Cancer Research AMC Telomeres
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aklilu, F.
Right arrow Articles by Rabbani, S. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aklilu, F.
Right arrow Articles by Rabbani, S. A.
[Cancer Research 60, 1753-1760, March 15, 2000]
© 2000 American Association for Cancer Research

Tumor Biology

Role of Mitogen-activated Protein Kinases in the Induction of Parathyroid Hormone-related Peptide1

Fasika Aklilu, Julienne Gladu, David Goltzman and Shafaat A. Rabbani2

Department of Medicine, McGill University, Montreal, Quebec, QC H3A 1A1 Canada


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor production of parathyroid hormone-related protein (PTHRP) is responsible for most cases of hypercalcemia of malignancy. The transplantable rat Leydig tumor H-500 is known to cause hypercalcemia in rats by the release of abundant PTHRP and to closely reproduce the human syndrome. We have demonstrated recently that Ras oncogene can stimulate PTHRP gene expression in Fr3T3 fibroblasts in vitro and cause hypercalcemia in vivo. Using rat Leydig tumor H-500 cells, we have investigated the role of effector pathways downstream of Ras in serum-induced PTHRP expression. The Ras inhibitors B-1086 and Lovastatin decreased PTHRP mRNA expression. i.p. administration of B-1086 (50–100 mg/kg/day) into H-500 tumor-bearing male Fischer rats resulted in a dose-dependent reduction in tumor volume, serum calcium, plasma PTHRP, and tumoral PTHRP mRNA expression. Transient transfection of dominant-negative Ras (Ras N17) and Raf (Raf C4B) reduced, whereas activated Raf-1 (Raf BXB) increased, basal expression of PTHRP in H-500 cells. A similar decrease in PTHRP production was seen with a mitogen-activated protein kinase kinase (MEK) inhibitor (PD 098059), implicating the involvement of Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway. In addition, stimulation with UV light, which can activate c-Jun NH2-terminal kinase (JNK), or expression of an activated form of Rac (Rac V12) was sufficient to increase PTHRP mRNA. Moreover, a dominant-negative Rac (Rac N17) blocked serum-induced PTHRP gene expression. Collectively, these results demonstrate that PTHRP is induced via both Raf-ERK and Rac-JNK mediated pathways, effects which can be blocked by chemical inhibitors and dominant-negative mutants of these pathways in vitro and in vivo. Availability of selective inhibitors of Ras signaling molecules may therefore add to our existing armamentarium to control hypercalcemia of malignancy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PTHRP3 is the major pathogenetic factor in hypercalcemia of malignancy. In addition to its endocrine effects, several studies have demonstrated that increased expression of PTHRP in cancer is associated with accelerated tumor growth and a more malignant phenotype (1, 2, 3, 4) , suggesting that PTHRP may play a role in promoting tumor progression. Studies on gene regulation have shown that PTHRP is inducible by an array of substances including serum, growth factors, and cycloheximide (1 , 5, 6, 7) . Additionally, transfection of Ras and Met oncogenes into fibroblasts was shown to induce PTHRP gene expression through a Ras-dependent mechanism (8, 9, 10) .

The family of Ras proteins are GDP/GTP-regulated molecular switches that relay their signals from the cell surface receptor to the nucleus through activation of various downstream signal transduction pathways (11) . Among the targets of Ras, the best characterized are MAP kinases. MAP kinases are a family of serine/threonine kinases that are involved in transduction of cellular signals to the nucleus and in regulating a diverse range of biological processes including cell proliferation, differentiation, malignant transformation, inflammation, apoptosis, and cytoskeletal rearrangement (12, 13, 14) . Upon activation, MAP kinases translocate to the nucleus, where they activate transcription of genes that mediate the cellular response. Activation of Ras leads to the sequential activation of the serine/threonine kinase Raf-1, MAP kinase kinases (MEK1 and MEK2), and ERKs (ERK1 and ERK2; Refs. 15, 16, 17, 18 ). In addition to the ERK cascade, activated Ras has also been shown to activate the JNKs. JNKs, also known as stress-activated protein kinases, represent a group of MAP kinases that are activated by cytokines and exposure of cells to environmental stresses, such as UV light (14 , 19) . Ras, by activating Rac1 and CDC42, can generate the successive activation of MEK kinase (MEKK-1), JNKK, and ultimately JNK (20 , 21) .

We have shown previously that the mechanism of PTHRP expression in cancer is Ras dependent. In the current study, we have investigated the role of MAP kinase pathways leading to ERK and JNK activation in serum-stimulated induction of PTHRP expression. For these studies, we have chosen the rat hypercalcemic Leydig tumor cell line H-500, which produces large quantities of PTHRP and which, upon inoculation into host animals, is a suitable in vivo model that closely mimics the human syndrome of hypercalcemia of malignancy.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Tissue Culture.
Rice H-500 Leydig tumor cells were maintained in vitro in RPMI 1640 supplemented with 2 mM L-glutamine (BRL/GIBCO Canada, Burlington, Ontario, Canada), 10% FBS, 100 units/ml of penicillin-streptomycin sulfate (BRL/GIBCO), and 0.2% gentamicin (Sigma Chemical Co., St. Louis, MO). For transfection studies, cells were plated at 1 x 105 cells/60-mm dish 3 days before transfection and growth in 5% CO2 in RPMI 1640. At 70% confluence, the cells were incubated with 10 µg/ml lipofectin (BRL/GIBCO) and cultured from 6 h to overnight in serum-free RPMI culture medium with 0.1, 1, or 10 µg of plasmid DNA. Fresh culture medium containing 10% FBS was then added. Transient transfection assays were performed at 48 h after transfection. For stable transfections in studies involving the IPTG-inducible, dominant-negative Raf C4B, the cells were selected in G418 (400 µg/ml) for 15 days, and resistant colonies were pooled as polyclonal cell populations (22) . Polyclonal populations with low basal and high expression of Raf C4B were determined by Northern blot analysis. The plasmids encoding the dominant-negative N17Ras and the plasmid encoding Raf C4B N17Rac were described previously (23 , 24) . The plasmids encoding the activated forms Raf BXB and V12Rac and the plasmid encoding Raf C4B were the gifts of Dr. M. Park (McGill University, Montreal, Quebec, Canada; Refs. 22 , 25, and 26 ). The inducible Raf BXB was made using a mammalian IPTG-inducible expression system (Clontech, Palo Alto, CA). The MEK inhibitor PD 098059 (Biomol Research Laboratories, Plymouth Meeting, PA), the inhibitor of phosphatidylinositol 3-kinase wortmannin (Sigma), and the ras inhibitor B-1086 (Eisai Research Institute, Andover, MA) were dissolved in DMSO and stored at -80°C at stock concentrations of 50, 10, and 10 mM, respectively, and diluted to the desired concentrations immediately prior to use (10) .

