Inhibition by adenylyl cyclase-cAMP system of ET-1-induced HSP27 in osteoblasts

Daijiro Hatakeyama, Osamu Kozawa, Masayuki Niwa, Hiroyuki Matsuno, Kanefusa Kato, Norichika Tatematsu, Toshiyuki Shibata, and Toshihiko Uematsu

Departments of Pharmacology and Oral and Maxillo-Facial Surgery, Gifu University School of Medicine, Gifu 500 - 8705; and Department of Biochemistry, Aichi Human Service Center, Institute for Developmental Research, Kasugai, Aichi 480-0392, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously reported that endothelin-1 (ET-1) stimulates heat shock protein (HSP) 27 induction in osteoblast-like MC3T3-E1 cells and that p38 mitogen-activated protein (MAP) kinase acts at a point downstream from protein kinase C (PKC) in HSP27 induction. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on ET-1-stimulated induction of HSP27 in MC3T3-E1 cells. Dibutyryl-cAMP (DBcAMP) dose dependently inhibited the HSP27 accumulation stimulated by ET-1. Forskolin and cholera toxin significantly suppressed the ET-1-stimulated accumulation of HSP27. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the ET-1-induced HSP27 accumulation. Forskolin reduced the p38 MAP kinase phosphorylation induced by ET-1 or 12-O-tetradecanoylphorbol-13-acetate (TPA). PGE1, an extracellular agonist that activates cAMP production, reduced the ET-1-induced HSP27 accumulation. In addition, the phosphorylation of p38 MAP kinase induced by ET-1 or TPA was suppressed by PGE1. Forskolin, DBcAMP, and PGE1 suppressed the ET-1-stimulated increase in the mRNA level for HSP27. These results indicate that the adenylyl cyclase-cAMP system has an inhibitory role in ET-1-stimulated HSP27 induction in osteoblasts and that the effect is exerted at the point between PKC and p38 MAP kinase in osteoblasts.

adenosine 3',5'-cyclic monophosphate; endothelin-1; heat shock protein 27; mitogen-activated protein kinase; protein kinase C


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

WHEN CELLS ARE EXPOSED to various stressors such as heat stress and chemical stress, they produce heat shock proteins (HSPs; see Ref. 13). HSPs are generally divided into high-molecular-weight HSPs and low-molecular-weight HSPs according to apparent molecular sizes. Low-molecular-weight HSPs, with molecular masses, from 15 to 30 kDa such as HSP27 and alpha B-crystallin, have high homology in amino acid sequences (1). High-molecular-weight HSPs are well known to act as molecular chaperones in protein folding, oligomerization, and translocation. Although the functions of low-molecular-weight HSPs are less known than those of high-molecular-weight HSPs, it is speculated that they may act as molecular chaperones like high-molecular-weight HSPs (1, 13). In osteoblasts, heat induces the expression of HSP27, and estrogen reportedly facilitates the heat-induced HSP27 expression (2, 16). Additionally, the downregulation of proliferation has been shown to be accompanied by a transient increase of the expression of HSP27 mRNA (2, 16). We have recently shown that endothelin (ET)-1 stimulates HSP27 induction via the ETA receptor and that p38 mitogen-activated protein (MAP) kinase is involved in the HSP27 induction in osteoblast-like MC3T3-E1 cells (8). In addition, protein kinase C (PKC) activated by ET-1 acts at a point upstream from p38 MAP kinase in the HSP27 induction (8). However, the detailed regulatory mechanisms of HSP27 induction in osteoblasts and its roles have not yet been precisely clarified.

