Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M203039200 on April 30, 2002

J. Biol. Chem., Vol. 277, Issue 29, 25884-25892, July 19, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/29/25884    most recent
M203039200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Qi, X.
Right arrow Articles by Chen, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Qi, X.
Right arrow Articles by Chen, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The p38 and JNK Pathways Cooperate to trans-Activate Vitamin D Receptor via c-Jun/AP-1 and Sensitize Human Breast Cancer Cells to Vitamin D3-induced Growth Inhibition*

Xiaomei QiDagger §, Rocky PramanikDagger §, Jintang WangDagger , Richard M. Schultz, Ratan K. Maitra||, Jiahuai Han**, Hector F. DeLucaDagger Dagger , and Guan ChenDagger §§

From the Dagger  Department of Radiation Oncology and the  Division of Biochemistry of the Department of Cell Biology, Neurobiology, and Anatomy, Loyola University of Chicago, Maywood, Illinois 60153, the || Department of Virology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, the ** Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, and the Dagger Dagger  Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706-1544

Received for publication, March 28, 2002, and in revised form, April 26, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The signaling connection between mitogen-activated protein kinases(MAPKs) and nuclear steroid receptors is complex and remains mostly unexplored. Here we report that stress-activated protein kinases p38 and JNK trans-activate nuclear steroid vitamin D receptor (VDR) gene and increase vitamin D3-dependent growth inhibition in human breast cancer cells. Activation of p38 and JNK by an active MAPK kinase 6 stimulates VDR promoter activity independently of the ligand vitamin D3 and estrogen receptor expression. Moreover, stimulation of the endogenous stress pathways by adenovirus-mediated delivery of recombinant MAPK kinase 6 also activates VDR and sensitizes MCF-7 cells to vitamin D3-dependent growth inhibition. Both the p38 and JNK MAPK pathways and the downstream transcription factor c-Jun/AP-1 are required for the VDR stimulation, as revealed by application of their dominant negatives, the specific p38 inhibitor SB203580, and site-directed mutagenesis of the AP-1 element in the VDR promoter. The essential role of the p38 and JNK stress pathways in up-regulation of VDR expression is further confirmed by using the chemical stimulator arsenite. These results establish a signaling connection between the stress MAPK pathways and steroid hormone receptor VDR expression and thereby offer new insights into regulation of cell growth by the MAPK pathways through regulation of vitamin D3/VDR activity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

p38 and c-Jun NH2-terminal kinase (JNK)1 are the major MAPK pathways, which, together with the extracellular signal-regulated kinase (ERK) pathways, convert signals of various extracellular stimuli into expression of specific target genes through phosphorylation and activation of transcription factors (1-3). The ERK pathway, downstream of Ras, is predominantly activated by mitogenic stimuli, whereas the p38 and JNK pathways are preferentially stimulated by environmental stresses. Although biological consequence of p38 and JNK activation in many cases overlaps with that of the ERK (1-3), one of its principle functions is regulation of stress and the inflammatory response (4, 5). The mechanisms leading to the functional diversity and/or specificity of the cellular physiological outcome of MAPK signaling are largely unknown and may relate to regulation of their specific downstream target genes. Although the p38 and JNK pathways can regulate expression of many molecules (2, 6), only a few of the true target genes selectively responsive to these stress pathways have been identified.

The active form of vitamin D3, 1,25-dihydroxyvitamin D3, is a hormone with known activity in the regulation of calcium homeostasis, cell proliferation, and cell differentiation (7). Vitamin D3 binds to its nuclear steroid receptor (VDR), a transcription factor, which dimerizes with itself or other nuclear receptors such as retinoid X receptor (RXR) to regulate gene expression by specific vitamin D response elements (VDREs) through which vitamin D3 exerts its biological effects (7, 8). Vitamin D3 is well known for its therapeutic activity against breast cancer, which in many cases requires the presence of functional VDR (9, 10). Loss of VDR expression in human breast cancer cells is frequently associated with the resistance to vitamin D3-dependent growth inhibition (10), whereas its up-regulation by different means increases the sensitivity in different systems (11-14). Alteration of VDR expression consequently emerges as an important approach for the regulation of vitamin D3 activity in inhibition of cancer cell proliferation. The signaling mechanisms by which VDR is regulated, however, remain largely undiscovered.

The signaling connection between MAPK activation and nuclear steroid hormone receptors is complex. The classical model is that the steroid hormone, because of its hydrophobic property, diffuses across the cellular membrane and binds to its nuclear receptor to regulate target gene expression through specific steroid receptor-responsive regulatory elements in the target genes, unrelated to MAPK regulation (15, 16). Recent experimental evidence suggests that there exist multiple interactions between the ERK- and JNK-MAPK pathways and the estrogen and progesterone steroid receptors at many levels (15, 16). (i) The Ras-ERK pathway phosphorylates the estradiol/estrogen receptor (ER) and increases its transcription activity (17-19). (ii) In reverse, signaling through ER and progestins/progesterone receptor (PR) stimulates the Ras/ERK activity (20-22). (iii) The JNK kinase MEKK1 stimulates the transcriptional activity of PR in a manner dependent (23) and independent of the ligand (24). (iv) Interactions also can occur between transcription factors (c-Jun and c-Fos) activated by the MAPK pathways and the nuclear steroid receptors via direct binding (25, 26). Although direct phosphorylation of ER and PR by MAPKs is involved in many of these processes, it remains unknown whether MAPKs can regulate steroid hormone receptor gene expression. Knowledge about transcriptional regulation of steroid receptor expression is critical for understanding steroid hormone effects. This is particularly important for VDR, because in many cell/tissue types the VDR level is limiting (7), and its concentration consequently largely determines the magnitude of the hormone-induced biological responses.

It has been postulated that the Ras/ERK suppresses vitamin D3/VDR signaling, because ras-transformed cells have lower levels of VDR expression than their normal counterparts (27). Accordingly, ras-transformed keratinocytes are resistant to vitamin D3-induced growth inhibition in comparison with their normal counterparts (28, 29). Consistent with this observation, activation of the Ras-ERK pathway by phorbol 12-myristate 13-acetate (30), epidermal growth factor, and fibroblast growth factor (27) down-regulates VDR expression. Furthermore, elevated Ras/ERK activity was demonstrated to be correlated with the disruption of VDR/RXR heterodimer (31). Because the Ras/ERK and the p38 MAPK pathways frequently have an opposite role in many systems (32-34), the inhibition of VDR activity by Ras may be either a direct consequence of Ras-ERK activation or the p38 pathway inactivation. Consequently, it may be reasoned that p38 kinase might stimulate VDR activity.

In this report we have tested the hypothesis that the stress-activated protein kinase p38 stimulates VDR and thereby increases VDR activity and vitamin D3-induced growth inhibition. By application of genetic as well as chemical stimuli, our results establish that the signaling through both the p38 and JNK integrated at c-Jun/AP-1 is not only sufficient but essential to trans-activate VDR. The VDR activation by this pathway was further shown to sensitize human breast cancer MCF-7 cells to vitamin D3-induced growth inhibition.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

cDNA Constructs and Expression Plasmids-- Recombinant adenovirus vectors containing hemagglutinin-tagged MKK6 and MEK1 were constructed as described previously (35, 36). Briefly, hemagglutinin-tagged active MKK6 and MEK1 were subcloned into the vector pAd/RSV, and then were co-transfected with the adenovirus-packaging plasmid pJM17 into 293 cells. A control recombinant adenovirus lacking a cDNA insert was constructed using pAd/RSV and pJM17. High-titered stocks of the recombinant adenoviruses (~1 × 1010 plaque-forming units/ml) were produced and purified at the Virus Core Facility of the Cleveland Clinic Foundation. Mouse VDR promoter (0.5 kb) was cloned and inserted into a pGL2 basic vector (Promega) in front of the luciferase gene as described previously (37, 38). pCMV plasmids encoding constitutively active MEK (MEK/2E), constitutively active MEKK1 (truncated), pEBG-expressing GST-tagged ERK (p42MAPK), GST-tagged JNK (p54SAPK), and the bacterial expressing plasmid pGEX for GST-Jun were provided by Dennis Templeton (39, 40) and have been used previously in this laboratory (41). A mammalian expressing plasmid pCMV-Tam67 (c-Jun lacking the trans-activation domain, residues 2-122) was originally provided by Michael Birrer (42-44). pcDNA3-MKK6/E (MKK6) is a constitutively active mutant of MKK6 with Ser-207 and Thr-211 replaced by glutamic acid. This plasmid as well as the pCMV5 vectors containing FLAG-tagged wild type and kinase dead form of p38alpha (p38/AF) and pGEX for GST-ATF2 were kindly provided by Roger Davis (45). A mammalian expression plasmid pcDNA3-containing cDNA for myc epitope-tagged MK2 was supplied by Matthias Gaestel (46). pAC-CMV encoding activated Ha-Ras (Leu-61) was kindly provided by Christopher B. Newgard as described previously (41). An AP-1 luciferase construct, which was generated by cloning three AP-1 repeats into a luciferase reporter gene containing a minimal Fos promoter, was kindly provided by Craig Hauser (47).

