©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Growth Hormone Signaling Leading to CYP2C12 Gene Expression in Rat Hepatocytes Involves Phospholipase A(*)

Petra Tollet (§) , Mats Hamberg (1), Jan- Gustafsson , Agneta Mode

From the (1) Department of Medical Nutrition, Karolinska Institute, Huddinge University Hospital, F 60 Novum, S-141 86 Huddinge, Sweden and the Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institute, S-171 77 Stockholm, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The expression of CYP2C12 is liver-specific and regulated at the transcriptional level by growth hormone (GH). In attempts to elucidate the nature of signaling molecules mediating the GH regulation of this gene in rat hepatocytes, a role for phospholipase A (PLA) as a transducer of GH-induced levels of P4502C12 mRNA was investigated. GH was shown to induce tyrosyl-phosphorylation of p42 and p44 microtubule-associated protein (MAP) kinases and to reduce the electrophoretic mobility of a 100-kDa protein, immunologically related to cPLA. These events were observed in parallel with GH-stimulated release of [H]arachidonic acid ([H]AA) from cellular phospholipids of rat hepatocytes labeled with [H]AA. These rapid effects of GH action, as well as the GH-induced expression of CYP2C12, were inhibited in cells treated with the tyrosine kinase inhibitor herbimycin A. Similarly, when the GH-induced liberation of [H]AA was blocked by the PLA inhibitor mepacrine or the Ca channel blocker verapamil, GH-induced accumulation of P4502C12 mRNA was absent. These results suggest a correlation between PLA activity and GH regulation of the CYP2C12 gene. The inhibitory effect of mepacrine on GH induction of P4502C12 mRNA was reversed by AA addition, further supporting a role for eicosanoids in the regulation of CYP2C12. Finally, inhibitors of P450-mediated AA metabolism, SKF-525A and ketoconazole as well as eicosatetraynoic acid, blocked the GH-mediated induction of P4502C12 mRNA, whereas more specific inhibitors of cyclooxygenase or lipoxygenase metabolism did not. Based on these results, we suggest that GH signaling in rat hepatocytes, leading to increased expression of CYP2C12, involves PLA activation and subsequent P450-catalyzed formation of an active AA metabolite.


INTRODUCTION

The cytochrome P450 gene superfamily (CYP) encodes multisubstrate monooxygenases (1) , catalyzing the metabolism of various endogenous and exogenous hydrophobic compounds (2, 3, 4) . Members of the CYP2C subfamily encoding the rat liver specific enzymes P4502C7, 2C11, 2C12, and 2C13, are transcriptionally regulated by growth hormone (GH),() as demonstrated in vivo and in primary cultures of adult rat hepatocytes (5). The components of the GH signal transduction pathway and the nature of transcription factors mediating the GH regulation of these CYP2C genes are, however, largely unknown.

The cellular responses to GH are initiated by the binding of GH to its cell-surface receptor (6, 7) which belongs to the class I cytokine receptors (also known as hematopoietin receptors) (8, 9) . Several studies have demonstrated that GH signal transduction involves a tyrosine kinase-mediated cascade which leads to the phosphorylation of various cellular proteins (10, 11, 12, 13, 14) , and the tyrosine-specific protein kinase JAK2 was recently identified as a primary signaling molecule in the GH signal transduction pathway (15) . The JAK-STAT signaling pathway, first described for interferon (16) , is used by several cytokine receptors (17, 18, 19, 20, 21, 22, 23) including the GH receptor (24, 25, 26) . Another signaling pathway utilized in common by different cytokines is the activation of the kinase cascade of raf, MAP kinase kinase and MAP kinase (27, 28) . Downstream events of MAP kinase activation have recently been shown to include phosphorylation and activation of a cytosolic high molecular weight form of phospholipase A (cPLA) (29) . In line with this, several cytokine receptors have been demonstrated to activate cPLA upon ligand binding (30, 31, 32, 33) .

Since MAP kinase has been shown to become tyrosyl-phosphorylated and activated in response to GH in various cell types (34, 35, 36) including rat hepatocytes (26) , it is conceivable that GH could trigger the activation of cPLA. We have previously demonstrated that protein kinases play an important role for the GH-induced expression of P4502C12 mRNA in primary cultures of rat hepatocytes (37) . Here we have investigated whether PLA transduces GH signaling to the CYP2C12 gene in rat hepatocytes. We conclude that the GH induction of the CYP2C12 gene involves activation of PLA and, furthermore, depends on subsequent P450-catalyzed eicosanoid metabolism.