cAMP is produced from ATP by adenylyl cyclase and then causes cAMP-dependent protein kinase (protein kinase A) activation (3). In osteoblasts, it is well recognized that the adenylyl cyclase-cAMP system plays an important role in cell differentiation and proliferation (17). We previously reported that PGE1 stimulates alkaline phosphatase activity, a marker of the mature osteoblast phenotype (18), via cAMP production without affecting phosphoinositide-hydrolyzing phospholipase C in osteoblast-like MC3T3-E1 cells (6). Moreover, we have shown that PGE1 induces interleukin-6 synthesis via protein kinase A activation in these cells (21). Thus it is speculated that the effects of PGE1 are exerted via the adenylyl cyclase-cAMP system in osteoblasts. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on the induction of HSP27 stimulated by ET-1 in osteoblast-like MC3T3-E1 cells. Here, we report that the adenylyl cyclase-cAMP system inhibits ET-1-stimulated HSP27 induction at the point between PKC and p38 MAP kinase in these cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. ET-1 was obtained from the Peptide Institute (Minoh, Japan). Dibutyryl-cAMP (DBcAMP), forskolin, dideoxyforskolin, cholera toxin, 12-O-tetradecanoylphorbol-13- acetate (TPA), and PGE1 were purchased from Sigma Chemical (St. Louis, MO). Phosphospecific p38 MAP kinase antibodies (rabbit polyclonal IgG, affinity purified) and p38 MAP kinase antibodies (rabbit polyclonal IgG, affinity purified) were purchased from New England BioLabs (Beverly, MA). An enhanced chemiluminescence (ECL) Western blotting detection system was obtained from Amersham Japan (Tokyo, Japan). Other materials and chemicals were obtained from commercial sources. Forskolin, dideoxyforskolin, and PGE1 were dissolved in ethanol. TPA was dissolved in dimethyl sulfoxide. The maximum concentrations of ethanol or dimethyl sulfoxide were 0.1%, which did not affect immunoassay of HSP27, Northern blot analysis of HSP27, or Western blot analysis.

Cell culture. Cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria (19) were maintained as previously described (9). In brief, the cells were cultured in alpha -MEM containing 10% FCS at 37°C in a humidified atmosphere of 5% CO2-95% air. The cells were seeded in 35-mm-diameter dishes or 90-mm-diameter dishes in alpha -MEM containing 10% FCS. After 5 days, the medium was exchanged for 2 ml of alpha -MEM containing 0.3% FCS. The cells were used for experiments after 48 h. When indicated, the cells were pretreated with DBcAMP, forskolin, dideoxyforskolin, or PGE1 for 20 min. The pretreatment of cholera toxin was performed for 12 h.

Immunoassay of HSP27. The concentration of HSP27 in soluble extracts of cells was determined by a sandwich-type enzyme immunoassay, as described previously (5). The cultured cells were stimulated by ET-1 in alpha -MEM for the indicated periods. The cells were then washed two times with 1 ml of PBS and frozen at -80°C for a few days before analysis. The frozen cells on each dish were collected and suspended in 0.3 ml of PBS, and each suspension was sonicated and centrifuged at 125,000 g for 20 min at 4°C. The supernatant was used for the specific immunoassay of HSP27. In brief, we used an enzyme immunoassay system that employs polystyrene balls (3.2 mm in diameter; Immuno Chemicals, Okayama, Japan) carrying immobilized F(ab')2 fragments of antibody and the same Fab' fragments of antibody labeled with beta -D-galactosidase from Escherichia coli. A polystyrene ball carrying antibodies was incubated either with the purified standard for HSP27 or with an aliquot of one of the samples. This incubation was carried out at 30°C for 5 h in a final volume of 0.5 ml of 10 mM sodium phosphate buffer, pH 7.0, containing 0.3 M NaCl, 0.5% hydrolyzed gelatin, 0.1% BSA, 1 mM MgCl2, and 0.1% NaN3. After being washed, each ball was incubated at 4°C overnight with 1.5 mU of galactosidase-labeled antibodies in a volume of 0.2 ml with 10 mM sodium phosphate buffer, pH 7.0, containing 0.1 M NaCl, 1 mM MgCl2, 0.1% BSA, and 0.1% NaN3. The galactosidase activity bound to the ball was assayed using a fluorogenic substrate, 4-methylumbelliferyl-beta -D-galactoside.

Isolation of RNA and Northern blot analysis of HSP27. The cultured cells were stimulated by ET-1 in alpha -MEM for the indicated periods. Total RNA was isolated using a QuickPrep total RNA extraction kit (Pharmacia Biotech, Tokyo, Japan). Total RNA (20 µg) was then subjected to electrophoresis on a 0.9% agarose-2.2 M formaldehyde gel before being blotted on a nitrocellulose membrane. For Northern blot, the membrane was allowed to hybridize with a cDNA probe that had been labeled using a Multiprime DNA labeling system (Amersham, Buckinghamshire, UK), as described previously (7). A BamH I-Hind III fragment of cDNA for mouse HSP27 was kindly provided by Dr. L. F. Cooper of the University of North Carolina (2).