Other Reagents-- Minimum essential medium, L-glutamine, and antibiotics were supplied by Invitrogen. Fetal bovine serum was obtained from BioWhittaker. DNA was prepared using an Endofree kit from Qiagen. A DNA transfection kit (calcium phosphate) and a dual luciferase kit were purchased from Promega. SB203580 was purchased from Calbiochem. Glutathione agarose beads were from Sigma. Protein G-Sepharose 4B and protein A-Sepharose 4B beads were purchased from Zymed Laboratories. Goat anti-MKK6, rabbit anti-MEK1, rabbit anti-VDR, goat anti-JNK2, and rabbit antibodies against p38, c-Fos, c-Jun, and ATF2 (the ATF2 recombinant protein) were obtained from Santa Cruz Biotechnology. Anti-FLAG M2 affinity gel and MBP for ERK assay were bought from Sigma. Vitamin D3 was provided by Hoffmann-La Roche. GST-Jun for JNK assay was purified with glutathione agarose beads (48). [gamma -32P]ATP and dCTP and [methyl-3H]thymidine were from Amersham Biosciences.

Cell Culture, Transfection, Infection, and Assay for Cell Proliferation-- Human breast cancer cell lines MCF-7 and MDA-MB-468 were obtained from ATCC and maintained in minimal essential medium containing 10% fetal bovine serum and antibiotics at 37 °C with 5% CO2. An MCF-7 subline stably expressing Tam67 (MCF/Tam) was generated by transfecting MCF-7 cells with equal amounts of pCMV-Tam and pcDNA3 (neomycin-resistant expression plasmid) using the FuGENE 6 kit (Invitrogen) followed by selection with 0.6 mg/ml G418 for one month (44). The resistant clones were pooled, and early passages of these cells were used for experiments. Expression of the transfected c-Jun Tam67 was detected using a c-Jun antibody (sc-44-G, Santa Cruz Biotechnology) that recognizes the COOH-terminal DNA-binding region (residues 247-263) of c-Jun. For transient transfection, the protocol of calcium phosphate-mediated transfection from Promega followed. Cells were allowed to grow for 48 h in serum-free medium before being collected for various assays. For adenovirus infection, cells were typically plated for a day before and switched to serum-free medium the next day. Cells were incubated with adenovirus (50 plaque-forming units/cell) for 5 h and then further incubated for 24 (468) or 48 h (MCF-7) to allow expression of proteins of interest. For cell proliferation, cells were infected with the control virus or the adenovirus coding for MKK6 for 5 h in serum-free minimal essential medium in six-well plates as mentioned above. Following infection, cells were washed once with phosphate-buffered saline and fed with serum-free medium containing the vehicle or 0.1 µM of vitamin D3 for 48 h. For the last 4 h of cell culture, 1 µCi of [3H]thymidine (Amersham Biosciences) was added (49). After trypsinization, the amount of thymidine incorporated into cells was determined by scintillation counting using ScintiSafeTM Plus 50% (Fisher Scientific) in a Scintillation Counter (Beckman). The results of these proliferation studies and the following luciferase assay, Western blot analyses, Northern blot analyses, and kinase assays were analyzed with the Student's t test for the statistically significant difference.

Luciferase Assay and Site-directed Mutagenesis-- Cells were transfected with calcium phosphate according to the Promega manual for 5 h (total 3 µg of DNA per 2 ml/well in a six-well plate). By the end of the experiments (48 h after DNA removal), cells were washed with phosphate-buffered saline and collected in the lysis buffer. Luciferase activity of the promoter was assayed with a dual luciferase kit from Promega using pRL-TK (encoding Renilla luciferase) as a normalization control in a TD-20/20 Luminometer (Turner Designs). Mutations in the potential AP-1 binding site of mVDR-luc plasmid were performed with the QuikChangeTM site-directed mutagenesis kit from Stratagene as instructed in the user manual. The sequences of the PCR primers were M1: FW, 5'-CTCAGGTACGGGTGCTACACCTGGGGGAGGCG-3' and REV, 5'-CGTAAACGCCTCCCCCAGGTGTAGCACCCGTACCTGAG-3'; M2: FW, 5'-CTCAGGTACGGGTGCTAGGCCTGGGGGAGGCG-3' and REV, 5'-CTGTAAACGCCTCCCCCAGGCCTAGCACCCGTACCTGAG-3'. The size of the mutated plasmid was verified with restriction enzyme digestion analysis, and the correct sized plasmid was used for further confirmation by DNA sequencing for the desired mutations.

Western and Northern Blotting Analyses-- For Western blot analyses, cells were lysed in modified radioimmune precipitation buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EGTA, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml aprotinin, leupeptin, and pepstatin). Protein concentration was determined by the DC Protein Assay kit (Bio-Rad). Typically, 50 µg of protein was separated by SDS-PAGE, which was transferred to a nitrocellulose membrane for detection of the molecule of interest using ECL (Amersham Biosciences) as described previously (41). For Northern blot, total RNA was prepared by TRIzol (Invitrogen). Human VDR cDNA in pCMV vector (kindly provided by Leonard Freedman (50)) was digested with ApaI/XbaI to generate a fragment as the probe. The probe was labeled with [32P]dCTP using the High Prime kit (Roche Molecular Biochemicals) and purified with Quick Spin Columns (Roche Molecular Biochemicals). The Northern blots were standardized for equal application of RNA by comparing the UV fluorescence of the 18 S rRNA band.

Preparation of Nuclear Extract, Electrophoretical Mobility Shift Assay (EMSA)-- Following infection or arsenite (ARS) treatment, about 1 × 107 cells were lysed in 10 mM Tris, pH 8.0, containing 0.5% Nonidet P-40, 2 mM MgCl2, 5 mM KCl, and 1 mM phenylmethylsulfonyl fluoride on ice for 15 min after washing once with cold phosphate-buffered saline (51, 52). Cells then were scraped down, and the pellets were resuspended in 100 µl of nuclear lysis buffer (10 mM Tris, pH 8.0, 0.1% Nonidet P-40, 500 mM NaCl, 1 mM EDTA, and proteinase inhibitors (Cocktail, Roche Molecular Biochemicals). The mixture was incubated on ice for 30 min with vortexing every 5 min. Following spin, the supernatant was either stored at -80 °C (normally used within 2 weeks) or used immediately for EMSA.

For EMSA, 5 µg of nuclear protein was pre-incubated in DNA binding buffer (10 mM HEPES, pH 7.9, 60 mM KCl, 4% Ficoll, 1 mM dithiothreitol, 1 mM EDTA, 1 mg/ml of poly [d(I-C)] (Amersham Biosciences). In supershift assays, incubation was in the absence or presence of 1 µg of respective antibody (Santa Cruz Biotechnology: rabbit anti-ATF2, cat. no. sc-187 X; rabbit anti c-Jun, cat. no. sc-44 X) for 15 min. The mixture was then incubated with ~105 cpm-labeled probe in the absence or presence of 50-fold cold oligos on ice for 30 min. The double-stranded oligonucleotides were obtained from Santa Cruz Biotechnology with the sequence as follows: AP-1 consensus oligonucleotide, 5'-CGC TTG ATG ACT CAG CCG GAA-3'; AP-1 mutant oligonucleotide, 5'-CGC TTG ATG ACT tgG CCG GAA-3'; VDRE consensus oligonucleotide (DR-3), 5'-AGC TTC AGG TCA AGG AGG TCA GAG AGC-3'.

The oligonucleotides were labeled with [32P]ATP and T4 polynucleotide kinase for 45-60 min and purified by Qiaquick nucleotide remove kit (Qiagen). Following incubation, the mixture was subjected to a native 4.5% acrylamide gel in 0.5× Tris borate/EDTA buffer. The gels then were dried and subjected to autoradiography.

