A role for protein kinases in the growth hormone regulation of cytochrome P4502C12 and insulin-like growth factor-I messenger RNA expression in primary adult rat hepatocytes.

A Role for Protein Kinases in the Growth Hormone Regulation of Cytochrome P4502C12 and InsulinLike Growth Factor-I Messenger RNA Expression in Primary Adult Rat Hepatocytes

Petra Toilet, Catherine Legraverend, Jan-Ake Gustafsson, and Agneta Mode Department of Medical Nutrition Huddinge University Hospital F60 Novum Karolinska Institute S-14186 Huddinge, Sweden

GH is a major determinant of cytochrome P4502C12 and insulin-like growth factor-l (IGF-I) mRNA expression in rat liver. In the present study, a possible role for protein kinase C (PKC) in the GH-mediated regulation of these two genes was investigated. Addition of bovine GH (bGH) to cultured primary adult rat hepatocytes lead to the formation of diacylglycerol and subsequent induction of P4502C12 and IGF-I mRNA, indicating a PKC-dependent signal transduction. However, stimulation of PKC by phorbol 12myristate 13-acetate (PMA) or sn-1,2-dioctanoylglycerol treatment, in dose and time-course experiments in the presence or absence of ionomycin, failed to induce either P4502C12 or IGF-I mRNA. On the other hand, down-regulation of PKC by PMA treatment, i.e. 24 h pretreatment, attenuated the bGH induction of both P4502C12 and IGF-I mRNA. One hundred nanomolar PMA reduced the bGH-stimulated expression of both IGF-I mRNA and P4502C12 mRNA (-50%). Treatment with the potent kinase inhibitor staurosporine in combination with bGH caused a dose-dependent decrease of the bGH response with different sensitivities toward the inhibitor for the different mRNA species, IGF-I being less sensitive. These data indicate a permissive role for PKC in the GH-mediated induction of P4502C12 and IGF-I mRNA. When activators of protein kinase A, such as forskolin and 8-Br-cAMP were added to the culture medium opposite effects were observed on the mRNA levels of P4502C12 and IGF-I. The basal expression of P4502C12 mRNA decreased, whereas that of IGF-I increased, effects which were observed independent of the presence or absence of bGH.

Glucagon, a ligand of an adenylate cyclase coupled receptor was found to exert the same effects, indicating a role for a cAMP-dependent signaling pathway in the overall regulation of P4502C12 and IGF-I mRNA expression in hepatocytes. (Molecular Endocrinology 5: 1351-1358, 1991)

INTRODUCTION GH has a broad range of physiological actions, including major effects on somatic growth and intermediary metabolism (1, 2). A major target organ of GH action is the liver, where GH regulates the expression of a variety of proteins, ranging from hormone and growth factor receptors to secretory proteins and enzymes (3). Our interest has been focused on GH regulation of hepatic cytochrome P450 enzymes. We have recently been able to show that GH alone is able to induce P4502C12 expression at a pretranslational level in adult primary rat hepatocytes maintained in a serum-free medium (4, 5). The insulin-like growth factor-l (IGF-I) gene is also induced by GH in this system. However, the GH-induced accumulation of the two mRNA species showed different kinetics and a marked difference in cycloheximide sensitivity. The signaling pathway(s) by which GH mediates these actions in hepatocytes is not known, although recently, results have started to emerge from various laboratories that may help to gain some understanding of the mechanisms involved in GH action. A primary event after GH receptor occupancy seems to be increased phosphorylation of the receptor on tyrosyl residues (6). The tyrosine kinase activity is evidently tightly associated with the receptor, and the association of GH receptors with tyrosine kinase activity has been demonstrated in various cell types from dif-

