -20 °C prior to use.

Cell Culture

Hepatic Ito cells were isolated from Sprague-Dawley male rats by previously described methods and subcultured on tissue culture flasks precoated with type I calf collagen(29) .

MAPK Immunoblotting

Ito cell lysates were prepared by washing subconfluent flasks with iced phosphate-buffered saline (PBS) times 2 followed by incubation in lysis buffer (PBS + 0.5% Triton X-100, 1 mM orthovanadate, 5 mM EDTA, 5 mM EGTA, 10 µg/ml leupeptin, 10 µg/ml aprotonin, 300 µg/ml phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, 10 mMp-nitrophenyl phosphate, 50 mM beta-glycerophosphate, (pH 7.5)) for 15 minutes at 4 °C(30) . The lysates were then scraped, disrupted by aspiration three times through a 25-gauge needle, and centrifuged (10,000 times g times 20 min) to remove insoluble cellular debris. Protein in the supernatants was then quantitated with the Bio-Rad protein assay, subaliquoted, and mixed with 4 times solubilization buffer (4% SDS, 10% glycerol, 50 mM Tris/HCl (pH 6.8)), heated (95 °C times 4 min) and then separated by SDS-PAGE on a 10% resolving gel or stored at -70 °C. Alternatively, the cell lysates (600 µg) in lysis buffer were immunoprecipitated with a polyclonal MAPK antibody (Santa Cruz Biotech., Santa Cruz, CA; SC-93: anti-ERK-1) (1 µg/lysate) that was prebound to protein A-agarose. Following immunoprecipitation for 90 min at 4 °C, the immune complexes were washed three times in lysis buffer, eluted in 4 times solubilization buffer (following heating at 95 °C times 4 min), and separated by SDS-PAGE on a 10% resolving gel. The electrophoresed samples were then transferred to Immobilon-P (Millipore, Bedford, MA) membranes and probed with a MAPK monoclonal antibody (Transduction Laboratories, Lexington, KY; ER-16, 0.15 µg/ml) as described previously(31) . The ER-16 antibody is capable of recognizing several MAPK species, including MAPK (or ERK-1) and MAPK (or ERK-2). Antigen localization was carried out with a polyclonal peroxidase-conjugated anti-mouse antibody (Kirkegaard and Perry Laboratories; 04-18-15, 5 ng/ml) with detection by enhanced chemiluminescent technique (Amersham Corp.) as described previously (32) .

MAPK Immunolocalization

Subconfluent Ito cell cultures (on 24-well culture plates precoated with type I collagen) were maintained in media containing 0.4% fetal calf serum (FCS) for 24 h to induce a quiescent state. The cells were then exposed either to D3 (10M), 20% FCS, or ethanol diluent (<.1%) for 40 min (37 °C). The wells were then washed two times with PBS and fixed in methanol/acetone (50/50, v/v) times 20 min. Subsequently, the cells were blocked with PBS + 1% bovine serum albumin followed by normal horse serum as described previously and incubated with the ERK monoclonal antibody (10 µg/well) or a non-immune mouse IgG (10 µg/well) for 1 h at room temperature(32) . The wells were washed three times in PBS + 1% bovine serum albumin and incubated with a biotinylated polyclonal horse anti-mouse antibody as described previously(32) . All subsequent steps were carried out as described previously with diaminobenzidine used for final color development(32) .

Measurement of MAPK Activation

The detection of MAPK activity was done either with an in situ kinase renaturation assay or with a MBP solution kinase assay.

In Situ Kinase Renaturation Assay

Ito cells were cultured in media containing 0.4% fetal calf serum and then exposed to either D3 (10M), PDGF (20 ng/ml), or ethanol diluent for 10 min at 37 °C and then lysates were prepared as described above. Following heating in solubilization buffer at 95 °C for 4 min, the lysates were separated on a SDS-PAGE in a 8% resolving gel containing polymerized MBP (0.1 mg/ml) containing as described by Kameshita and Fujisawa(33) . Following electrophoresis, the kinases within the gel were renatured and incubated with [-P]ATP as described previously(34) . The gel was then dried and exposed for autoradiography.