Immunoprecipitation and Protein Kinase Assay-- Cells were collected by trypsinization, and pellets were quick-frozen in liquid nitrogen after washing and spinning. The pellet either was stored at -80 °C or was used immediately for protein preparation (resuspended in 100 µl of the lysis buffer and incubated on ice for 30 min with vortexing for 30 s every 10 min). Protein (200 µg) was incubated either with 30 µl of 50% glutathione-agarose beads (Sigma) for GST-tagged ERK or JNK or with 20 µl of anti-FLAG M2 affinity gel (Sigma) for FLAG-tagged p38 at 4 °C in a rotating plate overnight. For endogenous JNK and p38 kinase assay, the protein was incubated with 1 µg of polyclonal antibodies from Santa Cruz Biotechnology against JNK2 (sc-572) and p38 (sc-535) and with 30 µl of protein A-Sepharose (Zymed Laboratories Inc.) at a volume of 300 µl. All of the beads were washed four times with the lysis buffer before adding to the mixture.

The precipitates were washed twice with respective lysis buffer and twice with kinase binding buffer (20 mM HEPES pH 7.6, 50 mM NaCl, 0.05% Triton X-100, 0.1 mM EDTA, 2.5 mM MgCl2). The kinase reaction was carried out at 30 °C for 30 min in 25 µl of kinase reaction buffer (20 mM HEPES pH 7.6, 20 mM MgCl2, 15 µM ATP, 20 mM beta -glycerol- phosphate, 20 mM p-nitrophenyl phosphate, 0.5 mM Na3VO4, 2 mM dithiothreitol) as described previously (41, 49). [gamma -32P]ATP (5 µCi) and 1 µg of substrate protein were used for each sample. Substrates for the kinase reactions were MBP for ERK, GST-Jun for JNK, and ATF-2 for p38. Following a 30-min reaction at 30 °C, an equal volume of 2× Laemmli buffer was added to stop the reaction. The phosphorylated proteins were separated by SDS-PAGE. The gels were dried, scanned, and quantitated in a phosphorimager. Each kinase assay starting at transfection and ending with the determination of the protein kinase activity was repeated two to three times with similar results.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MKK6 Stimulates VDR Promoter Activity-- To test directly the hypothesis that p38 kinase stimulates VDR expression, the mouse VDR promoter in a luciferase reporter vector (VDR-Luc) (37) was expressed transiently in human breast cancer MCF-7 cells with a constitutively active MKK6 (MKK6/E), an upstream activator of p38 kinase (45), or an empty vector control. As a further control, a constitutively active MEK1 (MEK1/E), an ERK activator, was co-transfected with the VDR-Luc vector into MCF-7 cells. It was observed that MKK6/E expression stimulates the VDR-Luc promoter activity by 10-fold, whereas the transfection with MEK1 has no effect (Fig. 1A). Because vitamin D3 is known to induce its receptor, VDR, protein expression (53, 54), we examined whether the MKK6 activation is regulated by vitamin D3. Accordingly, MKK6/E- and VDR-Luc-transfected cells were incubated with 0.1 µM vitamin D3 for 48 h prior to the luciferase assay. The results showed that the VDR promoter activation in MCF-7 cells is independent of vitamin D3. This is consistent with the lack of a potential VDRE sequence in the VDR promoter (37).


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1.   Stimulation of VDR promoter activity by dominant active MKK6 in human breast cancer cells. Cells were transiently transfected with the VDR promoter reporter (VDR-Luc) together with the pCMV expression vector (Vect) with or without MKK6/E- or MEK1/E-containing plasmids. The luciferase activity was determined after a 48-h incubation in the absence or presence of 0.1 µM vitamin D3 by a dual luciferase kit as described under "Materials and Methods." The results are the mean of three to four separate experiments (bars, S.E.). A, stimulation of VDR promoter activity by MKK6/E but not MEK1/E in MCF-7 cells (p < 0.05 for MKK6 versus Vect alone, but p > 0.05 for MEK1 versus the vector control, and p > 0.05 for MKK6 versus MKK6 plus vitamin D3); B, activation of VDR promoter activity by MKK6/E and MEK1/E in 468 cells (p < 0.05 for MKK6 and MEK1 versus Vect alone, respectively, but p > 0.05 for Vect plus vitamin D3, MKK6 plus vitamin D3, and MEK1 plus vitamin D3 versus their respective control groups without vitamin D3).

Previous work has suggested that there are functional interactions between vitamin D3/VDR and estrogen signaling in breast cancer cells (55). It is, therefore, important to determine whether the VDR promoter stimulation by the p38 activation is ER-dependent in breast cancer cells. Fig. 1B shows that p38 activation by MKK6/2E expression stimulates the VDR promoter 6-fold in ER-negative human breast cancer MDA-MB-468 (468) cells (56). Activation of the ERK pathway by MEK1/E in these cells also increases the promoter activity by 4-fold, a phenomenon that was not seen in ER-positive MCF-7 cells. Similar to ER-positive MCF-7 cells, addition of vitamin D3 does not alter the pattern of VDR promoter activation, despite the fact that the promoter activity was decreased in the vector-, MKK6/E-, and MEK1/E-transfected groups, probably as a result of nonspecific effects of vitamin D3. Taken together, these results demonstrate that MKK6 expression is able to stimulate the VDR promoter activity in human breast cancer cells, independently of ER status and the presence of vitamin D3.

MKK6 Increases VDR Expression, VDRE Binding, and Vitamin D3-induced Growth Inhibition in Human Breast Cancer Cells-- In order to establish activation of endogenous VDR by MKK6, recombinant adenoviruses encoding MKK6/E (35, 36) were applied to assess its effect on VDR expression and its biological activities. The infection efficiency in these two human breast cancer cells is more than 90%, and the results of Fig. 2A showed overexpression of the MKK6 protein following infection with the ad-MKK6 vector. After ad-MKK6 infection, total RNA was prepared and hybridized with 32P-labeled VDR cDNA probe. Fig. 2B shows that there is an increase in the VDR mRNA induced by MKK6 in comparison with the vector control in both MCF-7 and 468 cell lines. The mRNA elevation correlates with an increased level of VDR protein assayed by Western blot analyses (Fig. 2C). Importantly, ad-MKK6 does not increase ER protein expression (data not shown), indicating a specific effect on the steroid receptor VDR but not ER.


View larger version (60K):
[in this window]
[in a new window]
  		Fig. 2.   Increases in VDR mRNA and protein induced by ad-MKK6 in MCF-7 and 468 cells. A, MKK6 overexpression mediated by adenovirus-mediated infection. Proteins (50 µg) from cell lysates transfected with either ad-vector or ad-vector-expressing MKK6 were separated by SDS-PAGE and examined for MKK6 protein levels by Western blot analyses. B, increase of VDR mRNA induced by ad-MKK6. Total RNA was prepared with TRIzol (24 h after infection for 468 cells and 48 h after infection for MCF-7 cells) and analyzed for VDR mRNA levels by Northern blot analyses. Fold increase over the vector control from four experiments was 1.97 ± 0.38, S.D., p < 0.05 versus Vect. (MCF-7 cells) and 2.55 ± 0.79, S.D., p < 0.05 versus Vect (468 cells). C, elevation of VDR protein levels induced by ad-MKK6. Cell lysates were prepared after infection with ad-MKK6/E and subjected for Western blot analyses for expression of VDR protein. Fold increase over the vector control of four experiments after normalization to actin was 2.22 ± 0.32, S.D., p < 0.05 versus Vect (MCF-7 cells) and 2.43 ± 0.53, S.D., p < 0.05 versus Vect (468 cells).

To determine whether the elevated VDR is active in binding to its specific sequence VDRE, EMSA was performed by incubation of the labeled specific VDRE-containing oligo (DR3, Santa Cruz Biotechnology) with the nuclear lysates of MCF-7 and 468 cells following ad-MKK6 infection. Ad-MKK6-infected cells showed more protein binding to the DR3 oligo, which was completely inhibited by pre-incubation with excess cold wild-type oligo (Fig. 3A), indicating that the elevated VDR protein expression by the MKK6 is functionally active with regard to VDRE binding.


View larger version (35K):
[in this window]
[in a new window]
  		Fig. 3.   Ad-MKK6 increases binding to a VDRE element and sensitizes MCF-7 cells to vitamin D3-dependent growth inhibition. A, increase of VDR binding activity by ad-MKK6. Nuclear lysates were incubated with the labeled VDRE probe (DR3, see "Materials and Methods"), and analyzed by EMSA. B, sensitization of vitamin D3-induced growth inhibitory effect by ad-MKK6 in MCF-7 cells. Cells were infected for 5 h and then incubated in the absence or presence of 0.1 µM vitamin D3 for an additional 48 h. DNA synthesis then was determined by [3H]thymidine incorporation. The results shown are the mean of three independent experiments. Bars, S.E., n = 3, p < 0.05 for MKK6 + VitD3 versus Vect, or versus MKK6, or versus VitD3, respectively.