Western blot analysis of p38 MAP kinase. The cultured cells were stimulated by ET-1 in alpha -MEM for the indicated periods. The cells were washed two times with PBS and then lysed, homogenized, and sonicated in a lysis buffer containing 62.5 mM Tris · HCl, pH 6.8, 2% SDS, 50 mM dithiothreitol, and 10% glycerol. The cytosolic fraction was collected as the supernatant after centrifugation at 125,000 g for 10 min at 4°C. SDS-PAGE was performed by the method of Laemmli (10) in 10% polyacrylamide gel. Western blot analysis was performed as described previously (8) by using phosphospecific p38 MAP kinase antibodies or p38 MAP kinase antibodies, with peroxidase-labeled antibodies raised in goat against rabbit IgG being used as secondary antibodies. Peroxidase activity on the nitrocellulose sheet was visualized on X-ray film by means of the ECL Western blotting detection system.

Other methods. Protein concentrations in soluble extracts were determined using a protein assay kit (Bio-Rad, Hercules, CA) with BSA as the standard protein. Rat HSP27, which was used as the standard for the immunoassay, was purified from skeletal muscle (5). The densitometric analysis was performed using Molecular Analyst/Macintosh (Bio-Rad Laboratories).

Statistical analysis. Each experiment was repeated three times with similar results. The data were analyzed by ANOVA followed by the Bonferroni method for multiple comparisons between pairs, and P < 0.05 was considered significant. All data are presented as means ± SD of triplicate determinations.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of DBcAMP on ET-1-induced HSP27 accumulation in MC3T3-E1 cells. It is well recognized that Gs functions as an intermediary in transmembrane signaling from the receptor to adenylyl cyclase, and the activation of adenylyl cyclase results in cAMP production (4). To clarify the role of the adenylyl cyclase-cAMP system in ET-1-stimulated HSP27 induction in osteoblast-like MC3T3-E1 cells, we examined the effects of each direct activator of the adenylyl cyclase-cAMP system on HSP27 accumulation stimulated by ET-1. DBcAMP, a permeable analog of cAMP, which alone had little effect on the basal level of HSP27, significantly reduced the HSP27 accumulation stimulated by ET-1 (Fig. 1). The inhibitory effect of DBcAMP on ET-1-induced HSP27 accumulation was dose dependent in the range between 0.1 and 3 mM. The maximum effect of DBcAMP was observed at 3 mM, a dose that caused an ~50% reduction in the effect of ET-1 (Fig. 1).


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Fig. 1.   Effect of dibutyryl-cAMP (DBcAMP) on endothelin (ET)-1-induced heat shock protein (HSP)27 accumulation in MC3T3-E1 cells. Cultured cells were pretreated with various doses of DBcAMP for 20 min and then stimulated by 3 nM ET-1 () or vehicle (open circle ) for 12 h. Each value represents the mean ± SD of triplicate determinations. Similar results were obtained with 2 additional and different cell preparations. *P < 0.05 vs. ET-1 alone.

Effects of forskolin or dideoxyforskolin on ET-1- induced HSP27 accumulation in MC3T3-E1 cells. Forskolin, a direct activator of adenylyl cyclase (14) that by itself did not affect the basal level of HSP27 inhibited ET-1-stimulated HSP27 accumulation (Fig. 2). The inhibitory effect of forskolin on HSP27 accumulation induced by ET-1 was dose dependent in the range between 30 nM and 50 µM. The maximum effect of forskolin was observed at 50 µM, a dose that caused an ~84% reduction in the effect of ET-1 (Fig. 2). However, dideoxyforskolin (10 µM), a forskolin derivative that does not activate cAMP (15), failed to reduce the HSP27 accumulation induced by ET-1 while forskolin (10 µM) significantly suppressed the accumulation (Table 1).


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Fig. 2.   Effect of forskolin on ET-1-induced HSP27 accumulation in MC3T3-E1 cells. Cultured cells were pretreated with various doses of forskolin for 20 min and then stimulated by 3 nM ET-1 () or vehicle (open circle ) for 12 h. Each value represents the mean ± SD of triplicate determinations. Similar results were obtained with 2 additional and different cell preparations. *P < 0.05 vs. ET-1 alone.


                              
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Table 1.   Effects of dideoxyforskolin or forskolin on ET-1-induced HSP27 accumulation in MC3T3-E1 cells

Effect of cholera toxin on ET-1-induced HSP27 accumulation in MC3T3-E1 cells. Cholera toxin is known to be a direct activator for Gs, a GTP-binding protein that is a stimulative regulator of adenylyl cyclase (4). Cholera toxin significantly inhibited the accumulation of HSP27 induced by ET-1 (Table 2).