EXPERIMENTAL PROCEDURES

Animals and Materials

Sprague-Dawley rats (Alab, Stockholm, Sweden), about 8 weeks of age, were maintained under standardized conditions of light and temperature, with free access to animal chow and water. The polyclonal anti-cPLA antibody (32) was a generous gift from Dr. G. Fürstenberger (German Cancer Research Center, Heidelberg, Germany). The mouse monoclonal anti-phosphotyrosine (PY-4G10) and anti-ERK2 antibodies were purchased from Upstate Biotechnology Inc. (Lake Placid, NY) and Transduction Laboratories (Lexington, KY), respectively. Polyvinylidene diflouride membranes, molecular weight markers, and anti-rabbit and anti-mouse IgG antibodies conjugated to horseradish peroxidase were from Bio-Rad. Collagenase (type XI), insulin (24.4 units/mg), arachidonic acid, mepacrine, A23187, indomethacin, and ETYA were purchased from Sigma. Recombinant bovine GH was a generous gift from American Cyanamid Co. (Princeton, NJ). Herbimycin A, verapamil, SKF-525A, and 5,8,11-eicosatrienoic acid were obtained from Calbiochem, [5,6,8,9,11,12,14,15-H]arachidonic acid (150-230 Ci/mmol), and the ECL detection system from Amersham International plc (Aylesbury, UK), protein A-Sepharose from Pharmacia LKB (Uppsala, Sweden), Proteinase K from Merck (Darmstadt, Germany), glass-fiber filters (Whatman GF/C) from Whatman Ltd. (Madistone, UK), RNase-A and RNase-T from Boehringer-Mannheim, and 20-hydroxy-5,8,11,14-eicosatetraenoic acid from Cascade Biochem Ltd. (Reading, UK). Reagents for in vitro transcription of cRNA probes were obtained from Promega Biotech Inc.

Hepatocyte Isolation and Cell Culture

Hepatocytes were isolated and cultured on matrigel in a modified Waymouth medium containing 0.1 µg of insulin/ml as described previously (38, 39) . Treatment of the cells was carried out after 66 h in culture. Details about treatments are described in the figure legends. At harvesting of cells, the medium was first aspirated, whereupon the plates were scraped with a rubber spatula in ice-cold phosphate-buffered saline, and the suspended cells were pelleted at 750 g for 5 min.

Immunoprecipitation of cPLA

Hepatocytes were harvested in ice-cold phosphate-buffered saline containing 5 mM EDTA, 50 mM NaF, 30 mM NaPO, and 0.2 mM NaVO. The cell suspension was incubated on ice for 1 h to dissolve the matrigel, pelleted by centrifugation for 5 min at 1000 g, and lysed in a buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 50 mM NaF, 30 mM NaPO, 0.2 mM NaVO, 0.2 mM phenylmethanesulfonyl fluoride, 5 µg/ml aprotinin, 2 µg/ml leupeptin, and 2 µg/ml pepstatin. The cell lysate was cleared by centrifugation for 30 min at 15,000 g. Immunoprecipitation was performed by incubation of 3 ml of lysate (8 mg of protein/ml) with 20 µl of a polyclonal anti-cPLA antibody overnight at 4 °C. The immune complexes were precipitated by incubation with 50 µl of protein A-Sepharose for 3 h.