Because functional VDR is required for vitamin D3-induced growth inhibition in breast cancer cells (9, 10), VDR activation by MKK6 is expected to increase the sensitivity of MCF-7 cells to vitamin D3-dependent growth inhibition. To directly test this possibility, MCF-7 cells were infected first with the control virus or ad-MKK6 for 5 h and were then fed with virus-free medium in the absence or presence of vitamin D3 for 48 h. Cell proliferation was determined by [3H]thymidine incorporation. Although MKK6 may activate additional target genes, under these conditions neither ad-MKK6 nor vitamin D3 alone had any obvious inhibitory effect on DNA synthesis (Fig. 3B, p > 0.05). However, growth was reduced almost 50% by vitamin D3 in combination with ad-MKK6 infection (p < 0.05 versus vitamin D3 or MKK6 alone). A similar sensitization by ad-MKK6 was also observed in 468 cells (data not shown). Together, these results demonstrate that infection with ad-MKK6 up-regulates endogenous VDR expression, increases its DNA binding activity, and sensitizes human breast cancer cells to vitamin D3-induced growth inhibition.

MKK6 Stimulates VDR through the p38/JNK/c-Jun/AP-1 Pathways-- MKK6 is known to specifically activate all isoforms of p38 (45, 57, 58). Recent observations suggest that MKK6 can also stimulate JNK in various occasions (59, 60).2 Experiments were carried out to determine whether MKK6 signals to VDR via the p38 and/or the JNK pathways. MCF-7 cells were chosen for these analyses because the VDR promoter stimulation in these cells is more significant and is selectively responsive to the p38 activation by MKK6 but not to the ERK activation by MEK1 (Fig. 1A). p38 activity was assessed by assay of transiently expressed FLAG-p38 together with MKK6/E or MEK1/E and was compared with the activities of transfected GST-tagged ERK and JNK. The kinase activities were determined by assessing the activity of the precipitates immunoprecipitated with anti-FLAG antibody or with GSH beads followed by addition of their respective substrates in vitro (MBP for ERK, GST-Jun for JNK, and GST-ATF2 for p38) as described previously (41, 49). The MKK6/E was shown to strongly activate p38, whereas the ERK activity was stimulated only by the MEK1/E and not MKK6/E (Fig. 4A). Of interest, MKK6 also increased the JNK activity, albeit by a moderate level. In support of the validity of the results with exogenously expressed MAPKs, infection with ad-MKK6 also stimulated endogenous p38 and JNK when these enzymes were purified with respective antibodies and assessed for their kinase activities (Fig. 4B). These results suggest that MKK6 may activate VDR through both p38 and JNK kinases.


View larger version (50K):
[in this window]
[in a new window]
  		Fig. 4.   Activation of the p38 and JNK pathways by MKK6/E in MCF-7 cells. A, MKK6/E stimulates exogenous p38 and JNK (but not ERK) pathways. MCF-7 cells were transiently transfected with MKK6/E or other activators (MEK1/E, MEKK1, and Ras) together with epitope-tagged ERK, JNK, or p38. The activity of the transfected MAPKs was determined in vitro with the respective substrates (MBP for ERK, c-Jun for JNK, and ATF-2 for p38) after immunoprecipitation of the MAPK with the corresponding anti-epitope antibody. B, activation of the endogenous p38 and JNK pathways by ad-MKK6. Following ad-MKK6 infection, the endogenous p38 or JNK2 were immunoprecipitated with respective antibodies and their kinase activity determined in vitro with the substrates ATF-2 and c-Jun, respectively. Fold increase over the vector control (Cont.) from four independent experiments: p38 activity, 2.56 ± 0.52, p < 0.05 versus Cont. alone; JNK activity, 2.23 ± 0.42, p < 0.5 versus Cont. alone.

AP-1 is a major transcription factor, which consists of homodimers of the Jun family (c-Jun, JunD, and JunB) or heterodimers of a Jun family member with any of the Fos family members (c-Fos, FosB, Fra-1, and Fra-2) or other transcription factors such as ATF2, CREB, and NFAT. Because all three MAPK pathways (ERK, JNK, and p38) can activate AP-1 (61), experiments were performed next to examine whether AP-1 functions as an integrating module transmitting the p38 and JNK signals from MKK6 to VDR gene. AP-1 activity was determined by transient expression of an AP-1 luciferase promoter (a minimal c-fos promoter containing three additional AP-1 element repeats) (47) to assay for AP-1-driven gene expression, together with the active MKK6/E-expressing plasmid and various inhibitors of the p38 and JNK pathways. As shown in Fig. 5A, activation of p38 and JNK by MKK6 expression increased AP-1 activity by a mechanism requiring both p38 and JNK activities, because co-expression of either dominant negative p38 (p38/AF) or JNK (JNK/AF) substantially reduced AP-1 trans-activation. The role of p38 is further confirmed by decreased AP-1 activity in the presence of a chemical p38 inhibitor SB203580. In a similar manner (Fig. 5B), the stimulation of VDR promoter by MKK6 is also inhibited by dominant negatives of either p38 (p38/AF, SB 203586) or JNK (JNK/AF), indicating a requirement of both the p38 and JNK pathways for the VDR activation. Together, these results suggest that the signaling from p38 and JNK activation is integrated at AP-1, which may contribute to the VDR trans-activation. Consistent with this conclusion, expression of a dominant active JNK kinase kinase MEKK1 also increases kinase activity of JNK and p38 (Fig. 4A) and stimulates AP-1 reporter (Fig. 5C) and VDR promoter activity (Fig. 5D).


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 5.   MKK6/E stimulates AP-1 trans-activation activity (A) and VDR promoter activity (B) in a manner dependent on the p38 and JNK pathways; AP-1 reporter (C) and VDR promoter (D) are stimulated by MEKK1. Cells were co-transfected with the pCMV plasmids containing the indicated wild-type or mutant genes and either the (AP-1)3-Fos-Luc vector (A) or the VDR-Luc vector (B), and the luciferase activity was determined 48 h later with Renilla luciferase (Promega) as an internal control. In some experiments 20 µM SB203580 was added to the culture for the last 24 h after transfection. Results are the mean of four separate determinations (bars, S.E., n = 3-4). MEKK1 increases AP-1 (C) and VDR promoter (D) activity (bars, S.E., n = 4 for AP-1 activity and n = 3 for VDR activity).

The above results suggest a potential role of AP-1 activity in the stimulation of VDR gene expression by p38 and JNK activation. To further confirm the involvement of AP-1 in VDR activation and analyze molecular mechanisms of the AP-1 activity, a mutant c-Jun, Tam67, was used in the co-transfection. Tam67 codes for c-Jun minus its activation domain, and its expression has been shown to quench AP-1 trans-activating activities (42-44). Expression of Tam67 in MCF-7 cells inhibited the MKK6/E-induced but not basal AP-1 activity (Fig. 5A). Importantly, Tam67 also suppressed the MKK6/E-triggered VDR promoter activation (Fig. 5B). These results suggest a potential involvement of c-Jun in AP-1 activation and further support the role of AP-1 activity in VDR stimulation.

To further confirm c-Jun involvement, the level of the major AP-1 components (c-Jun, c-Fos, and ATF-2) was determined at 24 and 48 h after infection with the ad-MKK6 by Western blot analysis. As shown in Fig. 6A, c-Jun was undetectable in MCF-7 cells in the control infection, which was in agreement with previous reports (62). Ad-MKK6 significantly induced c-Jun protein level at 48 h, which correlated with the increase in phosphorylated c-Jun as detected with a specific antibody against phosphorylated c-Jun at Ser-63 (data not shown). Levels of ATF-2 were not altered by ad-MKK6, nor were the levels of c-Fos. To further establish the role of c-Jun in AP-1 activity, MCF-7 cells were stably transfected with the Tam67 construct and selected for G418 expression and expression of the truncated 29-kDa c-Jun protein as described (Fig. 6B) (44). AP-1 binding activity to a synthesized AP-1 consensus sequence was determined by EMSA. Fig. 6C shows that the nuclear lysates from ad-MKK6-infected cells have higher AP-1 binding activity than in the vector-transfected MCF-7 cells (MCF/Vect). Binding specificity is suggested by the complete inhibition of protein binding by competing with 50-fold cold excess of wild-type oligo. In MCF-7 cells stably expressing the dominant negative c-Jun Tam67 (MCF/Tam), however, there was no increase in binding to the AP-1 sequence after the MKK6 infection. Similar to our results, an inhibition of nickel-induced AP-1 element binding by Tam67 in human bronchial epithelial cells recently was reported (63). Supporting the role of c-Jun in AP-1 activity, c-Jun antibody decreased the AP-1 element binding to the control level. Surprisingly, although ATF2 is a major substrate of p38 (1, 2), anti-ATF2 had no effect on the retarded protein band (Fig. 6D), indicating that p38 induction of AP-1 activity is ATF2-independent. Taken together, these data support the hypothesis that MKK6 activates VDR via a p38- and JNK-dependent c-Jun/AP-1 pathway.