                              
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Table 2.   Effect of CTX on ET-1-induced HSP27 accumulation in MC3T3-E1 cells

Effect of PGE1 on ET-1-induced HSP27 accumulation in MC3T3-E1 cells. PGE1 is known to stimulate cAMP production in osteoblasts (18). In addition, we previously found that PGE1 stimulates cAMP accumulation in MC3T3-E1 cells (6). Thus, to investigate if a physiological agonist such as PGE1 that stimulates cAMP production affects the ET-1-stimulated HSP27 induction, we examined the effect of PGE1 on ET-1-induced HSP27 accumulation. PGE1 actually significantly suppressed HSP27 accumulation stimulated by ET-1 (Table 3). PGE1 (10 µM) caused a 26% reduction in the effect of ET-1.

                              
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Table 3.   Effect of PGE1 on ET-1-induced HSP27 accumulation in MC3T3-E1 cells

Effects of forskolin, DBcAMP, or PGE1 on the ET-1-increased mRNA level for HSP27 in MC3T3-E1 cells. In a previous study (8), we have reported that the expression level of mRNA for HSP27 was increased 2 h after the stimulation of ET-1. We next examined the effect of forskolin on the ET-1-induced increase in the level of mRNA for HSP27 in MC3T3-E1 cells. Forskolin, which alone had little effect on the basal level of HSP27 mRNA, significantly suppressed the ET-1-increased level of mRNA for HSP27 (Fig. 3A). Furthermore, we examined the effect of DBcAMP or PGE1 on the ET-1-induced increase in the level of mRNA for HSP27. DBcAMP or PGE1, which alone had little effect on the basal level of HSP27 mRNA, markedly reduced the ET-1-increased level of mRNA for HSP27 (Fig. 3B). According to the densitometric analysis, forskolin caused an ~75% reduction in the ET-1-effect. DBcAMP and PGE1 caused an ~50 and 70% reduction, respectively (Fig. 3, A and B).


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Fig. 3.   Effects of forskolin, DBcAMP, or PGE1 on mRNA level for HSP27 in response to ET-1 in MC3T3-E1 cells. A: cultured cells were pretreated with 30 µM forskolin or vehicle for 20 min and then stimulated by 3 nM ET-1 or vehicle for 2 h. B: cultured cells were pretreated with 3 mM DBcAMP, 10 µM PGE1, or vehicle for 20 min and then stimulated by 3 nM ET-1 or vehicle for 2 h. The cells were harvested, and total RNA was isolated. RNA from each sample (20 µg) was subjected to electrophoresis and blotted on a nitrocellulose membrane. The membrane was then allowed to hybridize with the cDNA probe for HSP27. Bands of 28S RNA are shown for reference. The histogram shows quantitative representations of the ET-1-induced HSP27 mRNA level obtained from laser densitometric analysis of 3 independent experiments. Each value represents the mean ± SD of triplicate determinations. Similar results were obtained with 2 additional and different cell preparations. *P < 0.05 vs. ET-1 alone.

Effect of forskolin on phosphorylation of p38 MAP kinase induced by ET-1 or TPA in MC3T3-E1 cells. We have recently reported that ET-1 activates p38 MAP kinase in MC3T3-E1 cells and that p38 MAP kinase is involved in the HSP27 induction stimulated by ET-1 (8). To investigate whether or not the adenylyl cyclase-cAMP system affects ET-1-induced p38 MAP kinase activation in these cells, we examined the effect of forskolin on the ET-1-induced phosphorylation of p38 MAP kinase. Forskolin markedly suppressed the ET-1-stimulated phosphorylation of p38 MAP kinase (Fig. 4A). In a previous study, we have shown that TPA, a PKC direct-activating phorbol ester (11), phosphorylates p38 MAP kinase in MC3T3-E1 cells (8). Thus we investigated whether the adenylyl cyclase-cAMP system affects p38 MAP kinase activation at a point upstream or downstream from PKC in these cells. Forskolin significantly suppressed the TPA-induced p38 MAP kinase phosphorylation (Fig. 4B). According to the densitometric analysis, forskolin caused a 71% reduction in the effect of ET-1 and caused a 40% reduction in the effect of TPA.