Western Blotting

Whole cell lysates (100-200 µg of protein), prepared as described by Campbell et al.(35) and passed through a 23-gauge needle five times, or immunoprecipitated proteins, prepared as described above, were resolved by SDS-PAGE on 12 or 6% polyacrylamide gels and transferred to polyvinylidene difluoride membranes with a Trans-Blot SD semi-dry transfer cell (Bio-Rad). The membranes were blocked for 1 h in Tris-buffered saline (TBS; 10 mM Tris, pH 8.0, 150 mM NaCl) containing 0.05% (v/v) Tween-20 and 0.25% (w/v) gelatin (TBSTG), incubated with either anti-phosphotyrosine, anti-ERK2, or anti-cPLA antibodies in TBSTG for 1-2 h, washed three times for 15 min with TBSTG, and incubated with secondary antibodies for 1 h in TBSTG. After an additional washing step antibody binding was visualized using an ECL detection system from Amersham. Analysis of [H]Arachidonic Acid Release-Hepatocytes were labeled for 24 h with 1 µCi/ml [H]arachidonic acid ([H]AA) in Waymouth medium containing 0.1% bovine serum albumin. The mean total radioactivity incorporated at time 0 was 3.1 ± 0.4 10 dpm/dish. The labeling medium was withdrawn, and cells were extensively washed and equilibrated for 5 min prior to addition of GH. When the effects of herbimycin A, verapamil, or mepacrine were investigated, the cells were incubated for 20 h (herbimycin A), 60 min (verapamil), or 20 min (mepacrine) in the presence of the inhibitor prior to GH addition. Aliquots (0.2 ml) of medium (containing 0.1% bovine serum albumin) were subsequently removed, without replacement, at the indicated times. The samples were centrifuged, to clear the medium from cells withdrawn with the media, prior to quantification of released [H]AA and its metabolites by scintillation counting. At the conclusion of the experiment, the cells were harvested and solubilized for determination of total [H]AA incorporation. Counts were corrected for volume and total incorporation and, after subtracting the time 0 values, [H]AA release was expressed as disintegrations/min/dish.

DAG Mass Assay

The total amount of extractable DAG was analyzed by using a DAG mass assay system from Amersham, based on a method described by Preiss et al.(40) . The assay was performed according to the manufacturer, with lipid extracts prepared by the procedure of Bligh and Dyer (41) .

Solution Hybridization

Total nucleic acids were prepared from pooled cells from three to five culture dishes, as described previously (39) . The concentration of nucleic acids in total nucleic acid samples was measured spectrophotometrically, and the DNA concentration was quantified using a fluorometric assay (42) .

Levels of P4502C12 mRNA and IGF-I mRNA were analyzed using specific S-UTP-labeled cRNA probes in solution hybridization assays, as described previously (39, 43) . Quantification of the mRNA species was achieved by comparison with standard curves obtained from hybridizations to liver total nucleic acids, calibrated to known amounts of in vitro synthesized mRNA. Samples were analyzed in triplicate, and the results are expressed as attomoles of mRNA per µg of DNA. The interassay variations were controlled by using internal total nucleic acids standards prepared from normal livers. The interassay variation averaged 10%.

Statistical Analysis

All experiments were performed two to five times, with cells obtained from different rats. Results are expressed as the average of two experiments or, when more than two identical experiments were carried out, as the mean ± S.E. Ranges (broken error bars) are given where the results are expressed as the average of two experiments.

Data analysis was performed using Student's t test or one-way analysis of variance, followed by Fisher's least significant difference test, when more than two samples were compared. Differences were considered to be statistically significant at p < 0.05.


RESULTS

To investigate whether GH signaling in rat hepatocytes involves MAP kinase and cPLA, GH dependent phosphorylation of these enzymes was studied by immunoblotting of proteins from rat hepatocytes cultured in the absence or presence of GH. Western blot analysis with phosphotyrosine antibody revealed GH stimulated tyrosyl-phosphorylation of proteins with the apparent molecular masses of 42 (p42) and 44 (p44) kDa (Fig. 1A), indicating phosphorylation and activation of MAP kinases (ERK2 and ERK1, respectively). Indeed, these proteins comigrated with 42 and 44 kDa proteins which were recognized by an anti-ERK2 antibody (data not shown). This observation might implicate GH-mediated activation of signal transducers downstream of these kinases, such as cPLA. When immunoblot analysis of cPLA was performed, a 100-kDa protein (p100) was detected which showed reduced electrophoretic mobility in GH treated cells (Fig. 1B). Decreased electrophoretic mobility of cPLA has previously been observed in agonist treated cells (33, 44) , or upon in vitro phosphorylation of the cPLA protein by p42 MAP kinase (29, 45) . The predicted molecular mass of cPLA is 85 kDa (46, 47) ; however, the apparent size determined by gel electrophoresis has been reported to be slightly higher (95-110 kDa) (31, 32, 44, 48, 49, 50) . The GH-dependent decrease in electophoretic mobility of p100 was blocked in the presence of the tyrosine kinase inhibitor herbimycin A (Fig. 1B), previously shown to block GH-dependent tyrosyl-phosphorylation of the GH receptor (14) . The GH-stimulated tyrosyl-phosphorylation of p42 and p44 was also inhibited (Fig. 1A).