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ferent species (7), indicating a possible role of tyrosine kinase activity in the actions of GH. However, the question of whether the receptor possesses intrinsic kinase activity remains unanswered. Several investigations suggest that phospholipid hydrolysis is of importance in the GH signaling process. Studies of basolateral membranes isolated from canine kidney have shown that GH stimulates the formation of diacylglycerol (DAG) and inositol triphosphate (8), indicating that GH activates a phospholipase C (PLC)catalyzed hydrolysis of phosphatidylinositol bisphosphate. DAG formation in Ob 1771 mouse preadipocytes in response to GH has also been demonstrated, but in this cell line, GH induced breakdown of phosphatidylcholine rather than phosphatidylinositol (9). Furthermore, GH administration to freshly isolated rat hepatocytes has been reported to result in DAG formation without simultaneous formation of inositol phosphates (10). The accumulating evidence for GH-induced production of DAG indicates that protein kinase C (PKC) could be a mediator of GH effects. Indeed, the GH effects on both lipogenesis and lipolysis in rat adipose tissue have been shown to be blocked by inhibitors of PKC (11, 12). Furthermore, it has been reported that the GHinduced expression of the protooncogene c-fos is attenuated after inhibition of PKC in Ob 1771 (13) and 3T3-F442A (14) preadipocytes. The present study was undertaken in order to study a possible involvement of PKC in the GH-induced expression of P4502C12 and IGF-I mRNA in primary rat hepatocytes. We report here that the steady state levels of the mRNAs encoding these proteins are dependent on active PKC, but show differences in their sensitivity toward PKC inhibition. Neither P4502C12 mRNA nor IGF-I mRNA could, however, be induced by stimulation of PKC, indicating that another factor(s) is needed together with PKC to transduce the GH effect on these genes. In this context, a possible role for protein kinase A (PKA) was investigated. Stimulation of PKA inhibited P4502C12 mRNA and induced IGF-I mRNA expression, independently of simultaneous activation of PKC.

RESULTS AND DISCUSSION Bovine GH (bGH) Induces Expression of P4502C12 mRNA and IGF-I mRNA in Rat Hepatocytes Previous studies on the regulation of P4502C12 and IGF-I expression have mainly been carried out with human GH (hGH). However, hGH possesses both lactogenic and somatogenic properties in the rat, i.e. it binds to both the PRL and GH receptor (15). Furthermore, GH is the major inducer of PRL receptors in rat liver (16), i.e. hGH both induces and binds to the PRL receptor. There is no doubt that the hGH induction of P4502C12 (17) and IGF-I (18) constitutes a somatogenic effect, but in order to study the postreceptor

events of GH leading to induction of these two mRNA species a non-PRL receptor ligand, bGH, was used. Dose-response experiments with bGH in the primary hepatocytes showed a linear increase in P4502C12 mRNA and IGF-I mRNA up to 20 ng bGH/ml (data not shown). Subsequent experiments were carried out with 50 ng/ml. The time course of induction of P4502C12 mRNA and IGF-I mRNA by 50 ng bGH/ml is shown in Fig. 1A. After 2 h of GH treatment, increased steady state levels of P4502C12 mRNA and IGF-I mRNA were already apparent. From 4 h of GH treatment, a statistically significant induction of both P4502C12 mRNA (4fold) and IGF-I mRNA (3-fold) was evident (P < 0.001; n = 10; Student's t test). The magnitude of induction of the steady state mRNA levels of P4502C12 at 4 and 9 h was closely paralleled by the increase in the relative rate of transcription of the corresponding gene, as determined by nuclear run-on analysis (Fig. 1B). This provides direct evidence for a transcriptional activation by GH of the P4502C12 gene in primary hepatocytes and is in agreement with our previous finding that GH treatment does not increase the steady state mRNA level of P4502C12 in the presence of the transcription inhibitor actinomycin-D (5). Recently, we have also been able to show that several other members of the P4502C gene subfamily are regulated by GH at the level of transcription (Legraverend, C , A. Mode, S. Westin, A. Strom, H. Eguchi, P. G. Zaphiropoulos, and J. A. Gustafsson, manuscript in preparation), and Mathews et al. (19) have previously demonstrated that the IGF-I gene is regulated by GH at the transcriptional level. Interestingly, Yoon et al. (20) have presented evidence for a GH-activated hepatic nuclear factor interacting with a regulatory sequence in the serine protease inhibitor 2.1 gene. The activity of this as yet unidentified factor is not blocked by protein synthesis inhibition, suggesting that a preexisting factor mediates this effect of GH. This is in contrast to our findings regarding transcriptional activation of P4502C12. As shown in Fig. 1C, the presence of cycloheximide blocked the GHinduced transcription of P4502C12. If the background hybridization to the vector is taken into account, P4502C12 transcription is completely abolished in the presence of cycloheximide. We have previously shown an absolute requirement for on-going protein synthesis in the accumulation of P4502C12 mRNA (5). In the same experiments, the GH-stimulated induction of IGFI mRNA did not show cycloheximide sensitivity. Thus, it would appear that GH can activate more than one signaling pathway in hepatocytes. Bovine GH Stimulates Formation of DAG Treatment of the cultured primary hepatocytes with 50 ng/ml (2.2 nM) bGH for 30 sec resulted in a 5-fold increase in DAG production (Fig. 2). This effect of bGH on DAG formation was comparable to that observed with epidermal growth factor (Fig. 2), which has been shown to stimulate PLC and DAG production (21). In a recent study, Johnson et al. (10) demonstrated a 1.4-