View larger version (45K):
[in this window]
[in a new window]
  		Fig. 6.   Contribution of c-Jun to MKK6-induced AP-1 activity. A, induction of c-Jun expression by ad-MKK6. Cells were infected with the control virus or ad-MKK6, and cell lysates were prepared 24 or 48 h later for detection of c-Jun, c-Fos, or ATF-2 protein expression by Western blot analyses. B, overexpression of a truncated c-Jun (Tam67) protein in MCF-7 cells (MCF/Tam). MCF-7 cells transfected with the pCMV-TAM67 plasmid and the pcDMA3 plasmid (expressing the neomycin resistance gene), selected for G418 resistance, were analyzed by Western blot analyses with anti-c-Jun antibody. A truncated c-Jun protein with a band at 29 kDa was observed, which is the expected molecular mass for TAM67. No band for wild-type c-Jun at 39 kDa was observed in Tam-expressing cells. C, increase of AP-1 binding activity by ad-MKK6 in MCF/Vect but not in MCF/Tam cells. After infection with the ad-MKK6, the nuclear lysates were prepared and incubated with 32P-labeled AP-1 consensus oligonucleotides (see "Materials and Methods" for the details of EMSA). D, inhibition of ad-MKK6-induced AP-1 binding activity by anti-c-Jun antibody. The nuclear lysates were pre-incubated with anti-c-Jun or anti-ATF-2 antibody for 30 min on ice before mixing with the labeled oligo probe and EMSA analysis.

The AP-1 Sequence on the VDR Promoter Mediates Stimulation by MKK6-- In the experiments described below we directly tested effects of AP-1 enhancer activation by site-directed mutagenesis of the VDR promoter to further confirm the role of AP-1 on VDR promoter activity. There is a single potential AP-1 consensus sequence in the VDR promoter between -377 and -370 (TGACACA) (Fig. 7A), which differs by only one nucleotide from the consensus AP-1 site TGACTCA as found in the osteocalcin promoter (37) (the same sequence that was used as the probe for AP-1 binding in EMSA under "Materials and Methods"). The TGACACA sequence in the VDR promoter was mutated to TGCTACA, and the mutated promoter was cloned into a pGL2 basic luciferase vector to generate the M1 VDR luciferase reporter vector. A second mutant VDR reporter vector was made with double mutations at TGCTAGG to obtain the M2 VDR luciferase reporter (underline indicates mutated nucleotides). Site-directed mutagenesis was achieved using QuikChangeTM site-directed mutagenesis kit as instructed in the user's manual and confirmed with enzyme digestion and DNA sequencing. The effect of stimulation by MKK6/E was then determined with transient transfections. Expression of c-Jun and c-Fos by transient transfection to generate high cellular levels of AP-1 activity was used as a positive control. As shown in Fig. 7B, stimulation of VDR promoter activity by p38 and JNK activation by MKK6/E was dramatically decreased when the AC was changed to CT (the M1 promoter) and was almost completely abolished when additional nucleotides, CA, were changed to GG (the M2 promoter, p < 0.05 for M2 promoter versus wild-type promoter). The specificity of these mutations is further confirmed by a similar decrease of the luciferase activity in both mutants in the presence of AP-1 expression caused by the transfection with c-Jun plus c-Fos plasmids. A similar mutation in the AP-1 consensus sequence of cyclin D1 promoter was found to block c-Jun-mediated trans-activation (64). These results provide direct evidence that the AP-1 sequence in the VDR promoter is responsible for the increase in VDR expression stimulated by the p38 and JNK activities conferred by MKK6.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 7.   The AP-1 element requirement for MKK6 stimulation of VDR promoter in MCF-7 cells. A, diagram of some potentially important enhancer elements in the mouse VDR promoter with the AP-1 sequence at -377 are indicated. B, inhibition of the VDR promoter stimulation by MKK6/E and by c-Jun and c-Fos overexpression with site-directed mutagenic forms of the AP-1 sequence. pCMV plasmids containing MKK6/E, c-Jun and c-Fos, or vector alone were transiently co-transfected into MCF-7 cells with the wild-type or AP-1 mutant VDR promoter-Luc plasmid. The results shown are the mean of three separate experiments (bars, S.E.).

ARS Activates JNK/p38/AP-1 and Increases VDR Expression-- The above results clearly have demonstrated that signaling from the p38 and JNK pathways conferred by MKK6 expression can be integrated at c-Jun/AP-1, leading to trans-activation of the VDR gene via the AP-1 consensus sequence on the VDR promoter in human breast cancer cells. Although expression of genetic materials is essential for establishing a specific signal transduction pathway to control a target gene expression, efforts also were made to confirm and extend these results without MKK6 transfection to further examine the physiological relevance of this observation. Arsenite, a well known p38 and JNK stimulator in many cell lines (49, 65-67), was included here to investigate effects of activation of endogenous p38 and JNK on AP-1 activity and VDR expression in these human breast cancer cells. ARS in MCF-7 cells stimulated AP-1 reporter activity (Fig. 8A), AP-1 DNA binding (not shown), and VDR promoter activity (Fig. 8B). Following ARS treatment, cell lysates were collected, endogenous p38 and JNK were immunoprecipitated, and the kinase activities determined as described above (Fig. 4). Treatment with ARS strongly activated MKK6 (data not shown) and endogenous p38 and JNK (Fig. 8C). Most importantly, the same treatment also increased VDR protein expression (Fig. 8D). Although additional pathways also may be activated by ARS, at least part of the VDR protein induction by ARS can be ascribed to activation of the p38/JNK/AP-1 pathway (Fig. 9). These results, therefore, establish that activation of the endogenous stress pathways by a chemical stimulus ARS leads to AP-1 activation and to increased VDR protein expression.


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 8.   Increase of VDR expression by activation of the p38/JNK/AP-1 pathway with ARS in MCF-7 cells. A, ARS increases AP-1 activity. Cells were transfected with the AP-1 luciferase construct (AP1)3-Fos-Luc, subjected to a 30-min treatment with 2 mM ARS after 24 h, and collected for the luciferase assay 24 h later. The results shown are the mean of three separate experiments (bars, S.E.). B, ARS increases VDR promoter activity (bars, S.E., n = 3). C, ARS stimulates p38 and JNK. After treatment with ARS, the endogenous p38 or JNK was immunoprecipitated with the respective antibodies and assayed by the in vitro kinase assay as described. D, ARS increases VDR protein expression. Protein (50 µg) from the MCF-7 cell lysates was separated by SDS-PAGE 24 h after ARS treatment, and the levels of VDR protein were determined by Western blot analyses. Cont., control.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 9.   Activation of the p38/JNK pathways stimulates VDR transcription via c-Jun/AP-1 and increases vitamin D3-induced and VDR-dependent growth inhibition in human breast cancer cells. Signaling from MKK6/ARS through the p38/JNK pathway is integrated at c-Jun/AP-1 and trans-activates VDR expression via the AP-1 consensus sequence, leading to increased VDR expression and enhanced vitamin D3-induced growth inhibition in MCF-7 cells. The question mark (?) indicates that the mechanisms for MKK6 activation of JNK and p38 phosphorylation by c-Jun remain unclear.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results demonstrate that the stress-activated protein kinases p38 and JNK trans-activate VDR through c-Jun-dependent AP-1 and sensitize human breast cancer cells to vitamin D3-dependent growth inhibition. This conclusion is based on the following observations. 1) MKK6 expression stimulated the exogenous and endogenous p38 and JNK pathways, which elevated both AP-1 trans-activation and VDR promoter activities. 2) Both the p38 and JNK activities are required for the AP-1 and VDR activations as shown by the transfection of dominant negative JNK and p38 genes and by the specific chemical p38 inhibitor SB203580. 3) The central role of c-Jun/AP-1 in this stimulation was shown by the selective c-Jun activation following stimulation of endogenous p38 and JNK by ad-MKK6, by the inhibitory effect of the mutant c-Jun (Tam67) on the induced AP-1 and VDR activations, and by the inhibitory effect of site-directed mutagenesis of the AP-1 element in the VDR promoter. 4) The biological significance of the VDR activation by p38 and JNK kinases was shown by the sensitization of MCF-7 cells to vitamin D3-mediated growth inhibition after infection with ad-MKK6. 5) The signaling connection between these stress pathways and VDR activation was further supported independently by treatment of the cells with the stress stimulator ARS. This work thus shows a previously unknown connection between the p38 and JNK MAPK pathways and the steroid hormone receptor VDR signaling through activation of the transcription factor c-Jun/AP-1.