Northern Blot Analysis.
Total cellular RNA was isolated from the control and experimental cells by acid guanidinium thiocyanate-phenol-chloroform extraction. Twenty µg of total cellular RNA were electrophoresed on a 1.1% agarose-formaldehyde gel, transferred to a nylon membrane (Nytran; S&S, Keene, NH) by capillary blotting, and then fixed by drying and UV cross-linking for 10 min. The integrity of the RNA was assessed by ethidium bromide staining. Hybridization was carried out with [32P]PTHRP cDNA and with 32P-labeled 18S RNA probe using a [32P]dCTP labeling procedure, as described previously (9 , 10) . After a 24-h incubation at 42°C, filters were washed twice under low stringency conditions (1x SSC and 1% SDS; at room temperature for 40 min) and under high stringency conditions (0.1x SSC, 0.1% SDS (10x SSC is 1.5 M NaCl, 0.5 M sodium citrate, pH 7.0); at 55°C for 40 min). Autoradiography of filters was carried out at -70°C using XAR film with two intensifying screens (Eastman Kodak Co., Rochester, NY). The level of PTHRP expression was quantified by densitometric scanning using the Mac BAS V1.01 alias program.

Animal Protocols.
Inbred male Fischer 344 rats weighing 200–220 g were obtained from Charles River Breeding Laboratories (Wilmington, MA). Before inoculation, H-500 tumor cells growing in serum-containing medium were washed with Hanks’ buffer, trypsinized, and collected by centrifugation at 1500 rpm for 5 min. Cell pellets (1 x 106 cells) were resuspended in 200 µl of saline and injected using 1-ml insulin syringes into the right flank of rats. At day 5 after tumor cell inoculation, experimental animals were treated i.p. with either 50 or 100 mg/kg/day of B-1086 for 10 consecutive days (27) . Control animals received saline alone as vehicle control. All animals were numbered, kept separately, and monitored daily for the development of tumors. The tumor mass of control and experimental animals was measured in two dimensions by calipers (8 , 14) . Both control and experimental animals were sacrificed from day 15 after tumor cell inoculation; their primary tumors, serum, and plasma were collected for further analysis.

For RIA, conditioned medium (1.5 ml/well of a six-well cluster plate) was removed at the appropriate times. Duplicate aliquots of 200–500 µl of H-500 cell-conditioned culture medium were evaporated to dryness in a Speed-Vac (Savant Instruments, Hicksville, NY) and stored at -20°C until assayed. Dried medium was reconstituted with 300 µl of outdated blood bank plasma and analyzed by RIA, as described previously (9) , using 125I-labeled [Tyr0]PTHrP-1–34(1–34) as a tracer and PTHrP-1–34(1–34) as a standard. The detection limit of the assay is 0.1 ng/ml. Results are expressed as nanogram equivalents of PTHrP-1–34(1–34)/106 cells. PTHrP-1–34(1–34) was obtained from Bachem (Philadelphia, PA).

Plasma calcium levels were determined by atomic absorption spectrophotometry (model 703; Perkin-Elmer, Norwalk, CT).

ERK and JNK Activity Assays.
Control and experimental H-500 cells were washed twice with ice-cold PBS, scraped off plates, and harvested in lysis buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10 µg/ml phenylmethylsulfonyl fluoride, 30 µg/ml aprotinin, and 10 µg/ml sodium orthovanadate). The kinases were immunoprecipitated in lysis buffer using anti-JNK and anti-ERK antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitates were washed twice in lysis buffer and twice in kinase buffer [50 mM Tris-HCl (pH 8.0), 25 mM MgCl2, 0.5 mM EGTA, 100 mM sodium orthovanadate, 2 mM DTT, and 10% glycerol]. Kinase assay was performed by incubating the immunoprecipitates in 30 µl of kinase buffer containing 20 µM ATP, 1 µCi [{gamma}-32P] ATP (3000 Ci/mmol; Amersham), and 0.5 µg/ml MBP as ERK kinase substrate, or 2 µg/ml GST-Jun as JNK substrate. After 30 min at 30°C, the reaction was stopped by adding 10 µl of 0.6% HCl containing 1 mM ATP and 1% BSA. Thirty µl of the samples were then spotted on phosphocellulose paper, washed five times in 180 mM phosphoric acid, and the amount of radioactivity incorporated into the respective substrates was determined by liquid scintillation spectrometry.

Statistical Analysis.
Results are expressed as the mean ± SE of at least triplicate determinations, and statistical comparisons are based on the Student’s t test or ANOVA. A probability value of <0.05 was considered to be significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Inhibiting Ras Signaling on PTHRP Production in Vitro.
We have reported previously that PTHRP expression is increased >10-fold in a Ras V12-transformed Fisher rat fibroblast cell line (10) . To determine whether activation of Ras is necessary for overexpression of PTHRP, we used H-500 cells, which express high levels of PTHRP in response to 10% FBS (serum). As shown in Fig. 1Citation , within 2 h serum increased PTHRP mRNA expression by 3-fold as compared with PTHRP expression in cells free of serum. Pretreatment of H-500 cells for 12 h with lovastatin (2 µg/ml) and B-1086 (3 µg/ml), which are potent inhibitors of Ras processing, caused a significant reduction in PTHRP mRNA expression (Fig. 1Citation ; Refs. 28, 29, 30 ). This period of pretreatment was most effective in inhibiting PTHRP production, and both inhibitors used at these concentrations were not cytotoxic in H-500 cells, as determined by trypan blue staining (data not shown).