Solution Kinase Assay

Ito cells maintained in media with 0.4% fetal calf serum were treated with D3 for 1-10 min. In some experiments, the cells were pretreated with PMA (1 µM) or with calphostin C (0.1 µM times 15 min)(35, 36) . To assess a protein kinase C-mediated response, some cells were acutely treated with PMA (100 nM) for 10 min. APK was immunoprecipitated from Ito cell lysates as described above. After three washes in lysis buffer and two washes in kinase buffer (10 mM Hepes (pH 7.4), 5 mM MgCl(2), 1 mM MnCl(2), 10 mMp-nitrophenyl phosphate), the immune complexes were incubated in kinase buffer (50 µl/sample) containing MBP (10 µg/reaction), 25 µM ATP, and [P]ATP (2 µCi) (3 min, 30 °C). Preliminary studies demonstrated that the kinase reaction was in the linear range from 2 to 15 min at 30 °C. The reaction was terminated with preheated (at 95 °C) solubilization buffer, heated at 95 °C for 4 min, and the agarose beads removed by centrifugation. The eluted proteins were resolved by SDS-PAGE on a 14% resolving gel and then fixed, dehydrated in acetone (to prevent cracking), dried, and exposed for autoradiography. The radiolabeled MBP bands were quantitated either by laser scanning densitometry of the autoradiograms or, after excision, P incorporation into the MBP bands was measured by Caerenkov counting. Either quantitation method gave comparable results.

Ito Cell Transfection

Ito cells were passaged 24 h prior to transfection to produce a approx50% confluent culture (approx5 times 10^5 cells/75-cm^2 flask). The medium, containing 10% fetal calf serum, was replenished 1 h prior to transfection. The calcium phosphate-DNA complexes were layered on the cells with gentle agitation as described previously(37, 38) . The DNA complexes were incubated for 16 h at 37 °C, washed several times with PBS, and then fresh medium containing 10% FCS was applied. After 6 h, the media was changed to 0.4% FCS-containing media, and the cells were cultured for an additional 48 h. In some experiments, 6 h prior to harvesting, the cells were exposed to vitamin D (or ethanol diluent). All experiments contained 1 µg/flask of a CMV-driven beta-galactosidase plasmid (pCMV-beta-galactosidase) (kindly provided by Dr. V. Sukhatme) to monitor transfection efficacy. The egr-CAT reporter plasmid (5 µg/flask) was included in each transfection. This plasmid contains a 1.2-kilobase pair segment of the 5` upstream promoter region of the egr gene(39) . In some experiments, double transfections were performed. These experiments used 5 µg of a MAPK containing plasmid (pCMV-p41)(40) . To block Raf or MAPK function, double and triple transfection experiments were carried out that included either 15 µg of pMNC-301-1, a dominant negative Raf plasmid, or 15 µg of pCMV-p41(Ala-54/55), a dominant negative MAPK plasmid(13, 40) . Transfection with these plasmids has been shown to lead to the overproduction of raf or MAPK proteins with defective ATP binding sites(13, 40) . These mutated proteins effectively abolish Ras-induced Raf and MAPK function(13, 40, 41) . The Raf and MAPK plasmids are driven by the same CMV promoter(13, 40) . Comparable total plasmid DNA (30 µg/transfection) was present in all experiments using pMNC empty vector plasmid as needed. At the completion of the transfections, cell lysates were prepared after three freeze/thaw cycles and the solubilized cell protein quantitated using the Bio-Rad protein assay. Comparable amounts of cell protein were used for the the CAT diffusion assay as well as the beta-galactosidase assay which used chlorophenol red beta-D-galactopyranoside as substrate(42, 43) . All data were normalized for cell protein and beta-galactosidase efficacy. Each transfection was performed in duplicate and repeated three times using different plasmid preparations. In some experiments, transfection was performed with the use of Lipofectamine (Life Technologies, Inc.) using the manufacturer's instructions. In these experiments, the cells were cultured in 3.5-cm^2 wells.


