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Endocrinology Vol. 138, No. 5 1821-1828
Copyright © 1997 by The Endocrine Society

Articles

Prostaglandin F2{alpha} Stimulates Tyrosine Phosphorylation and Mitogen-Activated Protein Kinase in Osteoblastic MC3T3-E1 Cells via Protein Kinase C Activation

Yoshiyuki Hakeda, Miho Shiokawa, Hiroshi Mano, Takashi Kameda, Lawrence G. Raisz and Masayoshi Kumegawa

Department of Oral Anatomy, Meikai University School of Dentistry (Y.H., M.S., H.M., T.K., M.K.), Sakado, Saitama 350–02, Japan; and the Division of Endocrinology and Metabolism, University of Connecticut Health Center (L.G.R.), Farmington, Connecticut 06030

Address all correspondence and requests for reprints to: Yoshiyuki Hakeda, Ph.D., Department of Oral Anatomy, Meikai University School of Dentistry, Sakado, Saitama 350–02, Japan. E-mail: y-hakeda{at}dent.meikai.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PGF2{alpha} stimulates the proliferation of clonal osteoblastic MC3T3-E1 cells via PGF2{alpha} receptor linked to phospholipase C activation. To elucidate intracellular events elicited by this receptor, we examined the effects of PGF2{alpha} on tyrosine phosphorylation and mitogen-activated protein kinase (MAPK) activity in MC3T3-E1 cells. PGF2{alpha} rapidly raised the level of phosphotyrosine of cellular proteins with Mr values of 62, 68, 72, 76, 82, 125, and 150 kDa. This PGF2{alpha}-induced tyrosine phosphorylation of proteins (except for pp62) was blocked by down-regulating protein kinase C (PKC) by 12-O-tetradecanoylphorbol 13-acetate pretreatment and by GF 109203X, a potent specific PKC inhibitor. The addition of PGF2{alpha} also transiently activated MAPK in the same range of concentrations that stimulated tyrosine phosphorylation. In addition, PGF2{alpha} augmented the MAPK kinase kinase activity of Raf-1, whereas basal activity of MAPK/extracellular signal-regulated protein kinase kinase was less than that of Raf-1 and was little affected by PGF2{alpha}. Like the tyrosine phosphorylation, these activations of Raf-1 and MAPK activities were reduced by inhibition and down-regulation of PKC. Genistein, a potent inhibitor of tyrosine kinases, did not block the Raf-1 induced by PGF2{alpha}, indicating a tyrosine kinase-independent pathway for Raf-1 activation. However, the tyrosine kinase inhibitor partially inhibited the MAPK activity, suggesting an involvement of another Raf-1-independent kinase cascade for activation of MAPK by PGF2{alpha}. Fluprostenol, a specific agonist of PGF2{alpha} receptor, mimicked the actions of PGF2{alpha} consistent with a PGF2{alpha} receptor pathway. Thus, the action of PGF2{alpha} on osteoblastic MC3T3-E1 cells appears to involve a single receptor that uses diverse interacting signal transduction systems.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ACTIONS of PGs on osteoblasts are pleiotropic through diverse signal transduction systems, with respect to osteoblastic differentiation and proliferation (1, 2). Elevation of cAMP induced by PGE1 or PGE2 is involved in osteoblastic differentiation or maturation (3, 4, 5, 6, 7). In contrast to those adenylate cyclase-coupled PGs, phospholipase C (PLC)-coupled PGF2{alpha} is a potent osteoblastic mitogen (5, 6, 7, 8, 9). The stimulation of PLC induced by PGF2{alpha} leads to inositol triphosphate-induced elevation of intracellular calcium and diacylglycerol activation of protein kinase C (PKC) (10, 11, 12). We have demonstrated that the simulation of proliferation of clonal osteoblastic MC3T3-E1 cells by PGF2{alpha} is dependent upon the activation of PKC (5). In addition, PGF2{alpha} acts on the proliferation of the quiescent MC3T3-E1 cells as a competence growth factor, and the stimulatory effect of PGF2{alpha} requires interaction with endogenously produced insulin-like growth factor I to induce DNA synthesis (13). Recently, a PGF2{alpha} (FP) receptor has been cloned (14, 15, 16) and reported to be linked to G proteins in several cells (17, 18, 19, 20). However, the signaling pathways of PGF2{alpha}, from the G protein-coupled activation of PLC to the action as competence factor, remain obscure.