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Fig. 4.   Effect of forskolin on the phosphorylation of p38 mitogen-activated protein (MAP) kinase induced by ET-1 or 12-O-tetradecanoylphorbol-13-acetate (TPA) in MC3T3-E1 cells. A: cultured cells were pretreated with 30 µM forskolin or vehicle for 20 min and then stimulated by 3 nM ET-1 or vehicle for 20 min. B: cultured cells were pretreated with 30 µM forskolin or vehicle for 20 min and then stimulated by 1 nM TPA or vehicle for 90 min. The extracts of cells were subjected to SDS-PAGE against phosphospecific p38 MAP kinase antibodies or p38 MAP kinase antibodies. The histogram shows quantitative representations of the levels of ET-1- or TPA-induced phosphorylation of p38 MAP kinase obtained from laser densitometric analysis of 3 independent experiments. Each value represents the mean ± SD of triplicate determinations. Similar results were obtained with 2 additional and different cell preparations. *P < 0.05 vs. ET-1 or TPA alone.

Effect of PGE1 on p38 MAP kinase phosphorylation induced by ET-1 or TPA in MC3T3-E1 cells. We next examined the effect of PGE1 on the phosphorylation of p38 MAP kinase stimulated by ET-1 or TPA. PGE1 inhibited the ET-1-induced p38 MAP kinase phosphorylation (Fig. 5A). In addition, PGE1 suppressed the TPA-induced p38 MAP kinase phosphorylation (Fig. 5B). According to the densitometric analysis, PGE1 caused a 71% reduction in the effect of ET-1, and PGE1 almost completely reduced the effect of TPA.


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Fig. 5.   Effect of PGE1 on phosphorylation of p38 MAP kinase induced by ET-1 or TPA in MC3T3-E1 cells. A: cultured cells were pretreated with 10 µM PGE1 or vehicle for 20 min and then stimulated by 3 nM ET-1 or vehicle for 20 min. B: cultured cells were pretreated with 10 µM PGE1 or vehicle for 20 min and then stimulated by 1 nM TPA or vehicle for 90 min. The extracts of cells were subjected to SDS-PAGE against phosphospecific p38 MAP kinase antibodies or p38 MAP kinase antibodies. The histogram shows quantitative representations of the levels of ET-1- or TPA-induced phosphorylation of p38 MAP kinase obtained from laser densitometric analysis of 3 independent experiments. Each value represents the mean ± SD of triplicate determinations. Similar results were obtained with 2 additional and different cell preparations. *P < 0.05 vs. ET-1 or TPA alone.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In a previous study (8), we have reported that ET-1 stimulates HSP27 induction in osteoblast-like MC3T3-E1 cells. In the present study, we showed that cholera toxin suppressed ET-1-induced HSP27 accumulation in these cells. Gs, a GTP-binding protein that mediates stimulative signals from the receptor to adenylyl cyclase, induces the activation of this enzyme (4). It is well recognized that cholera toxin ADP-ribosylates the alpha -subunit of Gs and elicits continuous activation of Gs, resulting in cAMP production (4). Thus it seems that cAMP inhibits ET-1-stimulated HSP27 induction in osteoblast-like MC3T3-E1 cells. We next demonstrated that forskolin inhibited HSP27 accumulation stimulated by ET-1. Forskolin is well known as a direct activator of adenylyl cyclase (14), and we previously found that forskolin actually induces cAMP accumulation in MC3T3-E1 cells (9). Moreover, dideoxyforskolin, a forskolin derivative that does not activate cAMP (15), failed to reduce the ET-1-stimulated HSP27 accumulation. Thus our findings suggest that cAMP induced by forskolin suppresses the HSP27 induction stimulated by ET-1 in these cells. In addition, DBcAMP, a permeable analog of cAMP, reduced the ET-1-increased level of HSP27. Furthermore, we showed that forskolin and DBcAMP significantly suppressed the ET-1-increased level of mRNA for HSP27. Therefore, based on our findings, it is most likely that the adenylyl cyclase-cAMP system has an inhibitory role in ET-1-stimulated HSP27 induction in osteoblast-like MC3T3-E1 cells.