The ERK or JNK MAPK pathway has been shown to interact with and phosphorylate ER (17, 20, 22, 68) and PR (18, 19, 23) at multiple levels in different systems. Our results show that VDR is stimulated transcriptionally by the p38/JNK pathway stimulation of AP-1. To our knowledge, this is the first report to show regulation of a steroid hormone receptor expression by MAPK pathways. In MCF-7 cells, the signaling connection between p38/JNK to VDR appears to be specific because activation of the ERK pathway by the constitutively active MEK did not stimulate VDR promoter (Fig. 1A). Also, the stimulation of the p38 and JNK pathways by ad-MKK6 increased levels of VDR but not ER protein expression (Fig. 2C and data not shown). The p38/JNK signaling specificity in MCF-7 cells is further demonstrated by the observation that the VDR promoter stimulation by MKK6 expression is vitamin D3- and ER-independent, in contrast to observations in some other systems (53, 69). The cooperative role of p38 and JNK in stimulation of VDR is suggested by the fact that both MKK6 and ARS stimulate both kinases, that inhibition of one of them is sufficient to block the AP-1/VDR stimulation, and that activation of p38/JNK by an independent molecule (MEKK1) also increases AP-1 and VDR activity (Fig. 4 and Fig. 5, C and D). Because VDR can bind to the VDRE in a manner independent of its ligand vitamin D3 (70), these results suggest that the p38/JNK stress pathways may regulate VDR-dependent target gene expression through VDR trans-activation without a requirement for the binding of vitamin D3.

Although our data are consistent with the notion that increased VDR activity is caused by transcriptional activation by p38 and JNK activities through the AP-1 consensus sequence, additional mechanisms such as VDR phosphorylations and interactions with molecules of the MKK6/p38/JNK/c-Jun pathway may also be involved in the increased VDR binding and the enhanced vitamin D3-dependent growth inhibition. It has been shown, for example, that VDR can be phosphorylated by casein kinase II at Ser-208, resulting in an increased transcriptional activity; albeit this remains to be confirmed (71, 72). Moreover, transcription factor activity of the VDR protein may be regulated by the phosphorylation of its partner RXRalpha at Ser-260 by the Ras/ERK pathway, leading to a decrease in VDR trans-activation activity and vitamin D3-dependent growth inhibition (29). In addition, VDR can co-associate with other transcription factors including Smad3 (73, 74) and transcription factor IIB (TFIIB) (7). The in vitro direct binding of VDR to c-Jun but not c-Fos was also described recently (75). It is therefore possible that in addition to transcriptional regulation, potential direct interactions between VDR and the MKK6/p38/JNK/c-Jun pathway also may contribute to increased VDR activity and subsequent potentiation of vitamin D3-induced growth inhibition.

The VDR is generally expressed at relatively low levels in vivo, and consequently regulation of VDR levels is an important approach for the regulation of vitamin D3-responses in target cells (7). Indeed, efforts have been made to regulate VDR expression using chemical stimuli including forskolin (37) and phorbol 12-myristate 13-acetate (30). The outcome of these investigations, however, is limited by nonspecific properties of these reagents. Elucidation of the signal transduction pathways that control VDR expression thus has important implications in the regulation of vitamin D3 activity. VDR up-regulation by the activation of endogenous p38 and JNK pathways by both MKK6 expression and ARS treatment indicates the feasibility of this regulation. Because functional VDR has been demonstrated to be required for vitamin D3-induced growth inhibition in breast cancer and other malignant cell lines (10-14), VDR stimulation by the p38/JNK stress pathways may aid in developing new strategies to improve therapeutic efficacy of vitamin D3 and overcome its resistance in tumor cells.

    ACKNOWLEDGEMENTS

We thank Leonard Freedman, Dennis Templeton, Roger Davis, Michael Birrer, Matthias Gaestel, Isamil Kola, and Craig Hauser for providing various reagents that made this work possible, and we thank Dennis Stacey, Andrew Vaughan, and Divaker Choubey for discussion.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants CA91576 (to G. C.) and GM51417 (to J. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Both authors contributed equally to this work.

§§ To whom correspondence should be addressed: Dept. of Radiation Oncology, Loyola University of Chicago, 114B-Bldg. 1 (Hines), 2160 S. First Ave., Maywood, IL 60153. Tel.: 708-202-8387 (ext. 23398); Fax: 708-202-2019; E-mail: gchen1@lumc.edu.