Phosphorylation of tyrosine residues in cellular proteins is considered to mediate intracellular mitogenic responses to various growth factors, such as platelet-derived growth factor, epidermal growth factor, insulin-like growth factor I, and insulin (21). The autophosphorylation of tyrosine residues on their receptors (receptor tyrosine kinases) is produced by an interaction between ligands and receptors, and subsequently results in the activation of kinases toward downstream cellular components, including phosphatidylinositol 3'-kinase, PLC{gamma}, Ras, mitogen-activated protein kinase (MAPK) kinase such as Raf-1 (22). Recently, beside the receptor tyrosine kinases, nonreceptor tyrosine kinases have been demonstrated to be positively modulated by the activation of PKC linked to PLC in response to several agents, such as thrombin, collagen, and vasoactive peptides in some types of cells (23, 24, 25, 26). With respect to this cross-talk between PKC and tyrosine kinases, PGF2{alpha} has been most recently reported to stimulate tyrosine phosphorylation through nonreceptor tyrosine kinases induced by activated PLC in fibroblastic Swiss 3T3 cells (27). However, the cellular roles of such tyrosine phosphorylation through nonreceptor tyrosine kinases remain unclear.

MAPKs are activated by diverse extracellular agonists that promote cell growth in most cells (28, 29). The activated MAPK subsequently transmits mitogenic signals by phosphorylating downstream components such as transcription factors, including c-myc and c-jun, that contribute to the control of numerous cellular events (30, 31). Therefore, the MAPK is one of the several possible points of convergence for various receptor-initiated signal events. Receptor tyrosine kinases in response to mitogenic agents in general activate MAPK through the Ras/Raf-1 pathway (22). However, a different mechanism has also been reported, in which PKC-activated Raf-1 activity resulted in the activation of MAPK independent of tyrosine kinases (32). On the other hand, the other Ras/Raf-1-independent MAPK-activating pathway has been demonstrated through MAPK/extracellular signal-regulated protein kinase (MEK) kinases (MEKKs) that are coupled to a complex of receptor-heterotrimeric G proteins (33). Thus, the MAPK-activating pathway depends on the types of agonists and target cells.

The present study was undertaken to elucidate the downstream signalings of PGF2{alpha}-induced PKC on osteoblastic MC3T3-E1 cells, examining the effect of PGF2{alpha} on tyrosine phosphorylation of intracellular proteins and MAPK activity. We found that PGF2{alpha} stimulated both tyrosine phosphorylation and MAPK activity via a PKC-dependent mechanism. The activation of MAPK seems to be achieved mainly through the Raf-1 pathway activated by PKC that is independent of the tyrosine kinase cascade. Thus, the action of PGF2{alpha} on osteoblastic MC3T3-E1 cells is mediated through diverse signal transduction systems.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Monoclonal antibodies raised against phosphotyrosine-keyhole limpet hemocyanin and bovine PLC{gamma}1 were purchased from Upstate Biotechnology (Lake Placid, NY). The anti-PLC{gamma}-1 was shown to react with mouse PLC{gamma}-1. Polyclonal rabbit anti-Raf-1 and anti-MEKK antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and these antibodies were raised against synthetic peptides corresponding to the 12 and 22 amino acids mapping at the carboxyl-terminus of mouse Raf-1 and MEKK, respectively. Xenopus recombinant MAPK kinase and recombinant kinase-negative MAPK (glutathione-S-transferase fusion protein) (34) were provided by Dr. E. Nishida (Kyoto University, Kyoto, Japan). PGF2{alpha}, 12-O-tetradecanoylphorbol 13-acetate (TPA), and myelin basic protein (MBP) were obtained from Sigma Chemical Co. (St. Louis, MO); islet-activating protein (IAP) was purchased from Funakoshi Biochemicals (Tokyo, Japan). Bisindolylmaleimide (GF 109203X) and genistein were obtained from Wako Pure Chemicals (Osaka, Japan). Immunostaining reagents were purchased from Vector Laboratories (Burlingame, CA). [{gamma}-32P]ATP (110 TBq/mmol) and reagents for the p42/p44 MAPK enzyme assay system were obtained from Amersham International (Aylesbury, UK).

Cell culture
Clonal osteoblastic MC3T3-E1 cells (2 x 103, 2 x 104, and 2 x 105) were seeded and cultured in {alpha}-modified MEM ({alpha}MEM; Flow Laboratories, McLean, VA) containing 10% FBS in each well of a 48-well plate, 35-mm dishes, and 100-mm dishes, respectively, for 3 days until nearly confluent at 37 C in a humidified CO2 incubator (5% CO2-95% air) as described previously (13). Then, the cells were washed twice with {alpha}MEM and incubated in serum-free {alpha}MEM containing 0.1% BSA for 1 day before treatment with various agents.