View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Effects of B-1086 and lovastatin on serum-induced PTHRP gene expression. H-500 Leydig cells were grown to 70% confluence and incubated in serum-free culture medium, as described in "Materials and Methods" for 24 h. During the serum-free period, the cells were pretreated with lovastatin (2 µg/ml) and B-1086 (3 µg/ml) for 12 h. After pretreatment, the cells were stimulated with 10% FBS for 2 h and lysed. Fifteen µg of total cellular RNA were extracted from control and experimental cells and electrophoresed on a 1.1% agarose formaldehyde gel. Nylon filters with immobilized RNA were probed with a [32P]PTHRP cDNA or with a 32P-labeled 18 S RNA probe as described in "Materials and Methods." All blots were quantified by densitometric scanning (bottom). Results represent three different experiments; bars, SD. *, significant difference from control cells (P < 0.05).

 
To further investigate the role of Ras in the regulation of PTHRP expression, H-500 cells were transiently transfected with 10 µg of Ras N17 and 1 µg of control LTR-GH cDNA. As shown in Fig. 2ACitation , transient expression of Ras N17 led to a significant inhibition of serum-induced PTHRP expression. Inhibition of PTHRP mRNA expression was correlated with a pronounced suppression of MAP kinase activity, as determined by MBP phosphorylation in in vitro kinase assay (Fig. 2B)Citation . Transfection of similar concentrations of control LTR-GH cDNA on empty vector alone failed to inhibit PTHRP mRNA expression or to suppress MAP kinase activity (data not shown).



View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Effect of transiently expressing Ras N17 on PTHRP gene expression and ERK kinase. H-500 cells were grown to 70% confluence and transiently transfected with Ras N17 plasmid as described in "Materials and Methods." Untransfected and transfected cells were stimulated with 10% FBS. A, after stimulation for 2 h, 15 µg of total cellular RNA extracted from control and experimental cells were electrophoresed on a 1.1% agarose formaldehyde gel. Nylon filters with immobilized RNA were probed with a [32P]PTHRP cDNA or with a 32P-labeled 18 S RNA probe as described in "Materials and Methods." All blots were quantified by densitometric scanning (bottom). Results represent triplicate determinations; bars, SD. *, significant difference from controls (P < 0.05). B, after 10 min of 10% serum stimulation, the cells were lysed, and ERK1 and ERK2 proteins were immunoprecipitated. Enzymatic activity was determined by immune complex kinase assays using MBP as substrate, as described in "Materials and Methods." The results are representative of three different experiments and are plotted as fold increase over unstimulated activity. Bars, SD.

 
Effect of Inhibiting Ras Signaling on Tumor Volume, Serum Calcium, and PTHRP Production in Vivo.
On day 5 after H-500 tumor cell inoculation, animals were administered daily i.p. either vehicle alone or 50 mg/kg or 100 mg/kg of B-1086 for 10 days. The effect of this treatment was monitored by the daily determination of tumor volume of control and experimental animals. Control animals showed a progressive increase in their tumor volume throughout the course of this study. In contrast, administration of B-1086 showed a dose-dependent decrease in tumor of these experimental animals (Fig. 3A)Citation . At the end of this study on day 15, both control and experimental animals were sacrificed, and their serum calcium was determined. Tumor-bearing animals receiving vehicle alone showed a marked increase in serum calcium in plasma PTHRP levels, as determined by RIA, and in PTHRP mRNA levels in their tumors. In contrast, administration of B-1086 resulted in a significant reduction in both plasma PTHRP levels and in tumoral PTHRP mRNA expression (data not shown). B-1086 also produced a dose-dependent decrease in serum calcium with the highest dose (100 mg/kg), producing near normalization of serum calcium levels (Fig. 3B)Citation . A similar decrease in plasma PTHRP and tumoral PTHRP mRNA expression was seen in experimental animals receiving B-1086 as compared with control animals injected with vehicle alone (data not shown).



View larger version (18K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Effect of B-1086 on the tumor volume and serum calcium of H-500 tumor-bearing animals. A, on day 5 after tumor cell inoculation, animals were administered daily i.p. either vehicle, 50 mg/kg B-1086, or 100 mg/kg B-1086, and the tumor volume was determined at daily intervals. Comparison was made with the tumor volume of animals receiving vehicle alone (untreated). B, H-500 tumor-bearing animals were infused with B-1086, and at the end of this study, at day 15, both control (Ctl) and experimental animals were sacrificed, and their serum calcium was then determined. Serum calcium in non-tumor-bearing animals is also shown (N). Results represent the means of at least three animals in each group in three different experiments; bars, SE. *, significant differences in tumor volume and serum calcium from the control group (P < 0.05).

 
Effect of an Activated Form of Raf-1 on PTHRP mRNA Expression and ERK Activity in Vitro.
Serum- and Ras-induced gene expression involves a signal cascade that leads to activation of multiple signaling pathways, including the Raf-MEK-ERK pathway. Raf-1 was shown to be required for serum- and Ras-induced promoter activation (22) . To examine whether activation of Raf alone could increase expression of the PTHRP gene, H-500 cells were transiently transfected with 10 µg of Raf BXB plasmid for 48 h, followed by inoculation in serum-free medium for 24 h. Transfection of Raf BXB caused a significant increase in PTHRP mRNA expression in H-500 cells as compared with untransfected wild-type cells on empty vector-transfected cells (Fig. 4A)Citation . After lysis of H-500 cells, ERK1 and ERK2 activity, determined by immunoprecipitation using MBP as substrate, showed a marked increase in MBP phosphorylation as compared with wild-type H-500 cells (Fig. 4B)Citation .



View larger version (29K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Effect of a truncation-activated form of Raf-1 (Raf BXB) on PTHRP mRNA expression and ERK activity. Semiconfluent H-500 cells were transiently transfected with 10 µg of Raf BXB construct as described in "Materials and Methods." Forty-eight h after transfection, cells were cultured in serum-free media for 24 h and assayed. A, control and transfected cells were lysed, and 15 µg of total cellular RNA extracted from the cells were electrophoresed on a 1.1% agarose formaldehyde gel. After immobilization of RNA, nylon filters were probed with [32P]PTHRP cDNA or with a 32P-labeled 18 S RNA probe as described in "Materials and Methods." All blots were quantified by densitometric scanning (bottom). Results represent triplicate determinations; bars, SD. *, significant difference from controls (P < 0.05). B, the cells were lysed, and ERK1 and ERK2 proteins were immunoprecipitated. Enzymatic activity was determined by immune complex kinase assays using MBP as substrate, as described in "Materials and Methods." The results are representative of three different experiments and are plotted as fold increase over unstimulated activity. *, significant differences from control (P < 0.05).