Published, JBC Papers in Press, April 30, 2002, DOI 10.1074/jbc.M203039200

2 X. Qi, R. Pramanik, R. M. Schultz, J. Han, and G. Chen, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: JNK, c-Jun NH2-terminal kinase; VDR, vitamin D receptor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; RXR, retinoid X receptor; VDRE, vitamin D response element; ER, estrogen receptor; PR, progesterone receptor; MEK, MAPK kinase; MEKK, MEK kinase; MKK, MAPK kinase; GST, glutathione S-transferase; MBP, myelin basic protein; MEK1/E, constitutively active MEK1; MKK6/E, constitutively active MKK6; EMSA, electrophoretical mobility shift assay; ARS, arsenite.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Nebreda, A. R., and Porras, A. (2000) Trends Biochem. Sci. 25, 257-260[CrossRef][Medline] [Order article via Infotrieve]
2. Ono, K., and Han, J. (2000) Cell. Signal. 12, 1-13[CrossRef][Medline] [Order article via Infotrieve]
3. Chang, L., and Karin, M. (2001) Nature 410, 37-40[CrossRef][Medline] [Order article via Infotrieve]
4. Han, J., Lee, J. D., Bibbs, L., and Ulevitch, R. J. (1994) Science 265, 808-811[Abstract/Free Full Text]
5. Lee, J. C., Laydon, J. T., McDonnel, P. C., Gallagher, T. F., Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W., Strickler, J. E., McLaughlin, M. M., Siemens, I. R., Fisher, S. M., Livi, G. P., White, J. R., Adams, J. L., and Young, P. R. (1994) Nature 372, 739-746[CrossRef][Medline] [Order article via Infotrieve]
6. Ichijo, H. (1999) Oncogene 18, 6087-6093[CrossRef][Medline] [Order article via Infotrieve]
7. Jones, G., Strugnell, S. A., and Deluca, H. F. (1998) Physiol. Rev. 78, 1193-1231[Abstract/Free Full Text]
8. Schrader, M., Muller, K. M., and Carlberg, C. (1994) J. Biol. Chem. 269, 5501-5504[Abstract/Free Full Text]
9. Berger, U., McClelland, R. A., Wilson, P., Greene, G. L., Haussler, M., Pike, J. W., Colston, K., Easton, D., and Coombes, R. C. (1991) Cancer Res. 51, 239-244[Abstract/Free Full Text]
10. Buras, R., Schumaker, L. M., Davoodi, F., Brenner, R. V., Shabahang, M., Nauta, R. J., and Evans, R. T. (1994) Breast Cancer Res. Treat. 31, 191-202[CrossRef][Medline] [Order article via Infotrieve]
11. Agadir, A., Lazzaro, G., Zheng, Y., Zhang, X., and Mehta, R. (1999) Carcinogenesis 20, 577-582[Abstract/Free Full Text]
12. Davoust, N., Wion, D., Chevalier, G., Garabedian, M., Brachet, P., and Couez, D. (1998) J. Neurosci. Res. 52, 210-219[CrossRef][Medline] [Order article via Infotrieve]
13. Musiol, I. M., and Feldman, D. (1997) Endocrinology 138, 12-18[Abstract/Free Full Text]
14. Park, M., Bae, H., Heydemann, A., Roberts, A. B., Dotto, P., and Sporn, M. B. (1994) Cancer Res. 54, 6087-6089[Abstract/Free Full Text]
15. Chen, H. C., and Farese, R. V., Jr. (1999) Curr. Biol. 9, R478-R481[CrossRef][Medline] [Order article via Infotrieve]
16. Olefsky, J. M. (2001) J. Biol. Chem. 276, 36863-36864[Free Full Text]
17. Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawashima, H., Metzger, D., and Chambon, P. (1995) Science 270, 1491-1494[Abstract/Free Full Text]
18. Lange, C. R., Shen, T., and Horwitz, K. B. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1032-1037[Abstract/Free Full Text]
19. Bagowski, C. P., Myers, J. W., and Ferrell, J. E., Jr. (2001) J. Biol. Chem. 276, 37708-37714[Abstract/Free Full Text]
20. Migliaccio, A., Domenico, M. D., Castoria, G., de Falco, A., Bontempo, P., Nola, N., and Auricchio, F. (1996) EMBO J. 15, 1292-1300[Medline] [Order article via Infotrieve]
21. Migliaccio, A., Piccolo, D., Castoria, G., Di, D. M., Bilancio, A., Lombardi, M., Gong, W., Beato, M., and Auricchio, F. (1998) EMBO J. 17, 2008-2018[CrossRef][Medline] [Order article via Infotrieve]
22. Improta-Brears, T., Whorton, A. R., Codazzi, F., York, J., Meyer, T., and McDonnel, D. P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4686-4691[Abstract/Free Full Text]
23. Shen, T., Horwitz, K. B., and Lange, C. R. (2001) Mol. Cell. Biol. 21, 6122-6131[Abstract/Free Full Text]
24. Abreu-Martin, M., Chari, A., Palladino, A. A., Craft, N. A., and Sawyers, C. (1999) Mol. Cell. Biol. 19, 5143-5154[Abstract/Free Full Text]
25. Duan, R., Xie, W., Burghardt, R. C., and Safa, S. (2001) J. Biol. Chem. 276, 11590-11598[Abstract/Free Full Text]
26. Lee, S. K., Kim, H. J., Na, S. Y., Kim, T. S., Choi, H. S., Im, S. Y., and Lee, J. W. (1998) J. Biol. Chem. 273, 16651-16654[Abstract/Free Full Text]
27. Escaleira, M. T. F., and Brentani, M. M. (1999) Breast Cancer Res. Treat. 54, 123-133[CrossRef][Medline] [Order article via Infotrieve]
28. Sebag, M., Henderson, J., Rhim, J., and Kremer, R. (1992) J. Biol. Chem. 267, 12162-12167[Abstract/Free Full Text]
29. Solomon, C., White, J. H., and Kremer, R. (1999) J. Clin. Invest. 103, 1729-1735[Medline] [Order article via Infotrieve]
30. Krishnan, A., and Feldman, D. (1991) Mol. Endocrinol. 86, 2071-2079
31. Solomon, C., Sebag, M., White, E., Rhim, J., and Kremer, R. (1998) J. Biol. Chem. 273, 17573-17578[Abstract/Free Full Text]
32. Berra, E., Diaz, M. M., and Moscat, J. (1998) J. Biol. Chem. 273, 10792-10797[Abstract/Free Full Text]
33. Oh, C., Chang, S., Yoon, Y., Lee, S., Lee, Y., Kang, S., and Chun, J. (2000) J. Biol. Chem. 275, 5613-5619[Abstract/Free Full Text]
34. Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., and Greenberg, M. E. (1995) Science 270, 1326-1331[Abstract/Free Full Text]
35. Huang, S., Jiang, Y., Li, Z., Nishida, E., Mathias, P., Lin, S., Ulevitch, R. J., Nemerow, G. R., and Han, J. (1997) Immunity 6, 739-749[CrossRef][Medline] [Order article via Infotrieve]
36. Wang, X., McGowan, C. H., Zhao, M., He, L., Downey, J. S., Fearns, C., Wang, Y., Huang, S., and Han, J. (2000) Mol. Cell. Biol. 20, 4543-4552[Abstract/Free Full Text]
37. Jehan, F., and Deluca, H. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10138-10143[Abstract/Free Full Text]
38. Jehan, F., and Deluca, H. (2000) Arch. Biochem. Biophys. 377, 273-283[CrossRef][Medline] [Order article via Infotrieve]
39. Yan, M., Dal, T., Deak, J. C., Kyriakis, J. M., Zon, L. I., Woodgett, J. R., and Templeton, D. J. (1994) Nature 372, 798-800[Medline] [Order article via Infotrieve]
40. Yan, M., and Templeton, D. J. (1994) J. Biol. Chem. 269, 19067-19073[Abstract/Free Full Text]
41. Chen, G., Templeton, D., Suttle, D. P., and Stacey, D. (1999) Oncogene 18, 7149-7160[CrossRef][Medline] [Order article via Infotrieve]
42. Brown, P. H., Alani, R., Preis, L. H., Szabo, E., and Birrer, M. J. (1993) Oncogene 8, 877-886[Medline] [Order article via Infotrieve]
43. Brown, P. H., Chen, T. K., and Birrer, M. J. (1994) Oncogene 9, 791-799[Medline] [Order article via Infotrieve]
44. Janulis, M., Silberman, S., Ambegaokar, A., Gutkind, J. S., and Schultz, R. M. (1999) J. Biol. Chem. 274, 801-813[Abstract/Free Full Text]
45. Raingeaud, J., Whitmarsh, A. J., Barrett, T., Derijard, B., and Davis, R. (1996) Mol. Cell. Biol. 16, 1247-1255[Abstract]
46. Engel, K., Schultz, H., Martin, F., Kotlyarov, A., Plath, K., Hahn, M., Heinemann, U., and Gaestel, M. (1995) J. Biol. Chem. 270, 27213-27221[Abstract/Free Full Text]
47. Galang, C. K., Der, C. J., and Hauser, C. A. (1994) Oncogene 9, 2913-2921[Medline] [Order article via Infotrieve]
48. Smith, D. B., and Johnson, K. S. (1988) Gene 67, 31-40[CrossRef][Medline] [Order article via Infotrieve]
49. Chen, G., Hitomi, M., Han, J., and Stacey, D. W. (2000) J. Biol. Chem. 275, 38973-38980[Abstract/Free Full Text]
50. Liu, M., Lee, M. H., Bommakanti, M., and Freedman, L. P. (1996) Genes Dev. 10, 142-153[Abstract/Free Full Text]
51. Thomas, R. S., Tymms, M. J., McKinlay, L. H., Shannon, M. F., Seth, A., and Kola, I. (1997) Oncogene 14, 2845-2855[CrossRef][Medline] [Order article via Infotrieve]
52. Silberman, S., Janulis, M., and Schultz, R. M. (1997) J. Biol. Chem. 272, 5927-5935[Abstract/Free Full Text]
53. Wiese, R. J., Rhland-Smith, A., Ross, T. K., Prahl, J. M., and DeLuca, H. F. (1992) J. Biol. Chem. 267, 20082-20086[Abstract/Free Full Text]
54. Li, X. Y., Boudjelal, M., Xiao, J. H., Peng, Z. H., Asuru, A., Kang, S., Fisher, G. J., and Voorhees, J. J. (1999) Mol. Endocrinol. 13, 1686-1694[Abstract/Free Full Text]
55. Love-Schimenti, C. D., Gibson, D. F. C., Ratnam, A. V., and Bikle, D. D. (1996) Cancer Res. 56, 2789-2794[Abstract/Free Full Text]
56. Armstrong, D. K., Kaufmann, S. H., Ottaviano, Y. L., Furuya, Y., Buckley, J. A., Isaacs, J., and Davidson, N. E. (1994) Cancer Res. 54, 5280-5283[Abstract/Free Full Text]
57. Han, J., Lee, J., Jiang, Y., Li, Z., Feng, L., and Ulevitch, R. J. (1996) J. Biol. Chem. 271, 2886-2891[Abstract/Free Full Text]