Assay for tyrosine phosphorylation of cellular proteins
The quiescent MC3T3-E1 cells were treated with various agents for indicated times. Then, the cells were quickly washed with PBS containing 5 mM EDTA and 0.1 mM Na3VO4, and lysed with a lysis buffer consisting of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 10 mM NaH2PO4, 5 mM EDTA, 10% glycerol, 2 mM Na3VO4, 10 mM NaF, 1 mM aminoethyl-benzenesulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The protein concentration in the cell lysates was measured using a bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL). Each cell lysate containing equal amounts of proteins was subjected to 8% SDS-PAGE under reducing conditions, and proteins separated in the gel were subsequently electrotransferred onto a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). After being blocked with 5% skim milk, the membrane was incubated with monoclonal antibody against phosphotyrosine and subsequently with biotin-conjugated antimouse IgG antibody. Phosphotyrosine-containing proteins were visualized using avidin-biotin-peroxidase complex immunostaining regents (Vector) following the manufacturer’s instructions. For immunoprecipitation of PLC{gamma}, after preclearing by incubation with 50 µl protein G-Sepharose, the cell lysate from four 100-mm dishes was incubated with 5 µg monoclonal anti-PLC{gamma} at 4 C overnight. The immunoprecipitates were then collected by centrifugation and washed five times with the lysis buffer. The proteins were released by boiling the immunoprecipitates in Laemmli’s buffer, divided into two equal volume of aliquots, subjected to 8% SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane. The transfers were incubated with anti-PLC{gamma} or antiphosphotyrosine, and the immunoblots were stained by the avidin-biotin-peroxidase complex method as described above.

Assay for DNA synthesis
After various treatments for 24 h, the cells in each well of a 48-well plate were labeled with 14.8 kBq [3H]thymidine as described previously (13). At the end of the labeling period, the cells were treated with trichloroacetic acid (final concentration, 10%). The amount of [3H]thymidine incorporated into the trichloroacetic acid-insoluble materials was counted, and this was regarded as the level of DNA synthesis.

Measurement of MAPK activity
After various treatments for the indicated periods, the cells were scraped in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM dithiothreitol, 2 mM EGTA, 1 mM Na3VO4, 1 mM aminoethyl-benzenesulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin and sonicated for 10 sec. The cell lysates were centrifuged, and MAPK activity in the supernatant was measured by using a p42/p44 MAPK enzyme assay system (Amersham) according to the manufacturer’s instruction. The supernatants were incubated for 30 min at 30 C in 75 mM HEPES-NaOH (pH 7.5), 0.3 mM Na3VO4, 1.2 mM [{gamma}-32P]ATP (37 kBq), and a peptide sequencing KRELVEPLTPAGEAPNQALLR as a substrate for MAPK. After the incubation, the reaction was stopped by the addition of a solution containing 300 mM orthophosphate and 0.02% carmosine red. The reaction mixture was centrifuged, and 30 µl of the supernatant obtained from the reaction mixture were spotted onto a phosphocellulose paper disk. The paper disks were washed twice with 1% acetic acid, and after washing twice with distilled water, and amount of 32P incorporated into the substrate peptide was counted.

A MAPK detection assay in the MBP-containing gel (in-gel kinase assay) was performed according to the method of Kameshita and Fujisawa (35) with minor modifications. The supernatant of the cell lysate was electrophoresed onto a 12% SDS-polyacrylamide gel containing 0.5 mg/ml MBP. After electrophoresis, SDS in the gels was removed by washing the gel with 20% 2-propanol in 50 mM Tris-HCl (pH 8.0), and the proteins in the gel were denatured with 6 M guanidine HCl and then renatured in 50 mM Tris-HCl (pH 8.0) containing 0.04% Tween-40 and 5 mM 2-mercaptoethanol. Phosphorylation of MBP was carried out by incubating the gel in 10 ml of a reaction buffer consisting of HEPES-NaOH (pH 8.0), 0.1 mM EGTA, 20 mM MgCl2, 2 mM dithiothreitol, and 200 µM [{gamma}-32P]ATP (925 kBq) at 25 C for 30 min, and the gel was extensively washed with 7% acetic acid. The dried gel was exposed to an x-ray film at -80 C. 32P-Labeled MBP bands were quantitated by an image-analyzing densitometer (B. I. Systems Corp., Ann Arbor, MI).