 
Effect of a Dominant-Negative Form of Raf-1 on PTHRP mRNA Expression and ERK Activity in Vitro.
We next investigated whether Raf-1 might also play a role in serum-induced expression of PTHRP in H-500 cells. To determine whether activation of Raf-1 was required, we used H-500 cells stably transfected with an inducible Raf C4B expression construct. We found that the addition of IPTG into the culture medium rapidly induced expression of the dominant-negative Raf C4B, reaching peak levels of expression by 8 h (data not shown). An increase in PTHRP gene expression was observed 2 h after serum stimulation. This increase was attenuated, however, when Raf C4B was induced. The effect of Raf C4B on the expression of PTHRP was detected 4 h after addition of IPTG, and PTHRP expression was inhibited to near basal levels after 8 h of incubation (Fig. 5)Citation . These data are consistent with previous findings demonstrating that Raf-1 is a direct target of Ras, and expression of many Ras-induced genes is dependent on Raf activity.



View larger version (36K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Effect of inducing a dominant-negative mutant of Raf-1 (Raf C4B) on PTHRP gene expression and ERK activity. H-500 cells, stably transfected with an IPTG-inducible Raf C4B construct, were starved for 24 h in serum-free media prior to induction of Raf C4B expression with 100 µM IPTG for 2, 4, and 8 h. After incubation with IPTG for the times indicated, cells were stimulated with 10% serum for 2 h. At the end of this time period, 15 µg of total cellular RNA were extracted from untreated control and experimental cells and electrophoresed on a 1.1% agarose formaldehyde gel. After transfer of RNA, the filters were probed with a [32P]PTHRP cDNA or with a 32P-labeled 18 S RNA probe as described in "Materials and Methods." All blots were quantified by densitometric scanning (bottom). Results represent triplicate determinations; bars, SD. *, significant difference from controls (P < 0.05).

 
Effect of Inhibition of MEK on PTHRP Gene Expression and on ERK Activation in Vitro.
PD 098059 is a specific inhibitor of MEK and has been shown at 50 µM concentration to completely inhibit the activation of MEK in vitro and in vivo (31) . Serum has been shown to stimulate gene expression by activating the MEK-ERK pathway (13 , 22) . To determine whether MEK activation was required for serum-induced PTHRP expression, H-500 cells were first pretreated with 10 µM or 50 µM PD 098059 and then stimulated with serum. Pretreatment with PD 098059 (for 24 h) inhibited expression of PTHRP mRNA in a dose-dependent manner (Fig. 6A)Citation . The effect of PD 098059 on PTHRP expression was also associated with inhibition of ERK activity at the same concentrations (Fig. 6B)Citation .



View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Effect of MEK inhibitor, PD 098059, on PTHRP gene expression and on ERK activation. A, after 24-h preincubation with 10 or 50 µM PD 098059 in serum-free media, H-500 cells were stimulated with 10% serum for 2 h. Fifteen µg of total cellular RNA were extracted from control and experimental cells and electrophoresed on a 1.1% agarose formaldehyde gel. After transfer of RNA to nylon membranes, the filters were probed with [32P]PTHRP cDNA or with a 32P-labeled 18 S RNA probe as described in "Materials and Methods." All blots were quantified by densitometric scanning (bottom). Results represent triplicate determinations; bars, SD. *, significant difference from controls (P < 0.05). B, after 10 min of 10% serum stimulation, the cells were lysed, and ERK1 and ERK2 proteins were immunoprecipitated. Enzymatic activity was determined by immune complex kinase assays using MBP as substrate as described in "Materials and Methods." The results are representative of three different experiments and are plotted as fold increase over unstimulated activity. Bars, SD.

 
Effect of UV Irradiation on PTHRP Gene Expression in Vitro.
We next explored a role for stress-activated JNK pathway in stimulation of PTHRP expression by serum. Recent studies have demonstrated that JNKs can be phosphorylated and activated in response to environmental stress, UV light, and serum (19 , 21 , 32) . To approach this question, we first examined the effect of UV stimulation on PTHRP expression. Exposure of H-500 cells to 40 and 80 J/m2 UVC for 15 s induced an increase in JNK activation within 10 min (Fig. 7A)Citation . Serum-starved H-500 cells were stimulated with 40 J/m2 of UVC for 15 s and PTHRP expression was measured at 0.5, 1, and 2 h after stimulation. Stimulation of H-500 cells with UVC irradiation led to increased PTHRP mRNA expression in a time-dependent manner (Fig. 7B)Citation .



View larger version (21K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. Effect of UV irradiation on PTHRP gene expression. A, H-500 cells were serum starved for 24 h and were stimulated for 15 s with UVC irradiation. JNK activity was determined as described in "Materials and Methods." These results are representative of three such determinations and are plotted as fold increase over unstimulated activity. B, H-500 cells were serum starved for 24 h and were either left untreated (0) or exposed to 40 J/m2 of UVC irradiation for 15 s. The cells were lysed at 0.5, 1, and 2 h after UVC stimulation. Fifteen µg of total cellular RNA were extracted from unstimulated and experimental cells and electrophoresed on a 1.1% agarose formaldehyde gel. After RNA transfer to nylon membranes, the filters were probed with [32P]PTHRP cDNA or with a 32P-labeled 18 S RNA probe as described in "Materials and Methods." All blots were quantified by densitometric scanning (bottom). Results represent the three different experiments; bars, SD. *, significant difference from controls (P < 0.05).