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GH

pGEM

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P4502C12

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Fig. 1. Time Course of Induction of P4502C12 and IGF-I mRNAs by bGH in Cultured Primary Hepatocytes A, Steady state mRNA levels of P4502C12 mRNA p and • ) and IGF-I mRNA (O and • ) in the presence ( • and • ) or absence P and O) of bGH measured by solution hybridization. Results are expressed as attomoles of mRNA per ^g DNA. B, Relative rate of transcription of P4502C12 after 4 or 9 h of bGH treatment, as measured by nuclear run-on analysis. The autoradiographs of hybridized filters {bottom) were scanned, and the P4502C12/j8-actin transcript ratio was plotted after normalization against untreated cells (top). C, Transcriptional activation of the P4502C12 gene by bGH in the presence or absence of cycloheximide. Cycloheximide (2 ng/m\) was added together with bGH, and the cells were harvested 3 h later. Cells were treated with bGH (50 ng/ml) from 66 h of culture age and harvested at the indicated time points. Total nucleic acid samples or nuclei were prepared and analyzed as described in Materials and Methods.

fold increase in DAG formation when freshly isolated hepatocytes were treated with 100 nM hGH. The researchers did not find evidence for a simultaneous production of inositol phosphates, indicating a source of DAG other than phosphatidylinositol. In adipocytes, GH has been shown to stimulate PLC-catalyzed hydrolysis of phosphatidylcholine (9), but further studies are needed to find the source of DAG produced as an early response to GH in the liver. Down-Regulation of PKC Attenuates Induction of P4502C12 mRNA and IGF-I mRNA by GH The second messenger DAG is known to activate PKC, and in order to investigate whether PKC is involved in the GH-stimulated induction of P4502C12 and IGF-I mRNA, we took advantage of the modulatory effects of phorbol esters on PKC. Prolonged treatment with phorbol esters has been shown to reduce cellular levels of PKC (22, 23). When the primary hepatocytes were preincubated with 4/3-phorbol 12-myristate 13-acetate (PMA) for 24 h, the ability of GH to induce P4502C12 and IGF-I mRNA was reduced at both 4 and 8 h of GH treatment (Fig. 3). The effect of PMA was dose dependent, and approximately 50% inhibition of the GH effect {i.e. after subtracting the basal level of expression) was observed at 100 nM PMA for both P4502C12 and IGF-I (Fig. 3). When considering the results of down-modulation

experiments, the potential effects of phorbol ester pretreatment on cell functions other than PKC content must be taken into account. To exclude the possibility that a toxic effect of PMA was responsible for the obtained results, the cells were pretreated with equimolar concentrations of the 4a- or the 4/3-isomer of phorbol 12,13-didecanoate. The 4«-isomer, inactive in PKC modulation (24), did not interfere with the GH induction of P4502C12 mRNA or IGF-I mRNA, whereas the active 4j8-phorbol congener was as efficient as PMA in this context (data not shown). As shown in Fig. 4, the potent kinase inhibitor staurosporine (25) also interfered with the ability of GH to induce the two mRNA species. In contrast to PMA, the dose of staurosporine required for inhibition differed for P4502C12 mRNA and IGF-I mRNA. About 10-fold higher concentrations of staurosporine were required to inhibit IGF-I mRNA induction by GH to the same extent as that of P4502C12 mRNA. Treatment of the cells with GH for 4 h in the presence of staurosporine gave identical results as 8 h of treatment (data not shown). Cytotoxic effects of staurosporine cannot be excluded; however, the fact that the basal expression of IGF-I mRNA was not affected up to 1 HM of the kinase inhibitor makes these an unlikely explanation. Activation of PKC Does not Induce Expression of P4502C12 or IGF-I mRNA In light of the results from down-modulation or inhibition of PKC in the primary hepatocytes, attempts were made


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Fig. 2. Time Course of Induction of DAG Formation in bGHand Epidermal Growth Factor-Treated Rat Hepatocytes At 66 h of culture age, cells were incubated in the absence or presence of bGH (•; 50 ng/ml) or EGF (O; 50 ng/ml). Cells were harvested at the indicated time points, and extractable DAG was analyzed as described in Materials and Methods. Results are expressed as fold induction compared to that in untreated cells. Untreated hepatocytes contained 0.80 ± 0.09 nmol extractable DAG/five culture dishes (n = 5).