Immunoprecipitation and immune complex kinase assays
After cells were treated with various agents for indicated periods, the they were lysed in a radioimmunoprecipitation assay buffer consisting of PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM NaF, 1 mM EGTA, 1 mM Na3VO4, 1 mM aminoethyl-benzenesulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Raf-1 or MEKK activity was measured according to the method of Matsuda et al. (36). The cell lysate precleared by incubation in protein A-agarose was incubated with 2.5 µg polyclonal anti-Raf-1 or anti-MEKK antibody at 4 C overnight. Then, 20 µl protein A-agarose were added to the immunomixture, and the incubation was continued for an additional 2 h. The immunocomplex was washed five times with the cold radioimmunoprecipitation assay buffer and suspended in 15 µl of a kinase buffer consisting of 20 mM Tris-HCl (pH 7.5), 2 mM EGTA, 20 mM MgCl2, and 200 µM [{gamma}-32P]ATP (185 kBq) in the presence of 3 µg Xenopus recombinant MAPK kinase and 5 µg recombinant kinase-negative MAPK (KN-MAPK). The reaction mixture was incubated for 1 h at 30 C. The reaction was terminated by the addition of Laemmli’s sample buffer. Phosphorylated KN-MAPK was resolved by SDS-PAGE (12% gel), and the dried gel was exposed to x-ray film at -80 C. The phosphorylated MAPK bands were quantitated by an image-analyzing densitometer. Raf-1 or MEKK activity represented the MAPK kinase-activating activity that catalyzed the phosphorylation of KN-MAPK.

Statistical analysis
Data were obtained from two or three independent experiments and are shown as the mean ± SD; statistical differences were assessed by Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PGF2{alpha} stimulates tyrosine phosphorylation in MC3T3-E1 cells
The addition of PGF2{alpha} to quiescent osteoblastic MC3T3-E1 cells stimulated the tyrosine phosphorylation of cellular proteins with Mr values of 62,000 (pp62), 68,000 (pp68), 72,000 (pp72), 76,000 (pp76), 82,000 (pp82), 125,000 (pp125), and 150,000 (pp150) in dose- and time-dependent manners (Fig. 1Go, B and C). The maximal effect of PGF2{alpha} was observed at 280 nM. The level of tyrosine phosphorylation in the proteins between pp62 and pp82 was more intensive than that in pp120 and pp150. pp62 was strongly tyrosine-phosphorylated within 2 min after the addition of PGF2{alpha}, and thereafter, the level of phosphorylation gradually decreased. After 2 min, the tyrosine phosphorylation time-dependently shifted to proteins with Mr between 68–82 kDa, and the high level remained for 60 min. TPA also increased the contents of phosphotyrosine of the cellular proteins in MC3T3-E1 cells, and the protein profile of tyrosine phosphorylation induced by TPA was similar to that induced by PGF2{alpha}, whereas the tyrosine residue of pp62 was not phosphorylated by TPA (Fig. 2Go, A and B). In addition, the phosphorylation of pp68, pp72, pp76, and pp82 induced by PGF2{alpha} was attenuated by down-regulating PKC via 24-h pretreatment of TPA and by the addition of GF 109203X, a specific inhibitor of PKC (37). These results indicated that PGF2{alpha} induced the tyrosine phosphorylation of cellular proteins, except pp62, through activation of PKC.



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  		Figure 1. Effect of PGF2{alpha} on tyrosine phosphorylation in clonal osteoblastic MC3T3-E1 cells. The cells were treated with various concentrations of PGF2{alpha} for 5 min (A and B) or with 280 nM PGF2{alpha} for the indicated times (C). The cell lysate was subjected to SDS-PAGE. The immunoblots were performed with nonimmune mouse IgG (A) or with mouse monoclonal antiphosphotyrosine IgG (B and C). The open arrow on the right side indicates pp62; closed arrows point to pp68, pp72, pp76, and pp82; arrowheads show pp125 and pp150.

 


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  		Figure 2. Involvement of PKC in tyrosine phosphorylation induced by PGF2{alpha} in clonal osteoblastic MC3T3-E1 cells. A, The cells were pretreated with or without TPA (50 ng/ml) for 24 h before treatment with PGF2{alpha} (140 nM) or TPA (50 ng/ml) for 5 min. B, The cells were preincubated with GF 109203X (5 µg/ml) for 45 min before treatment with PGF2{alpha} (140 nM) or TPA (50 ng/ml) for 5 min in the presence or absence of GF 109203X. Immunoblots were carried out with antiphosphotyrosine antibody.