 
Effect of Expressing an Activated Mutant and an Inactivated Mutant of Rac 1 on PTHRP Gene Expression Levels and on JNK Activation in Vitro.
The signaling intermediates between Ras and JNK are not well understood. Recently, it has been shown that Rac-1 and CDC42 are involved in mediating the signals from Ras to JNK (20) . In addition, it has also been shown that activation of Rac 1 leads to the sequential activation of MEKK1, JNKK, and JNK (33, 34) . Consequently, we determined the effect of an activated mutant of Rac 1, Rac V12, on PTHRP expression. Serum-starved H-500 cells were transiently transfected with 10 µg of Rac V12. This lead to a 2-fold increase in PTHRP expression when compared with untransfected or vector-only transfected cells (Fig. 8A)Citation .



View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 8. Effect of expressing an activated mutant (Rac V12) and an inactivated mutant (Rac N17) of Rac on PTHRP gene expression levels and on JNK activation. H-500 cells were grown to semiconfluence and transiently transfected with either 10 µg of Rac V12 or Rac N17 constructs as described in "Materials and Methods." After transfection, cells were cultured for 24 h in serum-free media. Untransfected and Rac N17-transfected cells were then stimulated with 10% FBS for 2 h. A, 15 µg of total cellular RNA were extracted from control and transfected cells and electrophoresed on a 1.1% agarose formaldehyde gel. After RNA transfer to nylon membranes, the filters were probed with [32P]PTHRP cDNA or with a 32P-labeled 18 S RNA probe as described in "Materials and Methods." All blots were quantified by densitometric scanning (bottom). Results represent triplicate determinations; bars, SD. *, significant difference from controls (P < 0.05). B, H-500 cells cultured under serum-free conditions (CH) or transfected with Rac V12 plasmid were lysed. Alternatively, after 10 min of serum stimulation, untransfected (FBS) and Rac N17-transfected cells were lysed, and JNK-1 protein was immunoprecipitated. Enzymatic activity was determined by immune complex kinase assay using GST-jun fusion protein as substrate as described in "Materials and Methods." The results are representative of three different experiments and are plotted as fold increase over unstimulated activity. Bars, SD.

 
We then tested the possibility that Rac 1 may be mediating the Ras-dependent, serum induction of PTHRP expression. To determine the role of Rac 1, we transiently transfected 10 µg of the dominant-negative Rac N17 construct into H-500 cells. Expression of the dominant-negative Rac N17 lead to a 60% block of the effects of serum of PTHRP expression (Fig. 8A)Citation .

To assess the effect of Rac-1 on JNK activity in our system, we first tested the capacity of Rac V12 to stimulate JNK activation in H-500 cells. Transiently transfecting 10 µg of Rac V12 into H-500 cells lead to a 2-fold increase in JNK activity, as measured by the phosphorylation of a GST-jun substrate in immunocomplex kinase assays in vitro (Fig. 8B)Citation . In addition, transient transfection of the dominant-negative construct Rac N17 into H-500 cells caused a marked inhibition in JNK activity, as determined by in vitro kinase assay (Fig. 8B)Citation .

Effect of Dominant-Negative Rac N17 on Cycloheximide-induced PTHRP Gene Expression.
As is the case with many immediate early genes, PTHRP can be induced by cycloheximide (36) . Cycloheximide has been shown to induce gene expression through a JNK-dependent mechanism (37 , 38) . To determine whether Rac1 was involved in cycloheximide-induced PTHRP expression, the effect of cycloheximide on H-500 cells transiently expressing Rac N17 was assessed. As shown in Fig. 9Citation , cycloheximide increased PTHRP expression by 4-fold. We found, however, that expression of Rac N17 did not interfere with cycloheximide-induced PTHRP expression. This finding is consistent with reports that cycloheximide activates JNK through mechanisms independent of Rac1/CDC42.



View larger version (27K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 9. Effect of dominant-negative Rac N17 on cycloheximide (CHX)-induced PTHRP gene expression. Control cells and cells transiently transfected with 10 µg of Rac N17 were serum starved for 24 h, stimulated with 10 µg/ml CHX for 2 h, and lysed. Fifteen µg of total cellular RNA extracted from the cells were electrophoresed on a 1.1% agarose formaldehyde gel. RNA was transferred to nylon membranes, which were probed with [32P]PTHRP cDNA or with a 32P-labeled 18 S RNA probe as described in "Materials and Methods." All blots were quantified by densitometric scanning (bottom). Results represent three different experiments; bars, SD. *, significant difference from controls (P < 0.05).

 
Collectively, these data demonstrate that at least two pathways may be implicated in PTHRP induction in response to serum (Fig. 10)Citation . Either the Ras-Raf-1-MEK cascade leading to ERK activation or the activation of Rac1 leading to stimulation of MEKK1-JNKK-JNK kinases appears to modulate serum-induced PTHRP gene expression.



View larger version (22K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 10. A schematic diagram of MAP kinase signaling pathways involved in regulating the PTHRP gene. Positive (+) and negative (-) regulators of the Ras-Raf-MEK cascade leading to ERK activation and positive and negative regulators of the Rac-MEKK-SEK cascade leading to JNK activation were used to demonstrate that both signaling pathways can lead to increases in PTHRP gene expression.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PTHRP plays a critical role in normal physiology and in disease. Studies by us and other investigators have identified numerous exogenous agents, including serum growth factors (5 , 39 , 40) , various oncogenes, and phorbol esters (8 , 9 , 10 , 41 , 42) , which induce the synthesis of this protein. We have also demonstrated previously that receptor tyrosine kinases mediate their effect on this gene via Ras-dependent pathways (9) . Indeed, many stimulators of PTHRP gene expression are also activators of Ras. However, the mechanisms that connect Ras to nuclear events leading to induction of PTHRP gene expression are not known. To our knowledge, this is therefore the first demonstration of a role for ERK- and JNK-dependent signaling in mediating effects downstream of Ras on PTHRP expression.