500

Fig. 4. Effects of Staurosporine on bGH Induction of Steady State mRNA Levels of P4502C12 and IGF-I At 66 h of culture age, cells were incubated in the absence or presence of different concentrations of staurosporine. Twenty minutes later, bGH was added to the culture medium. Cells were harvested after 8 h, and tNA samples were prepared and analyzed for P4502C12 mRNA (•) and IGF-I mRNA (U) levels by solution hybridization. Results are expressed as fold induction compared to that in untreated cells.

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PMA(nM) Fig. 3. Effects of PMA Pretreatment on bGH Induction of Steady State mRNA Levels of P4502C12 and IGF-I At 66 h of culture age, cells were incubated in the presence of different concentrations of PMA. Twenty-four hours later, the cells were incubated with or without bGH for 4 or 8 h in the presence or absence of PMA. Cells were harvested, and tNA samples were prepared and analyzed for P4502C 12 mRNA (•) and IGF-I mRNA P ) levels by solution hybridization. Results are expressed as fold induction compared to that in untreated cells.

PMA

r DiC8

PMA+I

DiC8+l

Fig. 5. Effects of PKC Activators on Steady State mRNA Levels of P4502C12, IGF-I, and c-fos At 66 h of culture age, cells were incubated in the absence or presence of bGH (GH; 50 ng/ml), PMA (100 nM), DiC8 (100 nM), or ionomycin (I; 200 nM), or the combinations of PMA or DiC8 with ionomycin. Treated and untreated cells were harvested after 30 min or 8 h, and tNA samples were prepared and analyzed for P4502C12 mRNA (•), IGF-I mRNA P ; 8 h), and c-fos mRNA (•; 30 min) levels by solution hybridization. Results are expressed as fold induction compared to that in untreated cells. Inserted is the time course of induction of cfos mRNA expression by bGH and PMA.

to induce P4502C12 mRNA and IGF-I mRNA expression by stimulation of PKC. However, when the cells were treated with various doses of PMA (1 -100 nM) or with the more physiological PKC activator sn-1,2-dioctanoylglycerol (DiC8; 100 nM to 100 HM) (26) for 4 or 8 h, neither P4502C12 mRNA nor IGF-I mRNA expression was stimulated (Fig. 5). Since several of the characterized PKC isozymes have been shown to be acti-



vated by a synergistic action of an increase in the intracellular Ca2+ concentration and the formation of DAG (27), the Ca2+ ionophore ionomycin (200 nM) (28) was added together with PMA or DiC8. These combined treatments were also ineffective in inducing P4502C12 mRNA or IGF-I mRNA. To verify that the treatments strmulated PKC activity, cells were harvested at earlier time points and analyzed for c-fos mRNA expression, which is known to be stimulated by PKC activation (29). As shown in Fig. 5, c-fos was induced by the addition of PMA, DiC8, or ionomycin to the culture medium. GH was, likewise, found to elicit a transient expression of c-fos mRNA in the cells, an effect that previously has been demonstrated in preadipocytes (13,14) and osteoblasts (30). Also, in early stages of experimental rat liver carcinogenesis, GH has been shown to affect the expression of c-fos mRNA, albeit in the opposite direction (31). Multiple doses of DiC8 given over 24 h, as opposed to a single dose, have been shown to be required for posttranscriptional regulatory mechanisms of importance for the AP-1 enhancer activity in U937 monoblastic leukemic cells (32). Given the pulsatile secretion of GH (33) and the rapid turnover of its receptor (34), it is tempting to suggest that similar mechanisms might be involved in the GH-mediated effects, i.e. multiple GHstimulated increments in DAG formation might be required to induce P4502C12 or IGF-I mRNA expression. Therefore, repeated additions of DiC8 were made to the cell cultures (100 HM every third hour for 9 h) in the presence or absence of ionomycin. Again, no stimulatory effect on P4502C12 or IGF-I mRNA expression could be detected (data not shown). It cannot be excluded that some other regimen of PKC stimulation would trigger a response. However, the accumulated data would, rather, indicate a permissive role for PKC in mediating the GH induction of P4502C12 mRNA and IGF-I mRNA, and that another factor(s) is required together with PKC. Possibly, a PKC-independent pathway is triggered by GH, which in itself might be responsible for the small effect of GH observed in the PMApretreated cells (Fig. 3, compare 4 and 8 h). Activation of PKA Inhibits P4502C12 and Stimulates IGF-I mRNA Expression As different signal transduction pathways c?n interact to regulate cell function (35, 36), and since staurosporine also inhibits other kinases beside PKC (37), the possible involvement of cAMP-dependent protein kinase (PKA) was investigated. The combination of the PKA activator forskolin (20) and PKC activators (PMA, DiC8, and ionomycin) did not induce P4502C12 mRNA (data not shown); instead, forskolin alone reduced the basal expression of P4502C12 mRNA (Fig. 6). Furthermore, when forskolin was added together with GH, the level of P4502C12 mRNA only reached 50% of that obtained by GH treatment alone. In contrast, the IGF-I mRNA level was induced 2- to 3-fold by forskolin. This effect appeared additive to the GH stimulatory effect