Figure 1: GH mediated tyrosyl-phosphorylation of MAP kinases and mobility shift of cPLA in rat hepatocyte. Rat hepatocytes were cultured in the absence or presence of GH (50 ng/ml) for 30 min with or without herbimycin A (2 µM) (H) pretreatment, as indicated in the figure. A, cells were harvested in SDS-PAGE loading buffer, proteins subjected to SDS-PAGE (12%), transferred to polyvinylidene difluoride membrane, immunoblotted with a monoclonal phosphotyrosine antibody, and developed by using the Amersham ECL system. B, cells were harvested and lysed as described in the materials and methods section, immunoprecipitated with a polyclonal cPLA antibody, subjected to SDS-PAGE (6%), and immunoblotted using the same cPLA antibody.



Decreased electrophoretic mobility of cPLA has been shown to be associated with increased enzymatic activity (44) . As demonstrated in Fig. 2A, GH stimulated the release of [H]AA from rat hepatocytes labeled with [H]AA. A significant increase in the liberation of [H]AA from cellular phospholipids was evident 2 min after GH addition, and a 6-fold induction was observed after 20 min. The release of [H]AA into the medium was inhibited by herbimycin A (Fig. 2B), indicating a correlation between inhibited tyrosyl-phosphorylation of p42 and p44, blocked electrophoretic mobility shift of p100, and blocked PLA activity. Moreover, the GH-induced liberation of [H]AA was blocked when the hepatocytes were pretreated with the PLA inhibitor mepacrine (51) (Fig. 2B).


Figure 2: Release of [H]AA from hepatocytes treated with GH or A23187, or GH in the absence or presence of herbimycin A, verapamil, or mepacrine. Primary cultures of rat hepatocytes were labeled for 24 h with 1 µCi/ml [H]AA prior to the different treatments. A, release of [H]AA and its metabolites from control () and GH-treated (50 ng/ml) () cells was measured at different time-points. B, release of [H]AA from control (C) cells and cells treated for 20 min with A23187 (10 µM) and GH (50 ng/ml) in the absence or presence of herbimycin A (2 µM), verapamil (150 µM), or mepacrine (40 µM) was determined. The cells were pretreated for 20 h with herbimycin A (H), for 60 min with verapamil (V), and for 20 min with mepacrine (M) prior to the addition of GH. A23187 and herbimycin A were dissolved in MeSO, and verapamil and mepacrine were dissolved in ethanol. Inset, dose-response curve of mepacrine on GH-stimulated liberation of [H]AA. Data are given as disintegrations/min released [H]AA per dish. Each point is the mean ± S.E. of triplicate determinations in five (A) or four (B) separate experiments.



The requirement of Ca for full activity of most PLA enzymes is well documented (29, 44, 47, 49, 52) . The effect of the Ca channel blocker verapamil on the GH-induced liberation of [H]AA was therefore investigated. As shown in Fig. 2B, pretreatment of the cells with verapamil inhibited the GH-stimulated phospholipase activity, whereas treatment of the hepatocytes with the Ca-mobilizing agent A23187 increased the levels of [H]AA in the medium. Taken together, these results show that GH stimulates release of eicosanoids from primary cultures of rat hepatocytes, in a tyrosine kinase- and Ca-dependent manner, which could be due to increased cPLA activity.

To examine whether the observed GH-dependent increase in eicosanoid production is involved in the GH regulation of the CYP2C12 gene, the effect of mepacrine on GH-induced steady state mRNA levels of P4502C12 was investigated. The mRNA expression from another GH-regulated gene in hepatocytes, the IGF-I gene (39, 53) , was studied in parallel. The time courses of P4502C12 and IGF-I mRNA induction, in the absence or presence of mepacrine (40 µM), are shown in Fig. 3A. After 9 h of GH treatment, the induced expression of P4502C12 mRNA was reduced from 7.4-fold to 1.5-fold when mepacrine was present. The GH-dependent increase in IGF-I mRNA was also inhibited (from 5-fold to 2.5-fold). When dose-response studies were performed, a slight difference in mepacrine sensitivity was observed between the expression of P4502C12 and IGF-I mRNA (Fig. 3B). Pretreatment of the cells with 30 µM mepacrine caused a 67% reduction of the P4502C12 mRNA levels, whereas the IGF-I expression was reduced by 47%. As shown in the insert of Fig. 3B, the GH-independent induction of IGF-I mRNA by 8-bromo-cAMP (37) was not affected by mepacrine at concentrations up to 30 µM; however, at 40 µM, the mRNA levels were reduced by 82%. Thus, at the highest dose of mepacrine (40 µM) other cellular mechanisms than AA release might be affected.