We have shown previously that PTHRP is induced in response to serum in H-500 cells (1) . Serum is known to potently activate the MAP kinases ERKs and JNKs (13 , 21) , which convey the intracellular signal to the nucleus, and therein to promoters of the many serum-regulated genes. Our findings suggest that ERK and JNK pathways are both involved in serum-induced PTHRP expression, because inhibition of either pathway completely attenuates PTHRP expression. Furthermore, independent activation of either JNK or ERK cascades results in a detectable but relatively small increase in PTHRP expression compared with the activation of both pathways in response to serum. This suggests that these pathways may cooperate to regulate PTHRP gene expression. Supporting this notion, cross-talk between these pathways has been shown to occur in activation of nuclear transcription factors affecting AP-1 activity. Although ERKs phosphorylate TCF/Elk-1, they are unable to activate c-Jun or ATF2 (43) . JNKs, on the other hand, can phosphorylate and activate both c-Jun and ATF2 (19 , 44) .

We were surprised to find a role for JNK in the regulation of PTHRP expression by serum, because JNK, which was initially identified as activated in response to UV light and changes in osmolarity (19 , 31) , is widely perceived as a stress-activated kinase. Nevertheless, although these stress stimuli are indeed the most potent activators of JNK, serum has been shown to produce a 3-fold increase of JNK activation (21) .

Previously, PTHRP expression was shown to be induced by cycloheximide (7) . Subsequently, it has been shown that cycloheximide as well as other protein synthesis inhibitors can activate JNK (45 , 46) . However, induction of JNK activity by protein synthesis inhibitors has been suggested to involve mechanisms alternative to activation of Rac1/CDC42 (47 , 48) . In agreement with these studies, we report that Rac N17 does not inhibit cycloheximide-induced PTHRP expression. Whether cycloheximide induces PTHRP via the JNK pathway has yet to be demonstrated directly.

The current study may also provide a possible mechanism through which a diverse set of other stimuli can induce PTHRP gene expression. Indeed, many factors that also induce PTHRP gene expression, including G-protein coupled receptors (49) , phorbol esters (47) , cytokines (38 , 46) , and mechanical stretch (47 , 50 , 51) , have been shown to activate these pathways.

Regulation of genes by the ERK- and JNK-dependent pathways is in part through activation of activating protein-1 transcription factors (Fos and Jun proteins), the serum response factor/tertiary complex factor, and the c-sis inducible factor (52) . The PTHRP promoter contains binding elements for tertiary complex factor transcription factors, which have been shown to mediate retinoic acid-induced PTHRP expression (53) . However, the nature of the transcription factors involved in activation of the PTHRP promoter in response to serum and growth factors requires further investigation.

In conclusion, the results from our study show that PTHRP is regulated by MAP kinase signaling pathways and involve ERK and JNK activation. MAP kinases are molecular cross-roads where multiple signals converge and thus provide exciting possibilities as targets to reduce PTHRP expression in hypercalcemia of malignancy.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Medical Research Council of Canada Grants MT-10630, MT-12609, and MT-5775 and by a grant from the National Cancer Institute of Canada. Back

2 To whom requests for reprints should be addressed, Calcium Research Laboratory, Royal Victoria Hospital, Room H4.72, 687 Pine Avenue West, Montreal, Quebec, H3A 1A1 Canada. Phone: (514) 843-1632; Fax: (514) 843-1712. Back

3 The abbreviations used are: PTHRP, parathyroid hormone-related peptide; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; JNKK, JNK kinase; MEK, MAP kinase kinase; MEKK, MEK kinase; FBS, fetal bovine serum; IPTG, isopropyl-1-thio-ß-D-galactopyranoside; MBP, myelin basic protein; GST, glutathione S-transferase. Back