1355

GH

cA

F

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Fig. 6. Effects of PKA Activators on Steady State mRNA Levels of P4502C12 and IGF-I in the Presence or Absence of bGH At 66 h of culture age, cells were incubated with 8-Br-cAMP (cA; 100 MM), forskolin (F; 20 MM), or glucagon (20 Mg/ml) in the presence or absence of bGH. Cells were harvested after 8 h, and tNA samples were prepared and analyzed for P4502C12 mRNA (•) and IGF-I mRNA P) levels by solution hybridization. Results are expressed as fold induction compared to that in untreated cells.

on IGF-I mRNA expression, indicating two different pathways of IGF-I regulation. When the cAMP analog 8-bromo-cAMP (8-Br-cAMP; 100 HM) was used instead of forskolin, similar data were obtained (Fig. 6). Thus, in the primary hepatocytes the GH response per se does not seem to be affected by the second messenger cAMP, since the fold induction of P4502C12 mRNA and IGF-I mRNA caused by GH was the same in the presence or absence of cAMP stimulatory agents (Fig. 6). On the other hand, a direct effect of cAMP on the GH response still remains a possibility, since it has been shown in rat adipocytes that the GH receptor can undergo cAMP-dependent phosphorylation, which leads to reduced GH binding and, presumably, reduced cellular responses to GH (38). To our knowledge, a positive effect of cAMP on IGFI induction has not previously been described. To further study a possible PKA involvement in IGF-I regulation, 8-Br-cAMP was added to cells pretreated with PMA or staurosporine. Only staurosporine interfered with the cAMP-induced expression of IGF-I mRNA (Fig. 7). Since PMA specifically down-regulates PKC activity, whereas staurosporine also inhibits PKA activity (37), this provides further evidence for a PKA-stimulated expression of IGF-I mRNA in the hepatocytes. Glucagon Inhibits P4502C12 and Stimulates IGF-I mRNA Expression To investigate whether the different effects of increased cellular levels of cAMP on P4502C12 mRNA and IGF-I mRNA expression could be elicited by a plausible physiological ligand for an adenylate cyclase-coupled receptor, the cells were treated with glucagon (20 Mg/ml), alone or in combination with GH. Indeed, glucagon had


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Fig. 7. Effects of PMA or Staurosporine Pretreatment on 8Br-cAMP Induction of IGF-I mRNA Steady State Levels Cells were incubated with different concentrations of PMA (at 66 h of culture age) or staurosporine (at 90 h of culture age) for 24 h (PMA) or 20 min (staurosporine). 8-Br-cAMP (100 nM) was added to pretreated and untreated cells. Eight hours later, cells were harvested, and tNA were prepared and analyzed for IGF-I mRNA levels by solution hybridization. Results are expressed as fold induction compared to that in untreated cells.