Figure 3: Steady-state mRNA levels of P4502C12 and IGF-I in GH-treated hepatocytes in the absence or presence of mepacrine. A, Time course induction of P4502C12 ( and ) and IGF-I ( and ) mRNA expression by GH, in the absence ( and ) or presence ( and ) of mepacrine (40 µM). B, dose-response curves of mepacrine on GH-stimulated P4502C12 () and IGF-I () mRNA levels after 8 h of treatment. Inset, dose-response curve of mepacrine on 8-bromo-cAMP-stimulated IGF-I mRNA levels. Mepacrine, dissolved in ethanol, was added 20 min prior to GH or 8-bromo-cAMP (100 µM) treatment. Data are expressed as fold induction compared to control cells receiving vehicle only, and represent (A) average ± ranges, or (B) mean ± S.E. of triplicate determinations in two or four separate experiments, respectively.



Since higher doses of mepacrine have been demonstrated to inhibit the actions of both phospholipases A and C, by forming complexes with the phospholipid substrate (51) , we investigated the effect of mepacrine on phospholipase C activity. The dose of mepacrine (40 µM), which completely blocked the effect of GH on [H]AA release and P450 mRNA induction, had no significant effect on phospholipase C activity, as judged from the unaffected GH stimulation of DAG production (Fig. 4).


Figure 4: Time course of induction of DAG formation by GH in the absence or presence of mepacrine. The production of DAG in control cells () and cells treated with GH (50 ng/ml) in the absence () or presence () of mepacrine (40 µM) was analyzed at different time points. Results are expressed as nanomoles of DAG per dish. Each point is the mean ± S.E. of duplicate determinations in three separate experiments.



If PLA activation is involved in the GH regulation of P4502C12 or IGF-I, the inhibitory effect of mepacrine should be reversed by the product of PLA activity, AA. As shown in Fig. 5, the mepacrine-dependent inhibition of GH-stimulated P4502C12 mRNA expression was partially rescued by the addition of 10 µM AA to the cells. This was not observed if AA was substituted with linoleic acid or docosahexaenoic acid (data not shown). A complete restoration of the GH effect was observed with repetitive additions of AA to the cells, 100 nM added every 3 h over the 9-h treatment time. AA by itself, regardless of how it was added, did not affect the expression of CYP2C12 (data not shown). This may be due to activation of more than one signaling pathway by GH, which somehow converge on the CYP2C12 gene. The rescuing effect seen after AA addition to the cells was specific for P4502C12 since IGF-1 mRNA expression was not restored (Fig. 5). This may be interpreted to mean that the inhibitory effect of mepacrine on IGF-I mRNA expression involves other mechanisms in addition to PLA inhibition. Taken together, these results suggest an important role for PLA and AA in the GH-mediated regulation of P4502C12, whereas the GH induction of IGF-I might be more dependent on other signaling molecules.


Figure 5: Effect of AA on mepacrine inhibited GH-dependent P4502C12 and IGF-I mRNA expression. P4502C12 (black bars) and IGF-I (gray bars) mRNA levels were determined in cells treated for 8 h with GH and with combinations of mepacrine (40 µM) and AA, as indicated in the figure. Data are expressed as -fold induction over appropriate control, and represent the mean ± S.E. of four experiments. *p < 0.05, ***p < 0.001 compared to mepacrine/GH-treated cells.