 
Activation of MAPK by PGF2{alpha} in MC3T3-E1 cells
The addition of PGF2{alpha} transiently stimulated the activity of MAPK in osteoblastic MC3T3-E1 cells; the maximal increase at 2 min was about 100-fold over the control value (Fig. 3AGo). Thereafter, the stimulated activity gradually decreased. The stimulation of MAPK depended on the concentration of PGF2{alpha}, and the activity reached a plateau at around 1 µM PGF2{alpha} (Fig. 3BGo). Likewise, fluprostenol, a potent agonist of FP receptor, and PGE2 elevated MAPK activity (Fig. 4AGo). The potency of fluprostenol to activate MAPK was higher by about 100-fold than that of PGF2{alpha}, and the IC50 value of PGE2 was 10-fold less. In addition, this fluprostenol or PGF2{alpha} dose dependency of MAPK activity was similar to that of the DNA synthesis in the cells (Fig. 4BGo). The activation of MAPK induced by PGF2{alpha} was also confirmed by in-gel kinase assay containing MBP (Fig. 3CGo). Two protein bands with Mr of 42 and 44 kDa showed a marked increase in their kinase activity to phosphorylate MBP in response to PGF2{alpha}, fluprostenol, and TPA. The molecular masses of the two kinases induced by PGF2{alpha} corresponded with those of both isotypes of classical mammalian MAPKs (ERK1 and ERK2). The magnitude of the activation by PGF2{alpha}, fluprostenol, and TPA in-gel kinase assay was similar to that in the assay using a synthetic MAPK substrate. The activation of MAPK induced by PGF2{alpha}, fluprostenol, and TPA was diminished by 24-h pretreatment of TPA, which caused down-regulation of protein kinase C (Fig. 5AGo). In addition, GF 109203X completely abolished the stimulatory effect of PGF2{alpha} on MAPK activity (Fig. 5BGo).



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  		Figure 3. Effect of PGF2{alpha} on MAPK activity in osteoblastic MC3T3-E1 cells. A, The cells were treated with PGF2{alpha} (140 nM) for the indicated times; B, the cells were treated with various concentrations of PGF2{alpha} for 2 min. After the treatments, the cells were lysed, and the activity of MAPK was measured with a synthetic peptide sequencing KRELVEPLTPAGEAPNQALLR as a substrate for MAPK as described in Materials and Methods. Values are the mean ± SD from four dishes in a representative experiment. *, P < 0.01 vs. the culture at 0 min (A) or without PGF2{alpha} (B). C, In-gel kinase assay for MAPK. The cells were treated for 2 min without (lane 1) or with PGF2{alpha} (140 nM; lane 2), TPA (10 ng/ml; lane 3), and fluprostenol (10-8 M; lane 4). An aliquot of each cell lysate was subjected to the kinase detection assay within a polyacrylamide gel containing MBP as described in Materials and Methods. The autoradiograph was shown. Arrows indicate p42MAPK (ERK2) and p44MAPK (ERK1).

 


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  		Figure 4. Effect of fluprostenol or PGE2 on MAPK activity in clonal osteoblastic MC3T3-E1 cells. The cells were treated for 2 min with various concentrations of PGF2{alpha} ({circ}), fluprostenol ({triangleup}), or PGE2 (•) for determination of MAPK activity (A). For assay of DNA synthesis, the cells were incubated with PGF2{alpha} ({circ}) and fluprostenol ({triangleup}) for 24 h (B). The MAPK assay was performed with a synthetic MAPK substrate as described in Materials and Methods. Values are the mean ± SD from four dishes in a representative experiment. *, P < 0.01 vs. the culture without agents.

 


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  		Figure 5. PKC-dependent activation of MAPK in response to PGF2{alpha}. The MC3T3-E1 cells were pretreated without (open bars) or with (closed bars) TPA (A; 50 ng/ml) for 24 h or GF 109203X (B; 5 µg/ml) for 45 min before treatment for 2 min without (None) or with PGF2{alpha} (140 nM), fluprostenol (FLUP; 10-8 M), and TPA (50 ng/ml). MAPK activity was assayed with a synthetic MAPK substrate. Values are the mean ± SD from four dishes in a representative experiment. *, P < 0.01 vs. the culture without any agents.