RESULTS AND DISCUSSION

D3-stimulated MAPK Activity

To consider the possibility that D3 could stimulate MAPK activity, we exposed Ito cells to D3 for 10 min and then assayed whole cell lysates with the MBP kinase renaturation assay. As shown in Fig. 1, this assay identified two phosphoproteins in the 44-46-kDa range which co-migrated with two phosphoproteins present in cells stimulated with PDGF. PDGF has been identified previously to activate MAPK(14, 19) . This observation suggested that D3 did stimulate MAPK. The kinase renaturation assay also identified other phosphoproteins, but their apparent activities were not consistently altered by D3 and were not further studied.


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(3) (Vitamin D) (10M) 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 times Laemmli solubilization buffer, heated (95 °C times 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 (approx44 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(3) (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(3) (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: times 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 times solubilization buffer and heated (95 °C times 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.

Modulation of Egr Expression by Ras-induced Raf versus MAPK

To evaluate the need for Ras-induced Raf and MAPK in D3 action, we performed a series of transfection experiments which utilized dominant negative mutants of raf and MAPK. These previously characterized constructs produce kinase defective raf or MAPK that effectively abolish ras-induced raf or MAPK activity, respectively(13, 40, 41) . As a marker of D3 activity, we co-transfected the cells with a reporter plasmid which contains the 1.2-kilobase pair promoter of Egr linked to the chloramphenicol acetyltransferase (egr-CAT) gene. Egr is a transcription factor that is acutely induced by many mitogens, including D3(11, 39) . The egr gene is a member of the immediate early gene family and its promoter contains an activator protein-1 (AP-1) binding site(39) .

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-beta-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 beta-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 (times 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^2 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-beta-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 (10M) (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 times 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.


FOOTNOTES

*
This work was supported in part by the Liver Research Fund, University of Chicago and National Institutes of Health Grants DK 02022, DK 40223, DK 42086, and DK 07074-18. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Gastroenterology Section, Dept. of Medicine, MC 4076, 5841 S. Maryland Ave., University of Chicago, Chicago, IL 60637. Tel.: 312-702-1467; Fax: 312-702-2182.

(^1)
The abbreviations used are: D3, 1,25-dihydroxyvitamin D(3); PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; PAGE, polyacrylamide gel electrophoresis; PDGF, platelet-derived growth factor; IGF, insulin-like growth factor; MBP, myelin basic protein; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; ERK, extracellular signal -regulated kinase; CMV, cytomegalovirus; PBS, phosphate-buffered saline; FCS, fetal calf serum; CAT, chloramphenicol acetyltransferase.


ACKNOWLEDGEMENTS

We thank Dr. T. Brasitus for supplying vitamin D as well as advice and encouragement during this study. The authors thank Dr. U. Rapp (NCI), Dr. R. Davis (University of Massachusetts), and Dr. V. Sukhatme (Beth Israel Hospital, Boston) for supply of raf, MAPK, and egr-CAT plasmids, respectively. The technical assistance of J. Mullen and R. Espinal is gratefully acknowledged.