Since the GH-stimulated release of [H]AA was found to be inhibited by the tyrosine kinase inhibitor herbimycin A or the Ca channel blocker verapamil, these agents should also affect the GH-induced accumulation of P4502C12 mRNA if PLA is involved in the GH regulation of P4502C12. The GH- mediated increase in P4502C12 mRNA levels was dose-dependently inhibited by both herbimycin A (Fig. 6A) and verapamil (Fig. 6B). Interestingly, GH-stimulated expression of the IGF-I gene appeared more sensitive toward tyrosine kinase inhibition than the CYP2C12 gene, whereas the opposite was observed after blockage of Ca channels. Addition of 1 µM herbimycin A, prior to GH treatment of the cells, totaly inhibited the induction of IGF-I expression and reduced the CYP2C12 expression by 70%. Similar results were obtained with the tyrosine kinase inhibitor tyrphostin. The presence of herbimycin A (1 µM) did not affect the 8-bromo-cAMP-induced expression of IGF-I mRNA in the cells, only the GH-dependent induction was inhibited (data not shown).


Figure 6: Effects of herbimycin A and verapamil on GH-induced mRNA levels of P4502C12 and IGF-I. Dose-response curves of A, herbimycin A and B, verapamil on GH-stimulated P4502C12 () and IGF-I () mRNA levels after 8 h of treatment. Herbimycin A (dissolved in MeSO) was added 20 h prior to GH treatment, and verapamil (dissolved in ethanol) was added 60 min prior to GH treatment. Data are presented as -fold induction over appropriate control. Results are expressed as mean ± S.E. of triplicate determinations in three separate experiments.



Pretreatment of the cells with 200 µM verapamil almost completely blocked the GH induction of P4502C12 mRNA, whereas the IGF-I expression was reduced by only 50%. To further investigate the role of Ca in the GH regulation of P4502C12 and IGF-I, GH dose-response studies were performed in the absence or presence of the Ca-mobilizing agent A23187. As shown in Fig. 7, a dose-response shift in induction of CYP2C12 expression by GH was obtained by addition of A23187. P4502C12 mRNA was equally well induced by GH at 1 ng/ml in the presence of A23187 as by GH at 10 ng/ml in the absence A23187, suggesting a role for Ca in GH signaling to the CYP2C12 gene. Consistent with the observed difference in verapamil sensitivity between the GH induced expression of P4502C12 and IGF-I mRNA, respectively, no effect of A23187 on GH induced IGF-I mRNA levels could be observed (Fig. 7).


Figure 7: Effect of A23187 on GH induced P4502C12 and IGF-I mRNA expression. Dose-response curves of GH on P4502C12 ( and ) and IGF-I ( and ) mRNA expression in the absence ( and ) or presence ( and ) of A23187 (0.2 µM). Results are expressed as average ± ranges of triplicate determinations in two separate experiments.



AA is known to be metabolized to a spectrum of biologically active eicosanoids, which are thought to serve as regulators of intracellular events. Three distinct pathways of AA metabolism are known: the cyclooxygenase, the lipoxygenase (54, 55) and the cytochrome P450 pathways (56, 57) . By using different inhibitors of these pathways we investigated the need for active AA metabolizing enzymes in the GH-induced expression of P4502C12 and IGF-I mRNA. As demonstrated in Fig. 8A, ETYA, a nonselective inhibitior of AA metabolism (58) dose-dependently decreased the GH induced accumulation ofP4502C12 and IGF-I mRNA. Neither the cyclooxygenase inhibitor indomethacin, nor the lipoxygenase inhibitor 5,8,11-eicosatrienoic acid, shown to block the synthesis of prostaglandins (59) and leukotrienes (60) , respectively, blocked the GH-induced accumulation of these mRNA species. Instead, a small increase of the GH-induced P4502C12 mRNA levels was observed both in the presence of 10 µM indomethacin (from 5.1 ± 0.9 to 6.9 ± 0.5, when expressed as -fold induction over control) and 1 µM 5,8,11-eicosatrienoic acid (from 5.9 ± 0.5 to 7.1 ± 0.8). Pretreatment of the hepatocytes with the P450 inhibitor SKF-525A caused a dose-dependent decrease in the GH-stimulated P4502C12 and IGF-I mRNA expression (Fig. 8B). Again, a difference in sensitivity toward inhibition of AA metabolism was observed, with P4502C12 expression being more sensitive than that of IGF-I. Similar results were obtained with the P450 inhibitor ketoconazole, whereas metyrapone was less effective (data not shown). As shown in Fig. 9, the rescuing effect of AA on the mepacrine inhibited GH induction of P4502C12 mRNA (cf. above and Fig. 5) was blocked by ETYA or SKF-525A, indicating that the rescuing effect of AA is mediated by an eicosanoid formed via P450-catalyzed metabolism of AA.