the same effects as forskolin and 8-Br-cAMP (Fig. 6). Interestingly, glucagon has previously been shown to affect P450-catalyzed steroid metabolism in rat hepatocytes (39). In view of the opposed effects of glucagon and insulin on storage and metabolism of fuels in the liver (40), it is of potential interest that IGF-I mRNA was found to be induced by glucagon, since we have demonstrated that the GH-induced expression of IGF-I mRNA is potentiated 2-fold by insulin (5). Thus, glucagon, directly, and insulin, indirectly, affect IGF-I mRNA expression in the same direction. This could possibly be interpreted as a demand for relatively well maintained hepatic levels of IGF-I, which would not vary with nutritional status. Whether other cAMP stimulatory agonists acting on the liver also participate in the regulation of P4502C12 and IGF-I mRNA levels remains to be tested. At present, at least glucagon can be added to the list of hormones, including GH, insulin, IGF-I, and thyroid and glucocorticoid hormones, that all directly or indirectly affect the mRNA levels of P4502C12 and IGF-I mRNA (5). In summary, several lines of evidence indicate that GH can trigger more than one signaling pathway of importance for gene transcription. PKC activity appears permissive for the GH-mediated induction of P4502C12 and IGF-I mRNA, but some as yet unidentified factor(s)/ kinase(s) acts in concert with PKC. Importantly, a role for the cAMP-dependent signal transduction pathway can be inferred in the overall regulation of P4502C12 and IGF-I mRNA expression in hepatocytes. MATERIALS AND METHODS Animals and Materials Adult male 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. Collagenase (type IV) was purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant bGH was a generous gift from American Cyanamid Co. (Wayne, NJ). Insulin (24.4 U/mg), epidermal growth factor, cycloheximide, PMA, 4a-phorbol 12,13-didecanoate, 4/3-phorbol 12,13-didecanoate, staurosporine, sn-1,2-dioctanoylglycerol, forskolin, 8Br-cAMP, and glucagon were purchased from Sigma Chemical Co. lonomycin was obtained from Calbiochem (La Jolla, CA), proteinase-K from Merck (Darmstadt, Germany), glass-fiber filters (Whatman GF/C) from Whatman Ltd. (Madistone, Kent, United Kingdom), and RNase-A and RNase-Ti from Boehringer-Mannheim (Mannheim, Germany). Reagents for in vitro transcription of cRNA probes were obtained from Promega Biotech (Madison, Wl). For detection of c-fos mRNA, a cRNA probe was transcribed using human fos Amprobe, purchased from Amersham International pic (Aylesbury, Buckinghamshire, United Kingdom). Hepatocyte Isolation and Cell Culture Matrigel was prepared from Engelbreth-Holm-Swarm sarcoma propagated in C57BL/6 female mice and stored at - 2 0 C, as described previously (41). After thawing on ice, 100-200 n\ were evenly inoculated onto 60-mm plastic dishes and allowed to form a gel at room temperature before cell isolation. Hepatocytes were prepared by nonrecirculating collagenase perfusion through the portal vein of ether-anesthetized rats, according to the method of Bissell and Guzelian (42). Cells were seeded at a density of 3.5 x 106/dish in 3 ml standard serumfree medium (42). The medium was renewed daily. This medium is a modification of Waymouth's medium 752 containing amino acids, salts, vitamins, minerals (zinc and selenium), and insulin (1 M9/ml). All cell-medium constituents were of cell culture grade. Cultures were maintained in a humidified incubator at 37 C in an atmosphere containing 5% CO2. At harvesting of cells, the medium was aspirated from the plates, and cells were washed and scraped with a rubber spatula in ice-cold PBS and pelleted at 750 x g for 5 min. Treatment of the cells was carried out between 66-98 h of cell culture age. Solution Hybridization Total nucleic acids (tNA) were prepared from the pooled cells from five to seven culture dishes by lysis of the cells in 1 % (wt/vol) sodium dodecyl sulfate (SDS), 10 ITIM EDTA, and 20 mM Tris-HCI, pH 7.5. Digestion of samples with proteinase-K and subsequent extraction with chloroform and phenol have been described previously (43). The concentration of nucleic acids in tNA samples was measured spectrophotometrically, and the DNA concentration was quantitated using a fluorometric assay (44). Levels of P4502CI2 mRNA, IGF-I mRNA, and c-fos mRNA were analyzed using [35S]UTP-labeled cRNA probes, transcribed in vitro from respective cDNA vector construct, essentially according to the method of Melton et al. (45). The characterization of each probe and analysis of P-4502C12 (46), IGF-I (19), and c-fos (31) have previously been described. Briefly, hybridization of aliquots of tNA samples was performed in 40 MI 0.6 M NaCI, 22 mM Tris-HCI (pH 7.5), 5 mM EDTA, 0.1% (wt/vol) SDS, 1 mM dithiothreitol (DTT), formamide (probe-dependent concentration), and 15,000-20,000 cpm probe/incubation. After overnight incubation (probe-dependent temperature), the samples were exposed to RNases, and the hybrids were precipitated by the addition of 100 n\ 6 M trichloroacetic acid, collected on a glass-fiber filter, and counted in a liquid scintillation counter. Quantitations of P4502C12 mRNA and IGF-I mRNA were achieved by comparison with a standard curve obtained from hybridizations to liver tNA. This liver tNA standard was calibrated to a standard curve using known amounts of in vitro synthesized mRNA. Samples were analyzed in triplicate, and the results are expressed as attomoles of mRNA per ng DNA.