 
PGF2{alpha}-induced tyrosine phosphorylation and MAPK activation are independent of IAP-sensitive G protein and PLC{gamma}
IAP-sensitive G protein has been reported to be involved in PGF2{alpha}-induced phosphoinoside hydrolysis, by which PKC is activated in osteoblastic MC3T3-E1 cells (38). However, pretreatment with and after simultaneous addition of IAP did not affect tyrosine phosphorylation, MAPK activity, and DNA synthesis induced by PGF2{alpha} (Fig. 6Go). On the other hand, there is a possible tyrosine phosphorylation motif (YIL) in the amino acid sequence of FP receptor between 304 and 306 that might bind to PLC{gamma} (14, 15). Association of the SH2 domain of PLC{gamma} with phosphotyrosine in some receptors results in tyrosine phosphorylation and activation of PLC{gamma} (39). PGF2{alpha} stimulated the tyrosine kinases in the MC3T3-E1 cells as demonstrated in Fig. 1Go. Therefore, we next examined the relationship between PGF2{alpha} actions and PLC{gamma}. As shown in Western blot analysis with anti-PLC{gamma} or with antiphosphotyrosine antibody of anti-PLC{gamma} immunoprecipitates, we could not detect the elevation of tyrosine phosphorylation of PLC{gamma} in response to PGF2{alpha}, whereas platelet-derived growth factor (as a positive control) induced the tyrosine phosphorylation of the enzyme (Fig. 7Go). Taken together with the above two experiments, PGF2{alpha} signaling does not seem to be linked with IAP-sensitive Gi protein and PLC{gamma}.



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  		Figure 6. IAP-sensitive G protein is not involved in PGF2{alpha}-induced tyrosine phosphorylation, MAPK activity, or DNA synthesis in clonal osteoblastic MC3T3-E1 cells. The cells were pretreated for 24 h with or without IAP (500 ng/ml in A and B, or 100 and 500 ng/ml in C) before treatment with or without PGF2{alpha} (140 nM) for 2 min, 2 min, and 24 h in the presence or absence of IAP for assays for tyrosine phosphorylation (A), MAPK activity (B), and DNA synthesis (C), respectively. Open bars in C, Without PGF2{alpha}; closed bars in C, with PGF2{alpha}. MAPK activity was measured with a synthetic MAPK substrate. Values are the mean ± SD from four dishes in a representative experiment. *, P < 0.01 vs. the culture without any agents.

 


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  		Figure 7. Immunoprecipitation of cell extract of clonal osteoblastic MC3T3-E1 cells with anti-PLC{gamma} monoclonal antibody. The cells were treated without (control) or with PGF2{alpha} (140 nM) or platelet-derived growth factor (1 nM) for 2 min. After immunoprecipitation (IP) with anti-PLC{gamma} ({alpha}-PLC{gamma}), Western blot analysis (WB) of the immunoprecipitates was performed with anti-PLC{gamma} (left panel) or anti-phosphotyrosine ({alpha}-PY; right panel) antibody as described in Materials and Methods. The arrow on the right side of the left and right panels indicates PLC{gamma}; the arrowhead shows the position of mouse IgG heavy chain.

 
Involvement of Raf-1 and MEKK in MAPK activation by PGF2{alpha}
As two distinct MAPK-activating pathways via Raf-1 and MEKK have been reported (33), we next examined which pathway is most important for activating MAPK in osteoblastic MC3T3-E1 cells. As shown in Fig. 8Go, we could detect both proteins from MC3T3-E1 cell extract by immunoprecipitation and Western blots with anti-Raf-1 and anti-MEKK antibodies. The MAPK kinase-activating activity of Raf-1 immunoprecipitate was much greater than that of MEKK immunoprecipitate and was enhanced by the addition of PGF2{alpha}. These results suggest that PGF2{alpha} activates MAPK activity mainly via a Raf-1 signaling pathway. Then, we examined the relationship between PGF2{alpha}-induced Raf-1 activity and PKC or tyrosine phosphorylation. As was the case for MAPK and tyrosine phosphorylation (Figs. 2Go and 5Go), the MAPK kinase-activating activity of Raf-1 stimulated by PGF2{alpha} was reduced by the down-regulation or inhibition of PKC (Fig. 9Go, A and B), whereas the inhibition and down-regulation somewhat decreased the basal Raf-1 activity. On the other hand, genistein, a potent inhibitor of tyrosine kinases, did not inhibit but, rather, increased the PGF2{alpha}-induced Raf-1 activity. However, at the same dose of genistein, the inhibitor partially decreased the MAPK activity (Fig. 10Go), suggesting another MAPK-activating mechanism distinct from Raf-1 pathway.



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  		Figure 8. Effects of PGF2{alpha} on MAPK kinase kinase activity of MEKK and Raf-1 in clonal osteoblastic MC3T3-E1 cells. The cells were treated without (lanes 1 and 3 in C) or with (A and B, and lanes 2 and 4 in C) PGF2{alpha} (140 nM for 2 min). In A and B, the cell lysates were then immunoprecipitated with rabbit nonimmune IgG (lanes 1 in A and B), anti-MEKK (lane 2 in A), or anti-Raf-1 (lane 2 in B). After being resolved in SDS-PAGE, each immunoprecipitate was stained with anti-MEKK (A) and anti-Raf-1 (B). C, An autoradiogram for immune complex kinase assay of each immunoprecipitate with anti-MEKK (lanes 1 and 2 in C) or anti-Raf-1 (lanes 3 and 4 in C). The immune complex kinase assay was performed as described in Materials and Methods.