Received 9/ 1/99. Accepted 1/19/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Liu B., Goltzman D., Rabbani S. A. Regulation of parathyroid hormone-related peptide production in vitro by the rat hypercalcemic Leydig cell tumor H-500. Endocrinology, 132: 1658-1664, 1993.[Abstract/Free Full Text]
  2. Alipov G. K., Ito M., Nakashima M., Ikeda Y., Nakayama T., Ohtsuru A., Yamashita S., Sekine I. Expression of parathyroid hormone-related peptide (PTHrP) in gastric tumours. J. Pathol., 182: 174-179, 1997.[Medline]
  3. Ito M., Nakashima M., Alipov G. K., Matsuzaki S., Ohtsuru A., Yano H., Yamashita S., Sekine I. Gastric cancer associated with overexpression of parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor in relation to tumor progression. J. Gastroenterol., 32: 396-400, 1997.[Medline]
  4. Nagataki S., Nakashima M., Akino K., Ohtsuru A., Namba H., Kiriyama T., Ito M., Sekine I., Yamashita S. R. Parathyroid hormone-related peptide expression in endocrine tumors. Miner. Electrolyte Metab., 21: 148-151, 1995.[Medline]
  5. Sebag M., Henderson J., Rhim J., Kremer R. Relative resistance to 1,25-dihydroxyvitamin D3 in a keratinocyte model of tumor progression. J. Biol. Chem., 267: 12162-12167, 1992.[Abstract/Free Full Text]
  6. Allinson E. T., Drucker D. J. Parathyroid hormone-like peptide shares features with members of the early response gene family: rapid induction by serum, growth factors, and cycloheximide. Cancer Res., 52: 3103-3109, 1992.[Abstract/Free Full Text]
  7. Ikeda K., Lu C., Weir E. C., Mangin M., Broadus A. E. Regulation of parathyroid hormone-related peptide gene expression by cycloheximide. J. Biol. Chem., 265: 5398-5402, 1990.[Abstract/Free Full Text]
  8. Li X., Drucker D. J. Parathyroid hormone-related peptide is a downstream target for ras and src activation. J. Biol. Chem., 269: 6263-6266, 1994.[Abstract/Free Full Text]
  9. Aklilu F., Park M., Goltzman D., Rabbani S. A. Increased PTHRP production by a tyrosine kinase oncogene. Tpr-Met: a role of the Ras signaling pathway. Am. J. Physiol., 271: 277-283, 1996.
  10. Aklilu F., Park M., Goltzman D., Rabbani S. A. Induction of parathyroid hormone-related peptide by the Ras oncogene: role of Ras farnesylation inhibitors as potential therapeutic agents for hypercalcemia of malignancy. Cancer Res., 57: 4517-4522, 1997.[Abstract/Free Full Text]
  11. Barbacid M. Ras genes. Annu. Rev. Biochem., 56: 779-827, 1987.[Medline]
  12. Tanaka S., Ouchi T., Hanafusa H. Downstream of Crk adaptor signaling pathway: activation of Jun kinase by v-Crk through the guanine nucleotide exchange protein C3G. Proc. Natl. Acad. Sci. USA, 94: 2356-2361, 1997.[Abstract/Free Full Text]
  13. Troppmair J., Bruder J. T., Munoz H., Lloyd P. A., Kyriakis J., Banerjee P., Avruch J., Rapp U. R. Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation by oncogenes, serum, and 12-O-tetradecanoylphorbol-13-acetate requires Raf and is necessary for transformation. J. Biol. Chem., 269: 7030-7035, 1994.[Abstract/Free Full Text]
  14. Whitmarsh A. J., Davis R. J. Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J. Mol. Med., 74: 589-607, 1996.[Medline]
  15. van Aelst L., Barr M., Marcus S., Polverino A., Wigler M. Complex formation between RAS and RAF and other protein kinases. Proc. Natl. Acad. Sci. USA, 90: 6213-6217, 1993.[Abstract/Free Full Text]
  16. Vojtek A. B., Hollenberg S. M., Cooper J. A. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell, 74: 205-214, 1993.[Medline]
  17. Warne P. H., Viciana P. R., Downward J. Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro. Nature (Lond.), 364: 352-355, 1993.[Medline]
  18. Zhang X. F., Settleman J., Kyriakis J. M., Takeuchi-Suzuki E., Elledge S. J., Marshall M. S., Bruder J. T., Rapp U. R., Avruch J. Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1. Nature (Lond.), 364: 308-313, 1993.[Medline]
  19. Derijard B., Hibi M., Wu I. H., Barrett T., Su B., Deng T., Karin M., Davis R. J. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and x phosphorylates the c-Jun activation domain. Cell, 76: 1020-1028, 1994.
  20. Coso O. A., Chiariello M., Yu J. C., Teramoto H., Crespo P., Xu N., Miki T., Gutkind J. S. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell, 81: 1137-1146, 1995.[Medline]
  21. Minden A., Lin A., McMahon M., Lange-Carter C., Derijard B., Davis R. J., Johnson G. L., Karin M. Differential activation of ERK and JNK mitogen-activated protein kiinases by Raf-1 and MEKK. Science (Washington DC), 266: 1719-1722, 1994.[Abstract/Free Full Text]
  22. Bruder J. T., Heidecker G., Rapp U. R. Serum-, TPA-, and Ras-induced expression from Ap-1/Ets-driven promoters requires Raf-1 kinase. Genes Dev., 6: 545-556, 1992.[Abstract/Free Full Text]
  23. Feig L. A., Cooper G. M. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol. Cell. Biol., 8: 3235-3243, 1988.[Abstract/Free Full Text]
  24. Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell, 70: 401-410, 1992.[Medline]
  25. Kolch W., Heidecker G., Lloyd P., Rapp U. R. Raf-1 protein kinase is required for growth of induced NIH/3T3 cells. Nature (Lond.), 349: 426-428, 1991.[Medline]
  26. Qiu R-G., Chen J., Kirn D., McCormick F., Symons M. An essential role for Rac in Ras transformation. Nature (Lond.), 374: 457-459, 1995.[Medline]
  27. Rabbani S. A., Gladu J., Harakidas P., Jamison B., Goltzman D. Overproduction of parathyroid hormone-related peptide results in increased osteolytic skeletal metastasis by prostate cancer cells in vivo. Int. J. Cancer, 80: 257-264, 1999.[Medline]
  28. Sinensky M., Beck L. A., Leonard S., Evans R. Differential inhibitory effects of lovastatin on protein isoprenylation and sterol synthesis. J. Biol. Chem., 265: 19937-19941, 1990.[Abstract/Free Full Text]
  29. Nagasu T., Yoshimatsu K., Rowell C., Lewis M. D., Garcia A. M. Inhibition of human tumor xenograft growth by treatment with the farnesyl transferase inhibitor B956. Cancer Res., 55: 5310-5314, 1995.[Abstract/Free Full Text]
  30. Gibbs J. B., Pompliano D. L., Mosser S. D. Selective inhibition of farnesyl-protein transferase blocks ras processing in vivo. J. Biol. Chem., 268: 7617-7620, 1993.[Abstract/Free Full Text]
  31. Alessi D. R., Cuenda A., Cohen P., Dudley D. T., Saltiel A. R. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem., 270: 27489-27494, 1995.[Abstract/Free Full Text]
  32. Kyriakis J. M., Banerjee P., Nikolakaki E., Dai T., Rubie E. A., Ahmad M. F., Avruch J., Woodgett J. R. The stress-activated protein kinase subfamily of c-Jun kinases. Nature (Lond.), 369: 156-160, 1994.[Medline]
  33. Bagrodia S., Derijard B., Davis R. J., Cerione R. A. Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation. J. Biol. Chem., 270: 27995-27998, 1995.[Abstract/Free Full Text]