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The quantitations of c-fos mRNA are expressed as counts per min/^g DNA. The interassay variations were controlled by using internal tNA standards prepared from normal livers (P4502C12 and IGF-I) and from livers 30 min after partial hepatectomy (c-fos). The interassay variation averaged 10%. All experiments were performed at least twice, with cells obtained from different rats. Where the results are expressed as the average of two experiments, ranges are given (broken error bars), and when more than two identical experiments were carried out, the mean ± SD {solid error bars) are shown. Nuclear Transcription Run-On Analysis Nuclei were isolated from harvested cells by homogenization in 3 vol buffer A [15 HIM HEPES (pH 7.5), 60 mui KCI, 15 mM NaCI, 2 mM EDTA, 0.5 mM EGTA, 0.15 mM spermine, 0.5 mM spermidine, 0.3 M sucrose, 1 mM DTT, and 0.8% Nonidet P40] in an all glass Dounze B homogenizer, followed by a 10fold dilution in buffer B [20 mM HEPES (pH 7.5), 15 mM KCI, 2 mM EDTA, 0.15 mM spermine, 0.5 mM spermidine, 20% glycerol, and 1 mM DTT] and subsequent centrifugation at 550 x g for 8 min. The crude nuclei pellets were resuspended in buffer C (12 ml glycerol/100 ml buffer D) layered over a 0.25vol cushion of buffer D [10 mM HEPES (pH 7.5), 15 mM KCI, 2 mM EDTA, 0.15 mM spermine, 0.5 mM spermidine, 10% glycerol, 2 M sucrose, and 1 mM DTT] and centrifuged at 24,000 rpm for 45 min at 2 C in an SW28 rotor (Beckman Instruments, Inc., Palo Alto, CA). The pelleted nuclei were washed once in TGEM [50 mM Tris-HCI (pH 8.0), 40% glycerol, and 5 mM MgCI2], resuspended in the same buffer, and counted in a Biirker chamber. Purified nuclei were stored at -HOC. The transcription analysis was carried out essentially as described by Linial et al. (47). Nuclei (10x10 6 ) were pelleted by centrifugation at 800 x g for 5 min, carefully resuspended in 40 n\ 6 mM Tris-HCI (pH 8); 20% glycerol; 150 mM KCI; 3 mM MgCI2; 2 mM DTT; 3000 U/ml RNasin; 0.25 mM each of ATP, GTP, and CTP, and 100 MCi [«-32P]UTP (400 Ci/mmol; Amersham International). Nuclei were harvested by centrifugation after 20 min at 30 C, resuspended in 100 /i\ 40 mM Tris-HCI (pH 7.5), 6 mM MgCI2, 2 mM CaCI2, and 1 mM DTT and lysed by the addition of 300 n\ DNase mix [40 mM TrisHCI (pH 7.5), 6 mM MgCI2, 2 mM CaCI2,1 mM DTT, 150 U/ml RNasin, and 65 /ig/ml DNase-l (Bethesda Research Laboratories, Bethesda, MD)] and subsequent incubation at 37 C for 1 h. Proteinase-K digestion was carried out at 42 C for 1 h by adding 150 MI 0.45 M Tris-HCI (pH 7.5), 4.57% SDS, 18.3 mM EDTA, and 1.6 mg/ml proteinase-K. In vitro labeled transcripts were recovered by phenol:CHCI3:isoamylalcohol (25:24:1) extraction and isopropanol precipitation and thereafter hybridized with Gene-Screen Plus or Hybond N + filters for 18 h at 42 C in 50% formamide, 2% SDS, 0.2 M sodium phosphate (pH 7.2), and 1 mM EDTA. Each filter contained 5 ng denaturated DNA pGEM-blue (Promega Biotech), /3-actin (48) (the cDNA encoding j8-actin was subcloned from pBR322 to pGEM-blue), and P4502C12 (49), immobilized as previously described (50). Washings consisted of two changes of 2 x SSC (1 x SSC; 150 mM NaCI, 15 mM sodium citrate, pH 7.0) at room temperature, once in 0.5 x SSC-0.1% SDS at 68 C for 1 h and once in 0.1 x SSC-0.1% SDS at 69 C for 1 h. Filters were then subjected to autoradiography. Where applicable, numerical data for the run-on analysis were obtained by densitometric scanning of the radiograms by using Image version 1.22y (NIH) on a Macintosh Ci (Apple, Cupertino, CA). After subtraction of background hybridization due to bacterial plasmid (pGEMblue), values were normalized against /3-actin. All hybridizations were carried out with more than 106 cpm.