Figure 8: Dose-response of ETYA and SKF-525A on GH induced P4502C12 and IGF-I mRNA expression. P4502C12 () and IGF-I () mRNA levels were determined in hepatocytes treated with GH for 8 h in the presence or absence of different concentrations of A, ETYA or B, SKF-525A, both dissolved in ethanol. The inhibitors were added 20 min prior to GH treatment. Data are expressed as -fold induction over control and represent mean ± S.E. of three experiments.




Figure 9: Effects of ETYA and SKF-525A on AA induced P452C12 mRNA expression. P4502C12 mRNA levels were determined in cells treated for 8 h with GH alone or in combination with mepacrine (40 µM), AA (10 µM), ETYA (50 µM), or SKF-525A (50 µM) as indicated in the figure. Results are expressed as -fold induction over appropriate control and represent the average of two experiments with range.




DISCUSSION

It was recently reported that cPLA is serine-phosphorylated and activated by p42 MAP kinase in agonist-treated cells (29) . The GH-increased tyrosyl-phosphorylation of MAP kinase in rat hepatocytes (26) suggests that GH signaling in hepatocytes include MAP kinase, as well as activation of signal transducers downstream of these kinases, such as cPLA. Results obtained in this study demonstrate simultaneous GH-dependent tyrosyl-phosphorylation of p42 MAP kinase, reduced electrophoretic mobility of a 100-kDa protein immunologically related to cPLA, and liberation of [H]AA from labeled cells, indicating a role for cPLA in GH signal transduction in rat hepatocytes.

The GH-stimulated release of AA from cultured hepatocytes does not per se prove a GH-dependent activation of the cytosolic 85-kDa PLA. The increase in [H]AA release could result from any PLA activity but also from the sequential enzymatic actions of PLA and lysophospholipase, or from phospholipases C or D and diglyceride lipase. The PLA route of AA formation was favored by the observation that agents known to block the enzymatic activity of PLA, such as mepacrine and verapamil, inhibited the GH-stimulated release of AA from the cells. The most compelling evidence for cPLA involvement was the demonstration that the GH-stimulated liberation of AA was paralleled by a GH-induced mobility shift of a protein with the expected molecular mass of cPLA (95-110 kDa) in Western blotting with an anti-cPLA antibody.

cPLA has been shown to translocate from cytosol to membranes in a Ca-dependent manner (61) , and therefore Ca-dependent activation of cPLA presumably occurs through enhanced enzyme-substrate interaction. This is in line with our observation that liberation of AA could be induced by the Ca-mobilizing agent A23187 and inhibited by the Ca channel blocker verapamil. Similarly, the GH-induced expression of CYP2C12 was augmented by A23187 and blocked by verapamil. Thus, a correlation between Ca levels, eicosanoid production, and GH signaling to the CYP2C12 gene seems to exist. Interestingly, Schwartz et al.(62) have shown that GH increases intracellular levels of Ca in fat cells, an effect which has been suggested to be due, at least partially, to the activation of Ca channels of the L-type. These authors have also shown that verapamil inhibits metabolic effects of GH (63, 64). Whether GH affects Ca influx in hepatocytes has yet to be determined.

As discussed above, our results suggest that GH activates a cPLA in rat hepatocytes. However, it should be mentioned that a 14-kDa type II PLA has been cloned from rat liver, and the mRNA encoding this enzyme is detected in freshly isolated hepatocytes (65) . The type II 14-kDa PLA appears to function both as a cell-associated enzyme and extracellularly when released in response to proinflammatory mediators. It is well known that the catalytic activity of this secreted enzyme is dependent on Ca(61) . It can therefore not be excluded that also other types of PLA enzymes than cPLA contribute to the increased AA release after A23187 or GH treatment of hepatocytes.