In a previous study (8), we have demonstrated that ET-1 stimulated HSP27 induction via p38 MAP kinase activation in osteoblast-like MC3T3-E1 cells. To investigate whether or not the adenylyl cyclase-cAMP system affects ET-1-induced p38 MAP kinase phosphorylation in these cells, we next examined the effect of forskolin on the p38 MAP kinase phosphorylation stimulated by ET-1. Forskolin inhibited the phosphorylation of p38 MAP kinase induced by ET-1. Thus our results suggest that the adenylyl cyclase-cAMP system suppresses the ET-1-stimulated HSP27 induction at a point upstream from p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In a previous study (20), we have shown that ET-1 stimulates phosphatidylinositol-specific phospholipase C and phosphatidylcholine-specific phospholipase D in osteoblast-like MC3T3-E1 cells. Phosphatidylinositol-specific phospholipase C hydrolyzes phosphoinositides and results in the formation of diacylglycerol, which is well known to be a physiological activator of PKC (11). Phosphatidylcholine-specific phospholipase D results in the formation of phosphatidic acid from phosphatidylcholine, and phosphatidic acid is further degraded into diacylglycerol (12). We have previously shown that ET-1-stimulated p38 MAP kinase activation is dependent on PKC activation in osteoblast-like MC3T3-E1 cells (8). Therefore, we next examined whether or not forskolin affects the activation of p38 MAP kinase induced by TPA, a direct PKC activator in these cells (11). Forskolin reduced the TPA-stimulated phosphorylation of p38 MAP kinase. With our results taken into account, it is most likely that the adenylyl cyclase-cAMP system acts as a suppressor in the HSP27 induction stimulated by ET-1 in osteoblast-like MC3T3-E1 cells, and the effect on HSP27 induction is exerted at the point between PKC and p38 MAP kinase.

In a previous study (6, 21), we have shown that PGE1 stimulates alkaline phosphatase activity via cAMP production in osteoblast-like MC3T3-E1 cells and that PGE1 induces interleukin-6 synthesis via cAMP production. Thus we investigated whether PGE1, a physiological agonist, extracellulary affects the induction of HSP27 stimulated by ET-1 in these cells. PGE1 actually suppressed the HSP27 accumulation by ET-1, forskolin, DBcAMP, and cholera toxin. The expression of mRNA for HSP27 stimulated by ET-1 was reduced by PGE1. Additionally, PGE1 suppressed the phosphorylation of p38 MAP kinase induced by ET-1 or TPA in MC3T3-E1 cells. The potential effect of the adenylyl cyclase-cAMP system on HSP27 induction stimulated by ET-1 shown in this study is summarized in Fig. 6. According to densitometric analysis, PGE1 caused a 71% reduction in the phosphorylation of p38 MAP kinase induced by ET-1, whereas PGE1 almost completely reduced p38 MAP kinase phosphorylation induced by TPA. On the other hand, DBcAMP and PGE1 caused a partial inhibition (50 and 26%, respectively) of ET-1-induced HSP27 accumulation. Thus it seems that other pathways, which are not inhibited by PGE1 or the adenylyl cyclase-cAMP system, are involved in the ET-1-stimulated HSP27 induction in osteoblast-like MC3T3-E1 cells. We previously showed that ET-1 activates p44/p42 MAP kinase in addition to p38 MAP kinase, and its activation is not involved in ET-1- stimulated HSP27 induction in MC3T3-E1 cells (8). Further investigations are necessary to clarify the exact mechanism of ET-1-stimulated HSP27 induction in osteoblasts.


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Fig. 6.   Diagram of the regulatory mechanism by the adenylyl cyclase-cAMP system of ET-1-stimulated HSP27 induction in MC3T3-E1 cells. -, Negative modulation; PI, phosphatidylinositol; PC, phosphatidylcholine; PLC, phospholipase C; PLD, phospholipase D; PA, phosphatidic acid; DAG, diacylglycerol; PKC, protein kinase C.

In conclusion, these results strongly suggest that the adenylyl cyclase-cAMP system has an inhibitory role in ET-1-stimulated HSP27 induction in osteoblasts and that the effect is exerted at the point between PKC and p38 MAP kinase.


    ACKNOWLEDGEMENTS

We are grateful to Masaichi Miwa and Hidenori Kawamura for skillful technical assistance.


    FOOTNOTES

Address for reprint requests and other correspondence: O. Kozawa, Dept. of Pharmacology, Gifu Univ. School of Medicine, Gifu 500-8705, Japan (E-mail: okozawa{at}cc.gifu-u.ac.jp).

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.

Received 2 March 2001; accepted in final form 3 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Benjamin, IJ, and McMillan DR. Stress (heat shock) proteins: molecular chaperones in cardiovascular biology and disease. Circ Res 83: 117-132, 1998[Abstract/Free Full Text].