DAG Assay Measurements of DAG formation were achieved by using a DAG assay system, available from Amersham International. The assay was performed according to their protocol.

Note Due to an arithmetic mistake, the absolute levels of P4502C12 mRNA previously reported by us (Mode et al., Mol Endocrinol 3:1142-1147,1989; and Toilet et al., Mol Endocrinol 4:19341942, 1990) are 10-fold lower than the correct value. This does not affect the interpretation of any of our data.

Acknowledgments We are indebted to Mrs. Eva Floby for skillful technical assistance. We are grateful to Mr. I. C. Hart (American Cyanamid Co., Wayne, NJ) for the kind gift of bGH. Special thanks are due to Dr. Johan Lund for helpful discussions during the course of this study and to Per Egnell for help in preparing the figures in this paper.

Received April 29, 1991. Revision received June 24, 1991. Accepted June 26,1991. Address requests for reprints to: Dr. Agneta Mode, Department of Medical Nutrition, Huddinge Uniiversity Hospital, F60 Novum, Karolinska Institute, S-14186 Huddinge, Sweden. This work was supported by grants from the Swedish Medical Research Council (no. 03X-06807) and the Magnus Bergvalls Foundation.

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Growth hormone (GH) regulation of cytochrome P-450IIC12, insulin-like growth factor-I (IGF-I), and GH receptor messenger RNA expression in primary rat hepatocytes: a hormonal interplay with insulin, IGF-I, and thyroid hormone.

Nutrition and somatomedin. XXV. Regulation of insulinlike growth factor binding protein 1 in primary cultures of normal rat hepatocytes.

Expression of the growth hormone-binding protein messenger RNA in the liver and extrahepatic tissues in the rat: co-expression with the growth hormone receptor.

Ontogeny of messenger RNA for the rat growth hormone receptor and serum binding protein.

Hormonal regulation of growth hormone receptors in primary cultured rat hepatocytes.

Glucose regulation of growth hormone receptors in primary cultured rat hepatocytes.

Growth hormone regulation of hepatic cytochrome P450 expression in the rat.

Ontogeny of growth hormone releasing hormone and insulin-like growth factors-I and -II messenger RNA in rat placenta.

Tissue distribution, characterization, and regulation of messenger ribonucleic acid for growth hormone receptor and serum binding protein in the rat.

Regulation of cytochrome P-450 CYPIA1 gene expression and proto-oncogene expression by growth factors in primary hepatocytes.

Transcriptional regulation of rat insulin-like growth factor-binding protein-1 expression by growth hormone.

Pregrowth hormone: product of the translation in vitro of messenger RNA coding for growth hormone.

Growth hormone regulation in primary fetal and neonatal rat pituitary cell cultures: the role of thyroid hormone.

Regulation of growth hormone messenger RNA by thyroid and glucocorticoid hormones.

Growth hormone-releasing hormone messenger ribonucleic acid in the hypothalamus of the adult male rat is increased by testosterone.

Growth hormone-dependent and -independent regulation of cytochrome P-450 isozyme expression in streptozotocin-diabetic rats.

Expression of the growth hormone receptor and growth hormone-binding protein during pregnancy in the mouse.

Effect of growth hormone on rat hepatic cytochrome P-450f mRNA: a new mode of regulation.

Regulation of albumin expression in fetal rat hepatocytes cultured under proliferative conditions: role of epidermal growth factor and hormones.

Neonatal rat hepatocytes in primary tissue culture free from density-dependent regulation of growth.

Role of growth hormone in the regulation of aldosterone biosynthesis.

Lack of growth hormone effect on insulin-associated suppression of insulinlike growth factor binding protein 1 in humans.

Expression and localization of growth hormone-releasing hormone messenger ribonucleic acid in rat placenta: in vitro secretion and regulation of its peptide product.

Growth hormone receptor messenger ribonucleic acid distribution in the adult male rat brain and its colocalization in hypothalamic somatostatin neurons.