PLA enzymes catalyze the hydrolysis of the sn-2-acyl chain of phospholipid substrates to yield fatty acids and lysophospholipids. This reaction is of particular significance when the fatty acid is AA, since AA and its metabolites have been shown to act as first and second messengers affecting a number of cellular processes, including blood clotting, inflammation, vascular tone, renal function, and reproduction (55, 56, 66, 67) . Furthermore, eicosanoids have recently been implicated in the regulation of various genes, such as the murine stearoyl-CoA desaturase gene (68) , the rat CYP4A1(69) , and human heat shock genes (70). AA and eicosanoids, derived mainly through the cyclooxygenase and lipoxygenase pathways, have been shown to stimulate the expression of proto-oncogenes in various cell systems (71, 72, 73, 74, 75) . Neither products of the the cyclooxygenase nor the lipoxygenase pathway appears to be involved in the regulation of CYP2C12, since inhibitors of these pathways did not reduce GH-stimulated levels of P4502C12 mRNA. However, SKF-525A and ketoconazol, as well as ETYA, inhibitors of P450 dependent oxidation of AA into epoxidated and hydroxylated metabolites (76, 77) , significantly decreased the GH-induced expression of CYP2C12.

The liver has mainly been implicated in the inactivation of different eicosanoids but there is also evidence for the formation of active metabolites (56) . The P450 enzyme system has been shown to catalyze the formation of six regioisomeric cistrans-conjugated hydroxyeicosatetraenoic acids, four regioisomeric epoxyeicosatrienoic acids (EETs), as well as and -1 alcohols (57) . Some of the constitutively expressed P450 forms in rat liver which have been shown to metabolize AA (see Ref. 78 and references therein) are expressed in primary rat hepatocytes when cultured as in this study (79) . Biological effects exerted by eicosanoids derived through the P450 pathway have mainly been studied in extrahepatic tissues. However, 14,15-EET has been shown to increase cytosolic Ca concentrations and to stimulate glycogenolysis in rat hepatocytes (80) . In attempts to identify the eicosanoid(s) that mediates the GH signaling to the CYP2C12 gene, various eicosanoids that could be formed by P450-catalyzed metabolism of AA were added to the hepatocytes. Different concentrations of the four regioisomeric EETs, 5,6-, 8,9-, 11,12-, and 14,15-EET, the 14,15-dihydroxyeicosatrienoic acid, or 20-hydroxyeicosatetraenoic acid were added according to the protocols used for AA treatments (cf. above). In some but not all experiments, the 11,12- and the 14,15-EETs were found to have a similar effect as AA on the P4502C12 mRNA expression (data not shown). The lack of consistent results could be due to rapid inactivation of these compounds when added to the cells, and further work is required to resolve what eicosanoid is involved.

Based on results obtained in this study, we suggest that GH signaling in rat hepatocytes leading to increased expression of CYP2C12 includes cPLA activation and subsequent P450-catalyzed formation of an active AA metabolite. This PLA- and P450-dependent signaling molecule was shown to be necessary but not sufficient for GH regulation of P4502C12 mRNA levels, indicating the importance of other GH-activated factors, yet to be identified. Possible candidates for such factors include members of the family of STAT proteins, recently shown to be tyrosyl-phosphorylated and activated in response to GH (24, 25, 26) . The observation that GH regulation of IGF-I expression was more dependent on tyrosine kinase activity, and less sensitive toward inhibition of eicosanoid production or Ca influx, indicates that GH signaling leading to increased expression of the IGF-I gene is different from signaling to the CYP2C12 gene. It is obvious that GH can activate various signaling molecules and that the nature and importance of different signaling events have to be identified for each specific target gene. The existence of different GH receptor signal transduction mechanisms could be a prerequisite for the broad range of physiological actions exerted by GH.


FOOTNOTES

*
This work was supported by grants from the Swedish Medical Research Council (no. O3X-06807), the Novo Nordisk Foundation, the Magnus Bergvall Fund, and the Karolinska Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 46 8 779 71 31; Fax: 46 8 711 66 59.

The abbreviations used are: GH, growth hormone; MAP, microtubule-associated protein; PLA, phospholipase A; cPLA, cystolic phospholipase A; AA, arachidonic acid; PAGE, polyacrylamide gel electrophoresis; IGF-I, insulin-like growth factor I; ETYA, eicosatetraynoic acid; EET, epoxyeicosatrienoic acid; DAG, diacylglycerol.


ACKNOWLEDGEMENTS

We are indebted to Dr. G. Fürstenberger for the generous gift of the cPLA antibody.


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