REFERENCES

  1. Mitsuhasho, T., Morris, R. C., and Ives, H. E. (1991) J. Clin. Invest. 87, 1889-1895 [Medline] [Order article via Infotrieve]
  2. Drittanti, L., De Boland, A. R., and Boland, R. (1989) Biochim. Biophys. Acta 1014, 112-119 [Medline] [Order article via Infotrieve]
  3. Halline, A. G., Davidson, N. O., Skarosi, S. F., Sitrin, M. D., Tieze, C., Alpers, D. H., and Brasitus, T. A. (1994) Endocrinology 134, 1710-1717 [Abstract]
  4. Clemens, T. L., Garrett, K. P., Zhou, X.-Y., Pike, J. W., Haussler, M. R., and Dempster, D. W. (1988) Endocrinology 122, 1224-1230 [Abstract]
  5. Jurutka, P. W., Hsieh, J.-C., McDonald, P. N., Terpening, C. M., Haussler, C. A., Haussler, M. R., and Whitfield, G. K. (1993) J. Biol. Chem. 268, 6791-6799 [Abstract/Free Full Text]
  6. Wali, R. K., Baum, C. L., Sitrin, M. D., and Brasitus, T. A. (1990) J. Clin. Invest. 85, 1296-1303 [Medline] [Order article via Infotrieve]
  7. Tien, X.-Y., Brasitus, T. A., Qasawa, B. M., Norman, A. W., and Sitrin, M. D. (1993) Am. J. Physiol. 265, G143-G148
  8. Bissonnette, M., Tien, X.-Y., Niedziela, S. M., Hartmann, S. C., Frawley, B. P., Roy, H. K., Sitrin, M. D., Perlman, R. L., and Brasitus, T. A. (1994) Am. J. Physiol. 267, G465-G475
  9. Friedman, S. L., and Arthur, M. J. P. (1989) J. Clin. Invest. 84, 1780-1785 [Medline] [Order article via Infotrieve]
  10. Friedman, S. L., Yamasaki, G., Wong, L., and Buck, J. (1992) Hepatology 16, 143A
  11. Lissoos, T. W., Beno, D. W. A., and Davis, B. H. (1993) J. Biol. Chem. 268, 25132-25138 [Abstract/Free Full Text]
  12. Rapp, U. (1991) Oncogene 6, 495-500 [Medline] [Order article via Infotrieve]
  13. Kolch, W., Heidecker, G., Lloyd, P., and Rapp, U. R. (1991) Nature 349, 426-428 [CrossRef][Medline] [Order article via Infotrieve]
  14. Blenis, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5889-5892 [Abstract]
  15. Sozeri, O., Vollmer, K., Liyanage, M., Frith, D., Kour, G., Mark, G. E., and Stabel, S. (1992) Oncogene 7, 2259-2262 [Medline] [Order article via Infotrieve]
  16. Kolch, W., Heldecker, G., Kochs, G., Hummel, R., Vahldl, H., Mischak, H., Finkenzeller, G., Marme, D., and Rapp, U. R. (1993) Nature 364, 249-252 [CrossRef][Medline] [Order article via Infotrieve]
  17. Morrison, D. K., Kaplan, D. R., Rapp, U., and Roberts, T. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8855-8859 [Abstract]
  18. Schaap, D., van der Wal, J., Howe, L. R., Marshall, C. J., and van Blitterswijk, W. (1993) J. Biol. Chem. 268, 20232-20236 [Abstract/Free Full Text]
  19. Davis, R. J. (1993) J. Biol. Chem. 268, 14553-14556 [Free Full Text]
  20. Boulton, T. G., Nye, S. H., Robbins, D. J., Ip, N. Y., Radziejewska, E., Morgenbesser, S. D., DePinho, R. A., Panayotatos, N., Cobb, M. H., and Yancopoulos, G. D. (1991) Cell 65, 663-675 [Medline] [Order article via Infotrieve]
  21. Robbins, D. J., Cheng, M., Zhen, E., Vanderbilt, C. A., Feig, L. A., and Cobb, M. H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6924-6928 [Abstract]
  22. Seger, R., Ahn, N. G., Posada, J., Munar, E. S., Jensen, A. M., Cooper, J. A., Cobb, M. H., and Krebs, E. G. (1992) J. Biol. Chem. 267, 14373-14381 [Abstract/Free Full Text]