2.   Cooper, LF, and Uoshima K. Differential estrogenic regulation of small M(r) heat shock protein expression in osteoblasts. J Biol Chem 269: 7869-7873, 1994[Abstract/Free Full Text].

3.   Defer, N, Best-Belpomme M, and Hanoune J. Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase. Am J Physiol Renal Physiol 279: F400-F416, 2000[Abstract/Free Full Text].

4.   Gilman, AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem 56: 615-649, 1987[ISI][Medline].

5.   Inaguma, Y, Goto S, Shinohara H, Hasegawa K, Ohshima K, and Kato K. Physiological and pathological changes in levels of the two small stress proteins, HSP27 and alpha B crystallin, in rat hindlimb muscles. J Biochem (Tokyo) 114: 378-384, 1993[Abstract].

6.   Ito, Y, Suzuki A, Watanabe-Tomita Y, Oiso Y, and Kozawa O. Okadaic acid enhances prostaglandin E1-induced alkaline phosphatase activity in osteoblast-like cells: regulation at a point downstream from protein kinase A. Prostaglandins Leukot Essent Fatty Acids 55: 357-361, 1996[ISI][Medline].

7.   Kato, K, Ito H, Hasegawa K, Inaguma Y, Kozawa O, and Asano T. Modulation of the stress-induced synthesis of hsp27 and alpha B-crystallin by cyclic AMP in C6 rat glioma cells. J Neurochem 66: 946-950, 1996[ISI][Medline].

8.   Kawamura, H, Otsuka T, Matsuno H, Niwa M, Matsui N, Kato K, Uematsu T, and Kozawa O. Endothelin-1 stimulates heat shock protein 27 induction in osteoblasts: involvement of p38 MAP kinase. Am J Physiol Endocrinol Metab 277: E1046-E1054, 1999[Abstract/Free Full Text].

9.   Kozawa, O, Tokuda H, Miwa M, Kotoyori J, and Oiso Y. Cross-talk regulation between cyclic AMP production and phosphoinositide hydrolysis induced by prostaglandin E2 in osteoblast-like cells. Exp Cell Res 198: 130-134, 1992[ISI][Medline].

10.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[ISI][Medline].

11.   Nishizuka, Y. Studies and perspectives of protein kinase C. Science 233: 305-312, 1986[ISI][Medline].

12.   Nishizuka, Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 9: 484-496, 1995[Abstract/Free Full Text].

13.   Nover, L. Inducers of hsp synthesis. In: Heat Shock Response, edited by Nover L.. Boca Rotan, FL: CRC, 1991, p. 5-40.

14.   Seamon, K, and Daly JW. Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein. J Biol Chem 256: 9799-9801, 1981[Abstract/Free Full Text].

15.   Seamon, K, Vaillancourt R, Edwards M, and Daly JW. Binding of [3H]forskolin to rat brain membranes. Proc Natl Acad Sci USA 81: 5081-5085, 1984[Abstract].

16.   Shakoori, AR, Oberdorf AM, Owen TA, Weber LA, Hickey E, Stein JL, Lian JB, and Stein GS. Expression of heat shock genes during differentiation of mammalian osteoblasts and promyelocytic leukemia cells. J Cell Biochem 48: 277-287, 1992[ISI][Medline].

17.   Siddhanti, SR, and Ouarles LD. Molecular to pharmacologic control of osteoblast proliferation and differentiation. J Cell Biochem 55: 310-320, 1994[ISI][Medline].

18.   Stein, GS, Lian JB, and Owen TA. Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation. FASEB J 4: 3111-3123, 1990[Abstract].

19.   Sudo, H, Kodama HA, Amagai Y, Yamamoto S, and Kasai S. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 96: 191-198, 1983[Abstract].

20.   Suzuki, A, Oiso Y, and Kozawa O. Effect of endothelin-1 on phospholipase D activity in osteoblast-like cells. Mol Cell Endocrinol 105: 193-196, 1994[ISI][Medline].

21.   Watanabe-Tomita, Y, Suzuki A, Oiso Y, and Kozawa O. Prostaglandin E1 stimulates interleukin-6 secretion via protein kinase A in osteoblast-like cells. Cell Signal 9: 105-108, 1997[ISI][Medline].


Am J Physiol Endocrinol Metab 281(6):E1260-E1266
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society




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