 


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  		Figure 9. Involvement of PKC and tyrosine kinase in PGF2{alpha}-induced Raf-1 activity in clonal osteoblastic MC3T3-E1 cells. The cells were pretreated without (lanes 2 and 3) or with GF 109203X (5 µg/ml; lanes 4 and 5) for 45 min and with genistein (100 µg/ml; lanes 6 and 7) for 45 min and with TPA (50 ng/ml; lanes 8 and 9) for 24 h before 2-min treatment without (lanes 2, 4, 6, and 8) or with (lanes 3, 5, 7, and 9) PGF2{alpha} (140 nM). Then, the Raf-1 immune complex kinase activity of each extract was determined; lane 1 shows phosphorylated KN-MAPK in the absence of any cell lysate. After bands were measured densitometrically, each intensity was subtracted by that of lane 1 (absence of cell lysate). B shows the relative intensity of Raf-1 activity vs. untreated culture.

 


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  		Figure 10. Involvement of tyrosine kinases in PGF2{alpha}-induced MAPK activity in clonal osteoblastic MC3T3-E1 cells. The cells were pretreated for 45 min with or without genistein (Genist.; 100 µg/ml) before treatment for 2 min without (none) or with PGF2{alpha} (140 nM), fluprostenol (FLUP; 10-8 M), or TPA (50 ng/ml) in the absence or presence of genistein. MAPK activity was measured with a synthetic MAPK substrate. Values are the mean ± SD from four dishes in a representative experiment. *, P < 0.01 vs. the culture without any agents; **, P < 0.01 vs. the each culture without genistein pretreatment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The extensive evidence for cross-talk between tyrosine kinases and PKC has indicated that interactions between these kinase cascades play an important role in controlling mitogenesis in a variety of cells. In the present study we found that PGF2{alpha}, a potent mitogen for osteoblasts, stimulated PKC-dependent tyrosine phosphorylation and MAPK activities in clonal osteoblastic MC3T3-E1 cells. One pathway for the activation of the MAPKs was via genistein-insensitive Raf-1 activation evoked by PGF2{alpha}, whereas MEKK activity was not as important in the stimulation. The PGF2{alpha}-stimulated tyrosine kinase cascades, which were not linked to the Raf-1 activity, were also involved in the enhancement of MAPK activity. These activations were linked to a stimulation of PKC by PGF2{alpha} that was not associated with Gi or PLC{gamma}.

In our study, PGF2{alpha} elevated the levels of tyrosine phosphorylation of several cellular proteins. Among these phosphotyrosine-containing proteins, the proteins with Mr values between 68–82 kDa were more prominent. Proteins with higher Mr of 125 and 150 kDa were also tyrosine phosphorylated, but to a lesser extent; judging from the Mr, pp125 may be p125FAK. Indeed, in NIH-3T3 cells, the tyrosine phosphorylation of FAK has been reported to be induced by PGF2{alpha} (27). Since the tyrosine phosphorylation of PLC{gamma} was not enhanced by PGF2{alpha}, pp150 was distinct from this enzyme, although both have similar molecular masses. The profile of tyrosine phosphorylation of these proteins by PGF2{alpha} was the same as that for TPA and was down-regulated by PKC depletion and GF 109203X, a PKC-specific inhibitor (37), indicating that these tyrosine phosphorylations were dependent on the stimulation of PKC. In contrast, the tyrosine phosphorylation of pp62 evoked by PGF2{alpha} was resistant to PKC depletion and GF 109203X, and preceded the tyrosine phosphorylation of the other proteins, suggesting a PKC-independent pathway.