  34. Zhang S., Han J., Sells M. A., Chernoff J., Knaus U. G., Ulevitch R. J., Bokoch G. M. Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1. J. Biol. Chem., 270: 23934-23936, 1995.[Abstract/Free Full Text]
  35. Frost J. A., Xu S., Hutchison M. R., Marcus S., Cobb M. H. Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members. Mol. Cell. Biol., 16: 3707-3713, 1996.[Abstract]
  36. Ikeda K., Lu C., Weir E. C., Mangin M., Broadus A. E. Regulation of parathyroid hormone-related peptide gene expression by cycloheximide. J. Biol. Chem., 265: 5398-5402, 1990.
  37. Raingeaud J., Gupta S., Rogers J. S., Dickens M., Han J., Ulevitch R. J., Davis R. J. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J. Biol. Chem., 270: 7420-7426, 1995.[Abstract/Free Full Text]
  38. Chen Q., Lin T. H., Der C. J., Juliano R. L. Integrin-mediated activation of MEK and mitogen-activated protein kinase is independent of Ras. J. Biol. Chem., 271: 18122-18127, 1996.[Abstract/Free Full Text]
  39. Heath J. K., Southby J., Fukumoto S., O’Keefe L. M., Martin T. J., Gillespie M. T. Epidermal growth factor-stimulated parathyroid hormone-related protein expression involves increased gene transcription and mRNA stability. Biochem. J., 307: 159-167, 1995.
  40. Ferrari S. L., Rizzoli R., Bonjour J. P. Effects of epidermal growth factor on parathyroid hormone-related protein production by mammary epithelial cells. J. Bone Miner. Res., 9: 639-644, 1994.[Medline]
  41. Motokura T., Endo K., Kumaki K., Ogata E., Ikeda K. Neoplastic transformation of normal rat embryo fibroblasts by a mutated p53 and an activated ras oncogene induces parathyroid hormone-related peptide gene expression and causes hypercalcemia in nude mice. J. Biol. Chem., 270: 30857-30861, 1995.[Abstract/Free Full Text]
  42. Rodan S. B., Wesolowski G., Ianacone J., Thiede M. A., Rodan G. A. Production of parathyroid hormone-like peptide in a human osteosarcoma cell line: stimulation by phorbol esters and epidermal growth factor. J. Endocrinol., 122: 219-227, 1989.[Abstract/Free Full Text]
  43. Hipskind R. A., Buscher D., Nordheim A., Baccarini M. Ras/MAP kinase-dependent and -independent signaling pathways target distinct ternary complex factors. Genes Dev., 8: 1803-1816, 1994.[Abstract/Free Full Text]
  44. Gupta S., Campbell D., Derijard B., Davis R. J. Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science (Washington DC), 267: 389-393, 1995.[Abstract/Free Full Text]
  45. Schultz H., Engel K., Gaestel M. PMA-induced activation of the p42/44ERK- and p38RK-MAP kinase cascades in HL-60 cells is PKC dependent but not essential for differentiation to the macrophage-like phenotype. J. Cell. Physiol., 173: 310-318, 1997.[Medline]
  46. Takahashi-Tazuka M., Yoshida Y., Fukada T., Ohtani T., Yamanaka Y., Nishida K., Kakjima K., Hibi M., Hirano T. Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase. Mol. Cell. Biol., 18: 4109-4117, 1998.[Abstract/Free Full Text]
  47. Newton R., Stevens D. A., Hart L. A., Lindsay M., Adcock I. M., Barnes P. J. Superinduction of COX-2 mRNA by cycloheximide and interleukin-1ß involves increased transcription and correlates with increased NF{kappa}B and JNK activation. FEBS Lett., 418: 135-138, 1997.[Medline]
  48. Iordanov M. S., Pribnow D., Magun J. L., Dinh T. M., Pearson J. A., Chen S. L., Magun B. E. Ribotoxic stress response: activation of the stress-activated protein kinase JNK1 by inhibitors of the peptidyl transferase reaction and by sequence-specific RNA damage to the {alpha}-sarcin/ricin loop in the 28S rRNA. Mol. Cell. Biol., 17: 3373-3381, 1997.[Abstract]
  49. Coso O. A., Chiariello M., Kalinec G., Kyriakis J. M., Woodgett J., Gutkind J. S. Transforming G protein-coupled receptors potently activate JNK (SAPK). Evidence for a divergence from the tyrosine kinase signaling pathway. J. Biol. Chem., 270: 5620-5624, 1995.[Abstract/Free Full Text]
  50. Hamada K., Takuwa N., Yokoyama K., Takuwa Y. Stretch activates Jun N-terminal kinase/stress-activated protein kinase in vascular smooth muscle cells through mechanisms involving autocrine ATP stimulation of purinoceptors. J. Biol. Chem., 273: 6334-6340, 1998.[Abstract/Free Full Text]
  51. Tumminia S. J., Mitton K. P., Arora J., Zelenka P., Epstein D. L., Russell P. Mechanical stretch alters the actin cytoskeletal network and signal transduction in human trabecular meshwork cells. Investig. Ophthalmol. Vis. Sci., 39: 1361-1371, 1998.[Abstract/Free Full Text]
  52. Lewis T. S., Shapiro P. S., Ahn N. G. Signal transduction through MAP kinase cascades. Adv. Cancer Res., 74: 49-139, 1998.[Medline]
  53. Karperien M., Farih-Sips H., Lowik C. W., de Laat S. W., Boonstra J., Defize L. H. Expression of the parathyroid hormone-related peptide gene in retinoic acid-induced differentiation: involvement of ETS and Sp1. Mol. Endocrinol., 11: 1435-1448, 1997.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
R. E. Schweppe, T. H. Cheung, and N. G. Ahn
Global Gene Expression Analysis of ERK5 and ERK1/2 Signaling Reveals a Role for HIF-1 in ERK5-mediated Responses
J. Biol. Chem., July 28, 2006; 281(30): 20993 - 21003.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
N. Chattopadhyay
Effects of calcium-sensing receptor on the secretion of parathyroid hormone-related peptide and its impact on humoral hypercalcemia of malignancy
Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E761 - E770.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
J. Tfelt-Hansen, R. J. MacLeod, N. Chattopadhyay, S. Yano, S. Quinn, X. Ren, E. F. Terwilliger, P. Schwarz, and E. M. Brown
Calcium-sensing receptor stimulates PTHrP release by pathways dependent on PKC, p38 MAPK, JNK, and ERK1/2 in H-500 cells
Am J Physiol Endocrinol Metab, August 1, 2003; 285(2): E329 - E337.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
R. Linforth, N. Anderson, R. Hoey, T. Nolan, S. Downey, G. Brady, L. Ashcroft, and N. Bundred
Coexpression of Parathyroid Hormone Related Protein and Its Receptor in Early Breast Cancer Predicts Poor Patient Survival
Clin. Cancer Res., October 1, 2002; 8(10): 3172 - 3177.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Berkovich and D. Ginsberg
Ras Induces Elevation of E2F-1 mRNA Levels
J. Biol. Chem., November 9, 2001; 276(46): 42851 - 42856.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
L. Carpio, J. Gladu, D. Goltzman, and S. A. Rabbani
Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways
Am J Physiol Endocrinol Metab, September 1, 2001; 281(3): E489 - E499.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Aklilu, F.
Right arrow Articles by Rabbani, S. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Aklilu, F.
Right arrow Articles by Rabbani, S. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online