  23. Kyriakis, J. M., App, H., Zhang, X.-F, Banerjee, P., Brautigan, D. L., Rapp, U. R., and Avruch, J. (1992) Nature 358, 417-421 [CrossRef][Medline] [Order article via Infotrieve]
  24. Howe, L. R., Leevers, S. J., Gomez, N., Nakielny, S., Cohen, P., and Marshall, C. J. (1992) Cell 71, 335-342 [Medline] [Order article via Infotrieve]
  25. Kyriakis, J. M., Force, T. L., Rapp, U. R., Bonventre, J. V., and Avruch, J. 993) J. Biol. Chem. 268, 16009-16019
  26. Lange-Carter, C. A., Pleiman, C. M., Gardner, A. M., Blumer, K. J., and Johnson, G. L. (1993) Science 260, 315-319 [Medline] [Order article via Infotrieve]
  27. Moodie, S. A., Willumsen, B. M., Weber, M. J., and Wolfman, A. (1993) Science 260, 1658-1661 [Medline] [Order article via Infotrieve]
  28. Zheng, C.-F., and Guan, K.-L. (1993) J. Biol. Chem. 268, 23933-23939 [Abstract/Free Full Text]
  29. Davis, B. H., Pratt, B. M., and Madri, J. A. (1987) J. Biol. Chem. 262, 10280-10286 [Abstract/Free Full Text]
  30. Pages, G., Lenormand, P., L'Allemain, G., Chambard, J.-C., Meloche, S., and Pouyssegur, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8319-8323 [Abstract/Free Full Text]
  31. Davis, B. H., Rapp, U. R., and Davidson, N. O. (1991) Biochem. J. 278, 43-47 [Medline] [Order article via Infotrieve]
  32. Davis, B. H., Coll, D., and Beno, D. W. A. (1993) Biochem. J. 294, 785-792 [Medline] [Order article via Infotrieve]
  33. Kameshita, I., and Fujisawa, H. (1989) Anal. Biochem. 183, 139-143 [Medline] [Order article via Infotrieve]
  34. Beno, D. W. A., Rapp, U. R., and Davis, B. H. (1994) Biochim. Biophys. Acta. 1222, 292-300 [CrossRef][Medline] [Order article via Infotrieve]
  35. Kobayashi, Y., Britton, R. S., O'Neil, R., Li, S. C. Y., and Bacon, B. R. (1992) Hepatology 16, 143A
  36. Khalil, R. A., and Morgan, K. G. (1993) Am. J. Physiol. 265, C406-C411
  37. Lissoos, T. W., Beno, D. W. A., Espinal, R., Brady, L., and Davis, B. H. (1993) Hepatology 18, 161A
  38. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory, NY
  39. Rupprecht, H. D., Sukhatme, V. P., Lacy, J., Sterzel, R. B., and Coleman, D. L. (1993) Am. J. Physiol. 265, F351-F360
  40. Seth, A., Gonzalez, F. A., Gupta, S., Raden, D. L., and Davis, R. L. (1992) J. Biol. Chem. 267, 24796-24804 [Abstract/Free Full Text]
  41. Bruder, J. T., Heidecker, G., and Rapp, U. R. (1992) Genes & Dev. 6, 545-556
  42. Neumann, J. R., Morency, C. A., and Russian, K. O. (1987) BioTechniques 5, 444-448
  43. Rouet, P., Raquenez, G., and Salier, J.-P. (1992) BioTechniques 13, 700-701 [Medline] [Order article via Infotrieve]
  44. Lenormand, P., Sardet, C., Pages, G., L'Allemain, G., Brunet, A., and Pouyssegur, J. (1993) J. Cell Biol. 122, 1079-1088 [Abstract]
  45. Anderson, N. G., Li, P., Marsden, L. A., Williams, N., Roberts, T. M., and Sturgill, T. W. (1991) Biochem. J. 277, 573-576 [Medline] [Order article via Infotrieve]
  46. Chao, T. O., Foster, D. A., Rapp, U. R., and Rosner, M. R. (1994) J. Biol. Chem. 269, 7337-7341 [Abstract/Free Full Text]
  47. Pinzani, M., Abboud, H. E., and Aron, D. C. (1990) Endocrinology 127, 2343-2349 [Abstract]
  48. Bogoyevitch, M. A., Glennon, P. E., Andersson, M. B., Clerk, A., Lazou, A., Marshall, C. J., Parker, P. J., and Sugden, P. H. (1994) J. Biol. Chem. 269, 1110-1119 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.