We also showed thatPGF2{alpha} stimulated a PKC-dependent Raf-1/MAPK cascade in osteoblastic MC3T3-E1 cells. These results were consistent with observations by Siddhanti et al. (40), although they were unsuccessful in assessing the involvement of MEKK, another MAPK kinase kinase for activation of MEK (33) and a homolog of yeast Ste11 and Byr2 (41, 42), in the stimulation of MAPK induced by PGF2{alpha}. MEKK is considered to mediate primarily signals originating from receptors that activate G proteins and PKC (33). On the other hand, Raf-1 is activated by the GTP-bound active form of Ras that is strongly linked to receptor tyrosine kinases for many growth factors in mammalian cells (43). In MC3T3-E1 cells, the action of PGF2{alpha} is mediated by G protein-coupled PGF2{alpha} receptor and PLC and subsequently by activation of PKC (5). Therefore, in the case of PGF2{alpha} signaling to MAPK, MEKK was expected to be more important in activating MAPK than Raf-1. However, the basal MAPK kinase kinase activity of MEKK was less than that of Raf-1, and the activity was not increased by the addition of PGF2{alpha}, although the MEKK immunoprecipitate was detectable in the cells. These results indicated that Raf-1 is a principal MAPK kinase kinase in PGF2{alpha}-induced kinase cascades to MAPK. This involvement of Raf-1 in PKC-dependent MAPK activation has been recently reported in other types of cells (44). With respect to the mechanism for activation of Raf/MAPK, PGF2{alpha} may be PKC-dependently capable of stimulating nonreceptor tyrosine kinases, resulting in tyrosine phosphorylation of some kinase substrates, such as Sos (45), and after activation of Raf-1 through a tyrosine kinase/Ras interaction (46). However, in our study, because Raf-1 activity was not attenuated by genistein, a tyrosine kinase inhibitor, such as tyrosine kinase/Ras interaction, seems unlikely. As genistein somehow enhanced the stimulatory effect of PGF2{alpha} on Raf-1 activity, PGF2{alpha}-induced tyrosine kinases may interact with some tyrosine phosphatases (47). Recently, PGF2{alpha} has been reported to activate the Ras/MAPK pathway through the Gq protein-coupled pathway in NIH-3T3 cells (48). In addition, some PKC-stimulating agents, such as TPA, stimulate formation of the GTP-bound active form of H-Ras (49) and thereby activate the MAPK pathway. However, our efforts to determine the effect of PGF2{alpha} on GTP/GDP exchange on Ras in MC3T3-E1 cells have been not successful to date (data not shown). Alternatively, recent reports have shown the direct phosphorylation of Raf-1 by PKC{alpha}, indicating a pathway of Ras-independent Raf-1 activation (32). Therefore, PKC{alpha} may be involved in the activation of Raf-1 by PGF2{alpha} in MC3T3-E1 cells. On the other hand, whereas genistein had little effect on the PGF2{alpha}-induced MAPK kinase kinase activity of Raf-1, the inhibitor partially attenuated the MAPK response, indicating a possible MAPK activation pathway dependent on other kinases. Similarly, Quarles et al. have shown that genistein partially inhibited the DNA synthesis of MC3T3-E1 cells (9). Therefore, such a genistein-sensitive MAPK activation in response to PGF2{alpha} is likely to cause in part the stimulation of cell proliferation.

As fluprostenol, a specific agonist for the FP receptor (50), not only mimicked the effects of PGF2{alpha} on tyrosine phosphorylation and MAPK activity in MC3T3-E1 cells, but was more potent than PGF2{alpha}, the action of PGF2{alpha} on the cells can be considered to be mediated through FP receptors. Indeed, FP messenger RNA was detected in this cells (20). PG receptors are typically G protein-coupled receptors with seven transmembrane domains. Multiple receptors for PGs have been cloned from bovine and mouse complementary DNA libraries. The interaction between the FP receptor and G proteins is not clearly defined. In NIH-3T3 fibroblasts, PGF2{alpha} responses have been linked to phosphoinositide-specific PLC activation via an IAP-insensitive G protein, perhaps Gq (18). This was supported by the observation that transfection of a complementary DNA encoding a constitutively active mutant of Gq {alpha}-subunit mimicked PGF2{alpha}’s actions (48). In contrast, the involvement of an IAP-sensitive G protein such as Gi has been also reported in PGF2{alpha}-induced phosphatidylinositol hydrolysis in osteoblastic MC3T3-E1 cells (38) and fibroblastic cells (51). However, the blockage of phosphatidylinositol turnover by IAP in MC3T3-E1 cells was only partial. In the present study, IAP did not affect PGF2{alpha}-induced tyrosine phosphorylation, MAPK activity, or DNA synthesis. Thus, the PGF2{alpha} receptor is probably mainly coupled to a G protein other than Gi.

In conclusion, the action of PGF2{alpha} on osteoblastic MC3T3-E1 cells involves a single receptor of FP that uses diverse interacting signal transduction systems, including protein phosphorylation and MAPK.


   Acknowledgments
 
We thank Dr. S. Matsuda and E. Nishida (Institute of Virus Research, Kyoto University, Kyoto, Japan) for generously providing rMAPKK and KN-MAPK.

Received October 21, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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