58. Enslen, H., Raingeaud, J., and Davis, R. J. (1998) J. Biol. Chem. 273, 1741-1748[Abstract/Free Full Text]
59. Spigelman, V., Stavropoulos, P., Latres, E., Pagano, M., Ronai, Z., Slaga, T., and Fuchs, S. Y. (2001) J. Biol. Chem. 276, 27152-27158[Abstract/Free Full Text]
60. Wang, C., Deng, L., Hong, M., Akkaraju, G. R., Inoue, J., and Chen, Z. (2001) Nature 412, 346-351[CrossRef][Medline] [Order article via Infotrieve]
61. Karin, M. (1995) J. Biol. Chem. 270, 16483-16486[Free Full Text]
62. Smith, L., Wise, S. C., Hendricks, D. T., Sabichi, A. L., Bos, T., Reddy, P., Brown, P. H., and Birrer, M. (1999) Oncogene 18, 6063-6070[CrossRef][Medline] [Order article via Infotrieve]
63. Andrew, A. S., Klei, L. R., and Barchowsky, A. (2001) Am. J. Physiol. Lung Cell Mol. Physiol. 281, L616-L623[Abstract/Free Full Text]
64. Wisdom, R., Johnson, R. S., and Moore, C. (1999) EMBO J. 18, 188-197[CrossRef][Medline] [Order article via Infotrieve]
65. Cavigelli, M., Li, W., Lin, A., Su, B., Yoshioka, K., and Karin, M. (1996) EMBO J. 15, 6269-6289[Medline] [Order article via Infotrieve]
66. Ludwig, S., Hoffmeyer, A., Goebeler, M., Kilian, K., Hafner, H., Neufeld, B., Han, J. H., and Rapp, U. R. (1998) J. Biol. Chem. 273, 1917-1922[Abstract/Free Full Text]
67. Huang, C., Ma, W., Li, J., Goranson, A., and Dong, Z. (1999) J. Biol. Chem. 274, 14595-14601[Abstract/Free Full Text]
68. Kousteni, S., Bellido, T., Plotkin, L. I., O'Brien, C. A., Bodenner, D. L., Han, L., Han, K., DiGregorio, G. B., Katzenellenbogen, J. A., Katzenellenbogen, B. S., Roberson, P. K., Weinstein, R. S., Jilka, R. L., and Manolagas, S. C. (2001) Cell 104, 719-730[Medline] [Order article via Infotrieve]
69. Walters, M. R. (1981) Biochem. Biophys. Res. Commun. 103, 721-726[CrossRef][Medline] [Order article via Infotrieve]
70. Ross, T. K., Darwish, H. M., Moss, V. E., and DeLuca, H. F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9257-9260[Abstract/Free Full Text]
71. Jurutka, P. W., Hsieh, J. C., Nakajima, S., and Haussler, C. A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 3519-3524[Abstract/Free Full Text]
72. Hilliard, G. M., IV, Cook, R. G., Weigel, N. L., and Pike, J. W. (1944) Biochemistry 33, 4300-4311
73. Yanagi, Y., Suzawa, M., Kawabata, M., Miyazono, K., Yanagisawa, J., and Kato, S. (1999) J. Biol. Chem. 274, 12971-12974[Abstract/Free Full Text]
74. Yanagisawa, J., Yanagi, Y., Masuhiro, Y., Suzawa, M., Watanabe, M., Kashiwagi, K., Toriyabe, T., Kawabata, M., Miyazono, K., and Kato, S. (1999) Science 283, 1317-1321[Abstract/Free Full Text]
75. Towers, T. L., Staeva, T. P., and Freedman, L. (1999) Mol. Cell. Biol. 19, 4191-4199[Abstract/Free Full Text]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
A. Maiti, N. C. Hait, and M. J. Beckman
Extracellular Calcium-sensing Receptor Activation Induces Vitamin D Receptor Levels in Proximal Kidney HK-2G Cells by a Mechanism That Requires Phosphorylation of p38{alpha} MAPK
J. Biol. Chem., January 4, 2008; 283(1): 175 - 183.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. Qi, N. M. Pohl, M. Loesch, S. Hou, R. Li, J.-Z. Qin, A. Cuenda, and G. Chen
p38{alpha} Antagonizes p38{gamma} Activity through c-Jun-dependent Ubiquitin-proteasome Pathways in Regulating Ras Transformation and Stress Response
J. Biol. Chem., October 26, 2007; 282(43): 31398 - 31408.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
L. A. Gilad and B. Schwartz
Association of estrogen receptor {beta} with plasma-membrane caveola components: implication in control of vitamin D receptor
J. Mol. Endocrinol., June 1, 2007; 38(6): 603 - 618.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Q.-P. Li, X. Qi, R. Pramanik, N. M. Pohl, M. Loesch, and G. Chen
Stress-induced c-Jun-dependent Vitamin D Receptor (VDR) Activation Dissects the Non-classical VDR Pathway from the Classical VDR Activity
J. Biol. Chem., January 19, 2007; 282(3): 1544 - 1551.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
X. Qi, J. Tang, M. Loesch, N. Pohl, S. Alkan, and G. Chen
p38{gamma} Mitogen-Activated Protein Kinase Integrates Signaling Crosstalk between Ras and Estrogen Receptor to Increase Breast Cancer Invasion.
Cancer Res., August 1, 2006; 66(15): 7540 - 7547.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Shrivastava, M. Tiwari, R. A. Sinha, A. Kumar, A. K. Balapure, V. K. Bajpai, R. Sharma, K. Mitra, A. Tandon, and M. M. Godbole
Molecular Iodine Induces Caspase-independent Apoptosis in Human Breast Carcinoma Cells Involving the Mitochondria-mediated Pathway
J. Biol. Chem., July 14, 2006; 281(28): 19762 - 19771.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
X. Wang and G. P. Studzinski
The Requirement for and Changing Composition of the Activating Protein-1 Transcription Factor during Differentiation of Human Leukemia HL60 Cells Induced by 1,25-Dihydroxyvitamin D3.
Cancer Res., April 15, 2006; 66(8): 4402 - 4409.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
T. I. Ellison, D. R. Dowd, and P. N. MacDonald
Calmodulin-Dependent Kinase IV Stimulates Vitamin D Receptor-Mediated Transcription
Mol. Endocrinol., September 1, 2005; 19(9): 2309 - 2319.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Tang, X. Qi, D. Mercola, J. Han, and G. Chen
Essential Role of p38{gamma} in K-Ras Transformation Independent of Phosphorylation
J. Biol. Chem., June 24, 2005; 280(25): 23910 - 23917.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
L. A. Gilad, T. Bresler, J. Gnainsky, P. Smirnoff, and B. Schwartz
Regulation of vitamin D receptor expression via estrogen-induced activation of the ERK 1/2 signaling pathway in colon and breast cancer cells
J. Endocrinol., June 1, 2005; 185(3): 577 - 592.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Chen, M. T. Cencioni, D. F. Angelini, G. Borsellino, L. Battistini, and C. F. Brosnan
Transcriptional Profiling of {gamma}{delta} T Cells Identifies a Role for Vitamin D in the Immunoregulation of the V{gamma}9V{delta}2 Response to Phosphate-Containing Ligands
J. Immunol., May 15, 2005; 174(10): 6144 - 6152.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. Qi, J. Tang, R. Pramanik, R. M. Schultz, S. Shirasawa, T. Sasazuki, J. Han, and G. Chen
p38 MAPK Activation Selectively Induces Cell Death in K-ras-mutated Human Colon Cancer Cells through Regulation of Vitamin D Receptor
J. Biol. Chem., May 21, 2004; 279(21): 22138 - 22144.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
F. Barletta, P. Dhawan, and S. Christakos
Integration of hormone signaling in the regulation of human 25(OH)D3 24-hydroxylase transcription
Am J Physiol Endocrinol Metab, April 1, 2004; 286(4): E598 - E608.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. Qi, S. Borowicz, R. Pramanik, R. M. Schultz, J. Han, and G. Chen
Estrogen Receptor Inhibits c-Jun-dependent Stress-induced Cell Death by Binding and Modifying c-Jun Activity in Human Breast Cancer Cells
J. Biol. Chem., February 20, 2004; 279(8): 6769 - 6777.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y. Ji and G. P. Studzinski
Retinoblastoma Protein and CCAAT/Enhancer-Binding Protein {beta} Are Required for 1,25-Dihydroxyvitamin D3-Induced Monocytic Differentiation of HL60 Cells
Cancer Res., January 1, 2004; 64(1): 370 - 377.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Z. He, Y.-Y. Cho, G. Liu, W.-Y. Ma, A. M. Bode, and Z. Dong
p38 Mitogen-activated Protein Kinase Regulation of JB6 Cl41 Cell Transformation Promoted by Epidermal Growth Factor
J. Biol. Chem., July 11, 2003; 278(29): 26435 - 26442.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. Danilenko, Q. Wang, X. Wang, J. Levy, Y. Sharoni, and G. P. Studzinski
Carnosic Acid Potentiates the Antioxidant and Prodifferentiation Effects of 1{alpha},25-Dihydroxyvitamin D3 in Leukemia Cells but Does Not Promote Elevation of Basal Levels of Intracellular Calcium
Cancer Res., March 15, 2003; 63(6): 1325 - 1332.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Pramanik, X. Qi, S. Borowicz, D. Choubey, R. M. Schultz, J. Han, and G. Chen
p38 Isoforms Have Opposite Effects on AP-1-dependent Transcription through Regulation of c-Jun. THE DETERMINANT ROLE OF THE ISOFORMS IN THE p38 MAPK SIGNAL SPECIFICITY
J. Biol. Chem., February 7, 2003; 278(7): 4831 - 4839.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/29/25884    most recent
M203039200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Qi, X.
Right arrow Articles by Chen, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Qi, X.
Right arrow Articles by Chen, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement