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Regulation of hemopexin transcription by calcium ionophores and phorbol ester in hepatoma cells
Molecular and Cellular Endocrinology 204 (2003) 111 /116 www.elsevier.com/locate/mce
Regulation of hemopexin transcription by calcium ionophores and phorbol ester in hepatoma cells Susan E. Stred a, Deborah Cote b, Ruth S. Weinstock b, Joseph L. Messina c,* a
Cell and Molecular Biology Program and Department of Pediatrics, SUNY Upstate Medical University, Syracuse, NY 13210, USA b Department of Medicine, Veterans Administration Medical Center, Syracuse, NY 13210, USA c Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Volker Hall G019, 1670 University Blvd., Birmingham, AL 35294-0019, USA Received 12 February 2003; accepted 24 March 2003
Abstract Hemopexin (Hx) is an acute-phase hepatic protein, whose transcription is upregulated by IL-6. The transcription rate of Hx was found to be increased 11-fold by the calcium ionophore A23187, 25-fold by the calcium ionophore ionomycin, and 4 /5-fold by phorbol 12-myristate 13-acetate (PMA) in serum-starved H4IIE rat hepatoma cells. Insulin did not affect the transcription rate of Hx. These findings are consistent with involvement of intracellular calcium concentrations and activation of protein kinase C (PKC) action in the regulation of Hx. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hemopexin; Acute phase protein; Calcium signaling; Protein kinase C; Hepatoma cells (rat); Growth hormone; Gene expression
1. Introduction Our laboratories have been studying the regulation of gene transcription in rat H4IIE (H4) hepatoma cells by insulin, growth hormone (GH), the activation of protein kinase C (PKC), and changes in intracellular calcium concentrations, the latter induced by calcium ionophores. In searching for novel genes differentially regulated by insulin (Bortoff et al., 1997) or GH, two independent clones, originally isolated from rat liver, were studied. GIG-3 and GIG-7 represent two of the 13 cDNA clones isolated from liver of a hypophysectomized rat treated with GH. Both have been shown to be induced by GH in rat liver and in rat H4 hepatoma cells1. When sequenced, both clones were found to code for separate sequences of the gene for rat hemopexin, a Class 2 acute phase protein.
* Corresponding author. Tel.: /1-205-934-4921; fax: /1-205-9751126. E-mail address: [email protected] (J.L. Messina). 1 Stred, S.E. and Messina, J.L., 2003. Identification of hemopexin as a GH-regulated gene. MCE 204 (1 /2), 101 /110, this issue.
Hemopexin (Hx) is a heme-binding serum glycoprotein produced in the liver as part of the acute-phase response (Muller-Eberhard, 1988; Muller-Eberhard and Fraig, 1993). An important role of Hx is to bind and transport free heme to the liver where it is internalized, degraded, and the iron retained for re-utilization in the synthesis of iron-containing proteins (Muller-Eberhard and Fraig, 1993; Muller-Eberhard and Liem, 1974; Nikkila et al., 1991). Due to its avid binding of heme, Hx can function to remove heme from other proteins (Hanson et al., 1992). Many bacteria require iron for rapid growth, but iron in the form of heme/Hx cannot be used by most bacteria. By binding and transporting heme liberated from lysed erythrocytes, Hx sequesters heme from bacteria and inhibits their growth. Thus, Hx may help control or prevent bacterial infection. When heme, free or protein-bound, interacts with oxygen, it can catalyze the formation of oxygen radicals which can damage lipids and proteins. Hemopexin, by its avid binding of heme, markedly inhibits hemecatalyzed oxygen radical formation, preventing oxidative damage (Vincent et al., 1988). Finally, the carboxyterminal portion of porcine Hx represents the major hepatic hyaluronidase activity in that species (Zhu et al.,
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1994). Since hyaluronidase activity has been observed at sites of wound repair and tumor invasion, Hx may also play an important role in these processes. In cultured hepatoma cells, incubation with the cytokine, IL-6, increases Hx mRNA levels. Glucocorticoids augment the IL-6 effect, while IL-1, HGF and TGFb have minimal or no effect (Immenschuh et al., 1995, 1994; Nagae and Muller-Eberhard, 1992). Insulin in combination with dexamethasone had no acute effect on Hx protein levels (Immenschuh et al., 1995), but insulin has been reported to inhibit the induction of Hx mRNA expression normally seen after treatment with a combination of IL-6, IL-1 and dexamethasone (Campos and Baumann, 1992). We have recently demonstrated that the Hx gene is regulated by GH. This was unexpected, since, although the GH receptor is a member of the cytokine/growth factor receptor superfamily and GH is released during stress, GH has not previously been shown to participate in the acute phase reaction. Rat liver-derived H4 hepatoma cells have been employed extensively in the investigation of gene regulation by peptide hormones (Messina, 1989; Messina and Weinstock, 1994; Ooi et al., 1997) and in the study of the acute phase reaction (Richards et al., 1996). In the present studies, these cells were used to investigate the actions of calcium ionophores, which can regulate intracellular calcium concentrations, and a phorbol ester (PMA), which activates PKC, on the regulation of the transcription of the gene for the acute phase protein Hx. We found rapid increases in Hx transcription, suggesting that intracellular calcium concentrations and PKC activation may be involved in the control of Hx gene expression.
2. Materials and methods 2.1. Cell culture Rat H4IIE hepatoma cells (H4; American Type Culture Collection, Rockville, MD) were grown in Swim’s 77 medium with 5% fetal bovine serum and 5% horse serum in a 5% CO2 incubator at 95% humidity. Serum was withdrawn for 20/24 h before experiments, when the cells were approximately 60% confluent. Detailed methods have been published previously (Messina, 1989). 2.2. Isolation of nuclei and measurement of transcription To measure the elongation of transcripts in control or treated cells, nuclear run-on assays were performed as described previously (Messina, 1989). Briefly, nuclei from :/107 cultured H4 hepatoma cells were isolated by centrifugation through 1.8 M sucrose. Nuclei were
incubated with 100 mCi [a-32P]UTP for 45 min at 26 8C. Labeled RNA was then isolated by sequential extraction and precipitation, and analyzed by dot-blot hybridization to nitrocellulose (65 /70 h at 65 8C) onto which 3 mg of RNA coding for GIG-3, GIG-7, or mouse ß-tubulin had previously been applied. Use of the unregulated ßtubulin gene served as hybridization control. After autoradiography, the extent of hybridization of labeled mRNAs to the cDNA probes was quantified by densitometric scanning (Shimadzu) of the autoradiograms. The integrated densitometric scan of the control for each experiment was arbitrarily set to unity and the densities of the experimental samples were compared with untreated and solvent controls. 2.3. Statistical analysis All data was analyzed by ANOVA followed by Tukey post-tests, or t-tests where appropriate, using the INSTAT statistical program (version 3; GRAPHPAD Software, Inc., San Diego, CA).
3. Results Since GH may utilize changes in intracellular calcium concentrations as one of many signaling pathways to regulate gene expression (Tollet et al., 1991), we hypothesized that alterations of calcium concentrations generated by calcium ionophores would lead to induction of the GH-responsive Hx gene. Therefore, the transcription rate of Hx mRNA was studied using the two different cDNA clones, GIG-3 and GIG-7, which we have shown to be specific to different regions of the Hx gene2. Serum-starved H4IIE hepatoma cells were incubated with a concentration of the calcium ionophore A23187 (1 mM) previously demonstrated to alter intracellular calcium concentrations and increase mRNA transcription of insulin-responsive genes (Weinstock et al., 1992, 1993). At different time points following this addition, Hx transcription was measured using the two different probes, GIG-3 and GIG-7. Incubation with vehicle did not stimulate Hx gene transcription (Fig. 1, zero time point, plus data not shown). However, incubation with A23187 for 15 min generated a 3.6-fold (P B/0.01) and 3.9-fold (P /0.09) increase in Hx transcription as measured by the GIG-3 and GIG-7 cDNA probes, respectively. The effects of 1 mM A23187 to induce Hx transcription increased over time, reaching maximum levels of 8/10-fold at 90/120 min using both the GIG-3 and -7 probes (P B/0.05 /P B/0.001 for each time point). 2 Stred, S.E. and Messina, J.L., 2003. Identification of hemopexin as a GH-regulated gene. MCE 204 (1 /2), 101 /110, this issue.
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Fig. 1. Effect of A23187 on transcription rate of Hx. Serum-starved H4IIE hepatoma cells were treated with the calcium ionophore, A23187 (1 mM), for the times indicated. Nuclei were isolated, and rates of transcription for Hx using GIG-3 and -7 probes was measured. Panel A: Autoradiogram of a representative experiment. Panel B: Comparison of relative transcription rate of nuclei isolated from cells incubated with A23187 vs. those incubated with vehicle alone. The symbols indicate the mean increase in transcription rate over control / S.E. of 4 /7 (15 /60 min; P B/0.001 for GIG-3; P B/0.05 for GIG-7, 30 /60 min) or mean/range of 2 (90 /180 min; P B/0.05) separate experiments.
By 180 min, transcription rates had started to decrease, falling to 4.5 /5.5-fold above basal levels (Fig. 1, P B/ 0.05). The transcription rate of ß-tubulin was not increased following incubation with 1 mM A23187. To further study the effects of calcium ionophores on Hx gene expression, experiments were performed using another ionophore. Ionomycin (1 mM), also previously demonstrated to increase mRNA transcription of insulin-responsive genes in H4 cells (Weinstock et al., 1993), was added and changes in Hx transcription were measured over time using the GIG-3 and GIG-7 cDNA probes. As noted previously by Weinstock et al. (1992), ionomycin was sometimes slightly more effective in regulating gene expression than an equimolar concentration of A23187. By 15 min following addition of ionomycin, there was a 7 /10-fold induction of Hx transcription rates (P B/0.05 vs. control; Fig. 2), a 2fold greater induction than with A23187. There was also a 2-fold greater induction of Hx by ionomycin 30 min following its administration compared with A23187, to 23 /28-fold above basal values (P B/0.001 vs. control). As with A23187, ß-tubulin transcription was unaffected. In contrast to the modest but prolonged induction by A23187, the stimulatory effect of ionomycin had decreased by 60/75 min and by 90/105 min, transcription rates of Hx, as measured by both the GIG-3 and GIG-7 probes, had returned to basal values (Fig. 2). Similar to the data obtained following A23187 treatment, at all time points studied the fold induction of Hx as measured by the two GIG probes was essentially identical.
Fig. 2. Effect of ionomycin on transcription rates of Hx. Serumstarved H4IIE hepatoma cells were treated with the calcium ionophore, ionomycin (1 mM), for the times indicated. Nuclei were isolated, and rates of transcription for Hx using GIG-3 and -7 probes were measured. The symbols indicate the mean increase in transcription rate over control/S.E. of two to three separate experiments (15 and 30 min; all points P B/0.05) or a single pilot experiment ( /60 min).
Specific isozymes of PKC are activated by increases in intracellular calcium concentrations. Furthermore, PKC may mediate some of the calcium-sensitive effects of GH (Smal and De Meyts, 1987; Schwartz et al., 1991). To determine whether stimulation of PKC activity induces Hx transcription, we treated H4 cells with the phorbol
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Fig. 3. Effect of PMA and insulin on transcription rates of Hx. Serum-starved H4IIE hepatoma cells were treated with vehicle (ethanol), the inactive phorbol ester 4a-phorbol 12,13-didecanoate (4PDD; 1 mg/ml), the active phorbol ester PMA (1 mg/ml) or insulin (5 nM) for the times indicated. Nuclei were isolated, and rates of transcription for Hx using GIG-3 and -7 probes was measured. The bars indicate the mean increase in transcription rate over control/S.E. of 4 (15 min) or 6 (60 min) separate experiments for 4PDD and PMA, and 4 /9 separate experiments with insulin (except GIG-3, insulin 60 min, n /2).
ester, PMA. Using both GIG-3 and GIG-7 probes, a small increase in transcription rate was measured after a 15 min incubation with PMA, but it did not reach statistical significance (Fig. 3). However, by 60 min, PMA was able to significantly (4.5 /5-fold, P B/0.05) induce Hx transcription as measured with these two cDNA probes. This stimulatory effect was of lower magnitude than that observed with the calcium ionophores tested, and was decreasing by 75 min (data not shown). The biologically inactive phorbol ester, 4aphorbol 12,13-didecanoate (PDD; 1 mg/ml) was ineffective in altering Hx transcription at all time points tested (Fig. 3). H4IIE hepatoma cells are insulin-responsive (Messina and Weinstock, 1994). Therefore, we asked whether insulin acutely regulated expression of the Hx gene in these cells. At the four time points studied (15, 30, 60 and 120 min), no appreciable effects of insulin (5 nM) on Hx transcription rate were measurable when using either the GIG-3 or GIG-7 cDNAs (only data for 15 and 60 min are presented in Fig. 3). This is in agreement with the results of Campos and Baumann (1992), who studied the effects of insulin on H-35 cells, the parent cell line of the H4 hepatoma cells used in the present experiments. As has been previously published, transcription of the ß-tubulin gene was not altered by any of the treatments, and is thus an invariant control for differences in nuclear isolation, RNA labeling, and hybridization efficiency (Figs. 1 and 2, and data not shown; see Messina et al., 1992; Messina and Weinstock, 1994).
4. Discussion Transcription of the gene for the acute-phase hepatic protein hemopexin (Hx) increases in response to the cytokine IL-6, but not to several other cytokines such as IL-1, HGF and TGFb. We have recently demonstrated that the Hx gene is regulated by GH. This was unexpected, since, although the GH receptor is a member of the cytokine/growth factor receptor superfamily and GH is released during stress, GH has not previously been shown to participate in the acute phase reaction. As an initial step in elucidating the mechanism of hemopexin transcription by GH, we asked whether increases in intracellular calcium concentrations, in the absence of added GH, might also increase Hx mRNA transcription rate. We were particularly intrigued by this possibility, since IL-6, the primary cytokine to induce Hx mRNA, does not increase intracellular calcium (Qiu et al., 1995; Huang et al., 1995) while GH does. Addition of GH for 3 /5 min to GH-responsive cells results in sustained elevations of intracellular calcium concentrations (Schwartz et al., 1991; Ilondo et al., 1994; Billestrup et al., 1995). Furthermore, Billestrup et al. (1995) have proposed that calcium signaling is required for GH stimulation of the hepatic gene, Spi 2.1, and GHstimulable c-fos expression increases following treatment of rat hepatocytes with the calcium ionophore, ionomycin (Tollet et al., 1991), indicating that increased intracellular calcium concentrations can mediate GH’s regulation of at least some GH-sensitive genes.
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Rat hepatoma cells have been employed extensively in the investigation of the acute phase reaction. In addition, calcium ionophores are routinely used in numerous other laboratories to produce increased intracellular calcium concentrations in the study of gene regulation. Our labs have been studying gene regulation by peptide hormones in rat hepatoma cell lines and we have taken advantage of this method to explore the signaling pathways used by insulin in the regulation of hepatic gene expression studies (Weinstock et al., 1992, 1993) and in the current work, the regulation of Hx mRNA in this GH-sensitive cell line (Ji et al. 1999, 2002). Here we show that incubation of cultured hepatoma cells with calcium ionophores, agents which can rapidly increase intracellular calcium concentrations, results in increases in the transcription rate of this gene in the absence of any added cytokine or hormone. Thus, increases in intracellular calcium concentrations may represent a common pathway in the regulation of a number of GH-sensitive hepatic genes including Spi 2.1 and c-fos, as well as Hx. However, other GH-stimulateable hepatic genes, such as P4502C12 and IGF-I, are not induced by increased intracellular calcium concentrations alone (Tollet et al., 1991). This suggests that although changes in intracellular calcium concentrations may be important in GH regulation of some genes, not all GH-sensitive genes are modulated following changes in calcium concentrations, indicating the importance of other signaling pathways in GH-regulated gene expression. Additionally, there most likely are multiple response elements in the Hx gene, specific for different signal transduction pathways, that together are important for control of hepatic expression of the Hx gene. Exposure to GH modulates the activity of PKC (Nivet et al., 1993), a family of serine /threonine protein kinases, some of which are calcium and phospholipid dependent. Stimulation of PKC activity in the absence of GH has been reported to mimic some actions of GH (Gorin et al., 1990; Slootweg et al., 1991; Catalioto et al., 1992) and down-regulation of PKC inhibits some GH effects (Smal and De Meyts, 1987; Slootweg et al., 1991; Tollet et al., 1991; Nivet et al., 1993). Activation of PKC may be downstream of changes in intracellular calcium concentrations in the pathways leading to regulation of GH-sensitive genes (Doglio et al., 1989). H4IIE hepatoma cells have been used extensively to study the effect of PKC activation on transcription of several different genes (Messina et al., 1992; Messina and Weinstock, 1994; Onyia et al., 1994; Reks et al., 1998). In the present study, PMA-induced activation of PKC, in the absence of any hormones or growth factors, increased the transcription rate of Hx. This effect was modest compared with the much greater increases in transcription observed following treatment with calcium ionophores. This suggests that the larger effects of increased intracellular calcium concentrations on Hx
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transcription were not mediated solely by activation of PKC, and that large changes in calcium can activate additional pathways that lead to a larger-fold induction of Hx transcription. The H4 cell line used in the present studies has been used extensively to study insulin regulation of gene expression (Messina, 1989; Weinstock et al., 1992; Messina et al., 1992; Weinstock et al., 1993; Messina and Weinstock, 1994; Onyia et al., 1994; Bortoff et al., 1997; Ji et al., 1999). Insulin has been reported to inhibit the customary induction of Hx mRNA expression by a combination of IL-6 and dexamethasone. However, in the present studies, insulin alone was ineffective in acutely regulating Hx mRNA. In the current work on Hx, we used two different cDNA probes, coding for sequences separated by several hundred bp in the native rat Hx gene. As indicated in the figures in this paper, Hx transcription, as measured by both GIG-3 and GIG-7 probes, responded with the same fold-induction under all conditions tested. This suggests that there is no intragenic pausing in the intervening region between the sequences coded for by GIG-3 and GIG-7 in the native Hx gene, when transcription of this gene is stimulated by calcium ionophores or PMA. In summary, the transcription rate of Hx, a Class 2 acute phase protein, rises dramatically within the first hour of treatment of rat hepatoma cells with either of two separate calcium ionophores or the phorbol ester, PMA. We have recently shown this acute phase protein to be GH-sensitive. The findings in the current study support the hypothesis that changes in intracellular calcium concentrations and/or activation of PKC can play a role in the regulation of the Hx gene.
Acknowledgements The authors would like to thank Dr Kevin Lynch and acknowledge the technical assistance of Cathy Saville, Melleny Hale, and Christopher Weirs. This project was supported by NIH DK02114 (SES), DK54440 (J.L. Messina), the Central New York Children’s Health Fund (S.E. Stred), the Department of Veterans Affairs (R.S. Weinstock), and the American Diabetes Association (J.L. Messina).
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Nagae, Y., Muller-Eberhard, U., 1992. Identification of an interleukin6 responsive element and characterization of the proximal promoter region of the rat hemopexin gene. Biochem. Biophys. Res. Commun. 185, 420 /429. Nikkila, H., Gitlin, J.D., Muller-Eberhard, U., 1991. Rat hemopexin. Molecular cloning, primary structural characterization, and analysis of gene expression. Biochemistry 30, 823 /829. Nivet, V., Clot, J.P., Do, X.T., Barrault, V., Prelot, M., Durand, D., 1993. Evidence that growth hormone stimulates protein kinase C activity in isolated rat hepatocytes. Metabolism 42, 1291 /1295. Onyia, J.E., Halladay, D.L., Messina, J.L., 1994. Identification of bactin sequences necessary for induction by phorbol esters and calcium ionophores. Oncogene 9, 1713 /1722. Ooi, G.T., Cohen, F.J., Tseng, L.Y.H., Rechler, M.M., Boisclair, Y.R., 1997. Growth hormone stimulates transcription of the gene encoding the acid-labile subunit (ALS) of the circulating insulinlike growth factor-binding protein complex and ALS promoter activity in rat liver. Mol. Endocrinol. 11, 997 /1007. Qiu, Z., Parsons, K.L., Gruol, D.L., 1995. Interleukin-6 selectively enhances the intracellular calcium response to NMDA in developing CNS neurons. J. Neurosci. 15, 6688 /6699. Reks, S.E., Smith, P.H., Messina, J.L., Weinstock, R.S., 1998. Translocation of PKC delta by insulin in a rat hepatoma cell line. Endocrine 8, 161 /167. Richards, C.D., Langdon, C., Pennica, D., Gauldie, J., 1996. Murine cardiotrophin-1 stimulates the acute-phase response in rat hepatocytes and H35 hepatoma cells. J. Interferon Cytokine Res. 16, 69 / 75. Schwartz, Y., Goodman, H.M., Yamaguchi, H., 1991. Refractoriness to growth hormone is associated with increased intracellular calcium in rat adipocytes. Proc. Natl. Acad. Sci. USA 88, 6790 / 6794. Slootweg, M.C., deGroot, R.P., Herrmann-Erlee, M.P., Koornneef, I., Kruijer, W., Kramer, Y.M., 1991. Growth hormone induces expression of c-jun and jun B oncogenes and employs a protein kinase C signal transduction pathway for the induction of c-fos oncogene expression. J. Mol. Endocrinol. 6, 179 /188. Smal, J., De Meyts, P., 1987. Role of kinase C in the insulin-like effects of human growth hormone in rat adipocytes. Biochem. Biophys. Res. Commun. 147, 1232 /1240. Tollet, P., Legraverend, C., Gustafsson, J.-A., Mode, A., 1991. 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. Mol. Endocrinol. 5, 1351 /1358. Vincent, S.H., Grady, R.W., Shaklai, N., Snider, J.M., MullerEberhard, U., 1988. The influence of heme-binding proteins in heme-catalyzed oxidations. Arch. Biochem. Biophys. 265, 539 / 550. Weinstock, R.S., Manning, C.A., Messina, J.L., 1992. The regulation of p33 gene expression by insulin and calcium ionophores. Endocrinology 130, 616 /624. Weinstock, R.S., Saville, C.M., Messina, J.L., 1993. Role of cytosolic calcium in regulation of cytoskeletal gene expression by insulin. Am. J. Physiol. 264, 519 /525. Zhu, L., Hope, T.J., Hall, J., Davies, A., Stern, M., Muller-Eberhard, U., Stern, R., Parslow, T.G., 1994. Molecular cloning of a mammalian hyaluronidase reveals identity with hemopexin, a serum Heme-binding protein. J. Biol. Chem. 269, 32092 /32097.
Regulation of hemopexin transcription by calcium ionophores and phorbol ester in hepatoma cells Susan E. Stred a, Deborah Cote b, Ruth S. Weinstock b, Joseph L. Messina c,* a
Cell and Molecular Biology Program and Department of Pediatrics, SUNY Upstate Medical University, Syracuse, NY 13210, USA b Department of Medicine, Veterans Administration Medical Center, Syracuse, NY 13210, USA c Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Volker Hall G019, 1670 University Blvd., Birmingham, AL 35294-0019, USA Received 12 February 2003; accepted 24 March 2003
Abstract Hemopexin (Hx) is an acute-phase hepatic protein, whose transcription is upregulated by IL-6. The transcription rate of Hx was found to be increased 11-fold by the calcium ionophore A23187, 25-fold by the calcium ionophore ionomycin, and 4 /5-fold by phorbol 12-myristate 13-acetate (PMA) in serum-starved H4IIE rat hepatoma cells. Insulin did not affect the transcription rate of Hx. These findings are consistent with involvement of intracellular calcium concentrations and activation of protein kinase C (PKC) action in the regulation of Hx. # 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hemopexin; Acute phase protein; Calcium signaling; Protein kinase C; Hepatoma cells (rat); Growth hormone; Gene expression
1. Introduction Our laboratories have been studying the regulation of gene transcription in rat H4IIE (H4) hepatoma cells by insulin, growth hormone (GH), the activation of protein kinase C (PKC), and changes in intracellular calcium concentrations, the latter induced by calcium ionophores. In searching for novel genes differentially regulated by insulin (Bortoff et al., 1997) or GH, two independent clones, originally isolated from rat liver, were studied. GIG-3 and GIG-7 represent two of the 13 cDNA clones isolated from liver of a hypophysectomized rat treated with GH. Both have been shown to be induced by GH in rat liver and in rat H4 hepatoma cells1. When sequenced, both clones were found to code for separate sequences of the gene for rat hemopexin, a Class 2 acute phase protein.
* Corresponding author. Tel.: /1-205-934-4921; fax: /1-205-9751126. E-mail address: [email protected] (J.L. Messina). 1 Stred, S.E. and Messina, J.L., 2003. Identification of hemopexin as a GH-regulated gene. MCE 204 (1 /2), 101 /110, this issue.
Hemopexin (Hx) is a heme-binding serum glycoprotein produced in the liver as part of the acute-phase response (Muller-Eberhard, 1988; Muller-Eberhard and Fraig, 1993). An important role of Hx is to bind and transport free heme to the liver where it is internalized, degraded, and the iron retained for re-utilization in the synthesis of iron-containing proteins (Muller-Eberhard and Fraig, 1993; Muller-Eberhard and Liem, 1974; Nikkila et al., 1991). Due to its avid binding of heme, Hx can function to remove heme from other proteins (Hanson et al., 1992). Many bacteria require iron for rapid growth, but iron in the form of heme/Hx cannot be used by most bacteria. By binding and transporting heme liberated from lysed erythrocytes, Hx sequesters heme from bacteria and inhibits their growth. Thus, Hx may help control or prevent bacterial infection. When heme, free or protein-bound, interacts with oxygen, it can catalyze the formation of oxygen radicals which can damage lipids and proteins. Hemopexin, by its avid binding of heme, markedly inhibits hemecatalyzed oxygen radical formation, preventing oxidative damage (Vincent et al., 1988). Finally, the carboxyterminal portion of porcine Hx represents the major hepatic hyaluronidase activity in that species (Zhu et al.,
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1994). Since hyaluronidase activity has been observed at sites of wound repair and tumor invasion, Hx may also play an important role in these processes. In cultured hepatoma cells, incubation with the cytokine, IL-6, increases Hx mRNA levels. Glucocorticoids augment the IL-6 effect, while IL-1, HGF and TGFb have minimal or no effect (Immenschuh et al., 1995, 1994; Nagae and Muller-Eberhard, 1992). Insulin in combination with dexamethasone had no acute effect on Hx protein levels (Immenschuh et al., 1995), but insulin has been reported to inhibit the induction of Hx mRNA expression normally seen after treatment with a combination of IL-6, IL-1 and dexamethasone (Campos and Baumann, 1992). We have recently demonstrated that the Hx gene is regulated by GH. This was unexpected, since, although the GH receptor is a member of the cytokine/growth factor receptor superfamily and GH is released during stress, GH has not previously been shown to participate in the acute phase reaction. Rat liver-derived H4 hepatoma cells have been employed extensively in the investigation of gene regulation by peptide hormones (Messina, 1989; Messina and Weinstock, 1994; Ooi et al., 1997) and in the study of the acute phase reaction (Richards et al., 1996). In the present studies, these cells were used to investigate the actions of calcium ionophores, which can regulate intracellular calcium concentrations, and a phorbol ester (PMA), which activates PKC, on the regulation of the transcription of the gene for the acute phase protein Hx. We found rapid increases in Hx transcription, suggesting that intracellular calcium concentrations and PKC activation may be involved in the control of Hx gene expression.
2. Materials and methods 2.1. Cell culture Rat H4IIE hepatoma cells (H4; American Type Culture Collection, Rockville, MD) were grown in Swim’s 77 medium with 5% fetal bovine serum and 5% horse serum in a 5% CO2 incubator at 95% humidity. Serum was withdrawn for 20/24 h before experiments, when the cells were approximately 60% confluent. Detailed methods have been published previously (Messina, 1989). 2.2. Isolation of nuclei and measurement of transcription To measure the elongation of transcripts in control or treated cells, nuclear run-on assays were performed as described previously (Messina, 1989). Briefly, nuclei from :/107 cultured H4 hepatoma cells were isolated by centrifugation through 1.8 M sucrose. Nuclei were
incubated with 100 mCi [a-32P]UTP for 45 min at 26 8C. Labeled RNA was then isolated by sequential extraction and precipitation, and analyzed by dot-blot hybridization to nitrocellulose (65 /70 h at 65 8C) onto which 3 mg of RNA coding for GIG-3, GIG-7, or mouse ß-tubulin had previously been applied. Use of the unregulated ßtubulin gene served as hybridization control. After autoradiography, the extent of hybridization of labeled mRNAs to the cDNA probes was quantified by densitometric scanning (Shimadzu) of the autoradiograms. The integrated densitometric scan of the control for each experiment was arbitrarily set to unity and the densities of the experimental samples were compared with untreated and solvent controls. 2.3. Statistical analysis All data was analyzed by ANOVA followed by Tukey post-tests, or t-tests where appropriate, using the INSTAT statistical program (version 3; GRAPHPAD Software, Inc., San Diego, CA).
3. Results Since GH may utilize changes in intracellular calcium concentrations as one of many signaling pathways to regulate gene expression (Tollet et al., 1991), we hypothesized that alterations of calcium concentrations generated by calcium ionophores would lead to induction of the GH-responsive Hx gene. Therefore, the transcription rate of Hx mRNA was studied using the two different cDNA clones, GIG-3 and GIG-7, which we have shown to be specific to different regions of the Hx gene2. Serum-starved H4IIE hepatoma cells were incubated with a concentration of the calcium ionophore A23187 (1 mM) previously demonstrated to alter intracellular calcium concentrations and increase mRNA transcription of insulin-responsive genes (Weinstock et al., 1992, 1993). At different time points following this addition, Hx transcription was measured using the two different probes, GIG-3 and GIG-7. Incubation with vehicle did not stimulate Hx gene transcription (Fig. 1, zero time point, plus data not shown). However, incubation with A23187 for 15 min generated a 3.6-fold (P B/0.01) and 3.9-fold (P /0.09) increase in Hx transcription as measured by the GIG-3 and GIG-7 cDNA probes, respectively. The effects of 1 mM A23187 to induce Hx transcription increased over time, reaching maximum levels of 8/10-fold at 90/120 min using both the GIG-3 and -7 probes (P B/0.05 /P B/0.001 for each time point). 2 Stred, S.E. and Messina, J.L., 2003. Identification of hemopexin as a GH-regulated gene. MCE 204 (1 /2), 101 /110, this issue.
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Fig. 1. Effect of A23187 on transcription rate of Hx. Serum-starved H4IIE hepatoma cells were treated with the calcium ionophore, A23187 (1 mM), for the times indicated. Nuclei were isolated, and rates of transcription for Hx using GIG-3 and -7 probes was measured. Panel A: Autoradiogram of a representative experiment. Panel B: Comparison of relative transcription rate of nuclei isolated from cells incubated with A23187 vs. those incubated with vehicle alone. The symbols indicate the mean increase in transcription rate over control / S.E. of 4 /7 (15 /60 min; P B/0.001 for GIG-3; P B/0.05 for GIG-7, 30 /60 min) or mean/range of 2 (90 /180 min; P B/0.05) separate experiments.
By 180 min, transcription rates had started to decrease, falling to 4.5 /5.5-fold above basal levels (Fig. 1, P B/ 0.05). The transcription rate of ß-tubulin was not increased following incubation with 1 mM A23187. To further study the effects of calcium ionophores on Hx gene expression, experiments were performed using another ionophore. Ionomycin (1 mM), also previously demonstrated to increase mRNA transcription of insulin-responsive genes in H4 cells (Weinstock et al., 1993), was added and changes in Hx transcription were measured over time using the GIG-3 and GIG-7 cDNA probes. As noted previously by Weinstock et al. (1992), ionomycin was sometimes slightly more effective in regulating gene expression than an equimolar concentration of A23187. By 15 min following addition of ionomycin, there was a 7 /10-fold induction of Hx transcription rates (P B/0.05 vs. control; Fig. 2), a 2fold greater induction than with A23187. There was also a 2-fold greater induction of Hx by ionomycin 30 min following its administration compared with A23187, to 23 /28-fold above basal values (P B/0.001 vs. control). As with A23187, ß-tubulin transcription was unaffected. In contrast to the modest but prolonged induction by A23187, the stimulatory effect of ionomycin had decreased by 60/75 min and by 90/105 min, transcription rates of Hx, as measured by both the GIG-3 and GIG-7 probes, had returned to basal values (Fig. 2). Similar to the data obtained following A23187 treatment, at all time points studied the fold induction of Hx as measured by the two GIG probes was essentially identical.
Fig. 2. Effect of ionomycin on transcription rates of Hx. Serumstarved H4IIE hepatoma cells were treated with the calcium ionophore, ionomycin (1 mM), for the times indicated. Nuclei were isolated, and rates of transcription for Hx using GIG-3 and -7 probes were measured. The symbols indicate the mean increase in transcription rate over control/S.E. of two to three separate experiments (15 and 30 min; all points P B/0.05) or a single pilot experiment ( /60 min).
Specific isozymes of PKC are activated by increases in intracellular calcium concentrations. Furthermore, PKC may mediate some of the calcium-sensitive effects of GH (Smal and De Meyts, 1987; Schwartz et al., 1991). To determine whether stimulation of PKC activity induces Hx transcription, we treated H4 cells with the phorbol
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Fig. 3. Effect of PMA and insulin on transcription rates of Hx. Serum-starved H4IIE hepatoma cells were treated with vehicle (ethanol), the inactive phorbol ester 4a-phorbol 12,13-didecanoate (4PDD; 1 mg/ml), the active phorbol ester PMA (1 mg/ml) or insulin (5 nM) for the times indicated. Nuclei were isolated, and rates of transcription for Hx using GIG-3 and -7 probes was measured. The bars indicate the mean increase in transcription rate over control/S.E. of 4 (15 min) or 6 (60 min) separate experiments for 4PDD and PMA, and 4 /9 separate experiments with insulin (except GIG-3, insulin 60 min, n /2).
ester, PMA. Using both GIG-3 and GIG-7 probes, a small increase in transcription rate was measured after a 15 min incubation with PMA, but it did not reach statistical significance (Fig. 3). However, by 60 min, PMA was able to significantly (4.5 /5-fold, P B/0.05) induce Hx transcription as measured with these two cDNA probes. This stimulatory effect was of lower magnitude than that observed with the calcium ionophores tested, and was decreasing by 75 min (data not shown). The biologically inactive phorbol ester, 4aphorbol 12,13-didecanoate (PDD; 1 mg/ml) was ineffective in altering Hx transcription at all time points tested (Fig. 3). H4IIE hepatoma cells are insulin-responsive (Messina and Weinstock, 1994). Therefore, we asked whether insulin acutely regulated expression of the Hx gene in these cells. At the four time points studied (15, 30, 60 and 120 min), no appreciable effects of insulin (5 nM) on Hx transcription rate were measurable when using either the GIG-3 or GIG-7 cDNAs (only data for 15 and 60 min are presented in Fig. 3). This is in agreement with the results of Campos and Baumann (1992), who studied the effects of insulin on H-35 cells, the parent cell line of the H4 hepatoma cells used in the present experiments. As has been previously published, transcription of the ß-tubulin gene was not altered by any of the treatments, and is thus an invariant control for differences in nuclear isolation, RNA labeling, and hybridization efficiency (Figs. 1 and 2, and data not shown; see Messina et al., 1992; Messina and Weinstock, 1994).
4. Discussion Transcription of the gene for the acute-phase hepatic protein hemopexin (Hx) increases in response to the cytokine IL-6, but not to several other cytokines such as IL-1, HGF and TGFb. We have recently demonstrated that the Hx gene is regulated by GH. This was unexpected, since, although the GH receptor is a member of the cytokine/growth factor receptor superfamily and GH is released during stress, GH has not previously been shown to participate in the acute phase reaction. As an initial step in elucidating the mechanism of hemopexin transcription by GH, we asked whether increases in intracellular calcium concentrations, in the absence of added GH, might also increase Hx mRNA transcription rate. We were particularly intrigued by this possibility, since IL-6, the primary cytokine to induce Hx mRNA, does not increase intracellular calcium (Qiu et al., 1995; Huang et al., 1995) while GH does. Addition of GH for 3 /5 min to GH-responsive cells results in sustained elevations of intracellular calcium concentrations (Schwartz et al., 1991; Ilondo et al., 1994; Billestrup et al., 1995). Furthermore, Billestrup et al. (1995) have proposed that calcium signaling is required for GH stimulation of the hepatic gene, Spi 2.1, and GHstimulable c-fos expression increases following treatment of rat hepatocytes with the calcium ionophore, ionomycin (Tollet et al., 1991), indicating that increased intracellular calcium concentrations can mediate GH’s regulation of at least some GH-sensitive genes.
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Rat hepatoma cells have been employed extensively in the investigation of the acute phase reaction. In addition, calcium ionophores are routinely used in numerous other laboratories to produce increased intracellular calcium concentrations in the study of gene regulation. Our labs have been studying gene regulation by peptide hormones in rat hepatoma cell lines and we have taken advantage of this method to explore the signaling pathways used by insulin in the regulation of hepatic gene expression studies (Weinstock et al., 1992, 1993) and in the current work, the regulation of Hx mRNA in this GH-sensitive cell line (Ji et al. 1999, 2002). Here we show that incubation of cultured hepatoma cells with calcium ionophores, agents which can rapidly increase intracellular calcium concentrations, results in increases in the transcription rate of this gene in the absence of any added cytokine or hormone. Thus, increases in intracellular calcium concentrations may represent a common pathway in the regulation of a number of GH-sensitive hepatic genes including Spi 2.1 and c-fos, as well as Hx. However, other GH-stimulateable hepatic genes, such as P4502C12 and IGF-I, are not induced by increased intracellular calcium concentrations alone (Tollet et al., 1991). This suggests that although changes in intracellular calcium concentrations may be important in GH regulation of some genes, not all GH-sensitive genes are modulated following changes in calcium concentrations, indicating the importance of other signaling pathways in GH-regulated gene expression. Additionally, there most likely are multiple response elements in the Hx gene, specific for different signal transduction pathways, that together are important for control of hepatic expression of the Hx gene. Exposure to GH modulates the activity of PKC (Nivet et al., 1993), a family of serine /threonine protein kinases, some of which are calcium and phospholipid dependent. Stimulation of PKC activity in the absence of GH has been reported to mimic some actions of GH (Gorin et al., 1990; Slootweg et al., 1991; Catalioto et al., 1992) and down-regulation of PKC inhibits some GH effects (Smal and De Meyts, 1987; Slootweg et al., 1991; Tollet et al., 1991; Nivet et al., 1993). Activation of PKC may be downstream of changes in intracellular calcium concentrations in the pathways leading to regulation of GH-sensitive genes (Doglio et al., 1989). H4IIE hepatoma cells have been used extensively to study the effect of PKC activation on transcription of several different genes (Messina et al., 1992; Messina and Weinstock, 1994; Onyia et al., 1994; Reks et al., 1998). In the present study, PMA-induced activation of PKC, in the absence of any hormones or growth factors, increased the transcription rate of Hx. This effect was modest compared with the much greater increases in transcription observed following treatment with calcium ionophores. This suggests that the larger effects of increased intracellular calcium concentrations on Hx
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transcription were not mediated solely by activation of PKC, and that large changes in calcium can activate additional pathways that lead to a larger-fold induction of Hx transcription. The H4 cell line used in the present studies has been used extensively to study insulin regulation of gene expression (Messina, 1989; Weinstock et al., 1992; Messina et al., 1992; Weinstock et al., 1993; Messina and Weinstock, 1994; Onyia et al., 1994; Bortoff et al., 1997; Ji et al., 1999). Insulin has been reported to inhibit the customary induction of Hx mRNA expression by a combination of IL-6 and dexamethasone. However, in the present studies, insulin alone was ineffective in acutely regulating Hx mRNA. In the current work on Hx, we used two different cDNA probes, coding for sequences separated by several hundred bp in the native rat Hx gene. As indicated in the figures in this paper, Hx transcription, as measured by both GIG-3 and GIG-7 probes, responded with the same fold-induction under all conditions tested. This suggests that there is no intragenic pausing in the intervening region between the sequences coded for by GIG-3 and GIG-7 in the native Hx gene, when transcription of this gene is stimulated by calcium ionophores or PMA. In summary, the transcription rate of Hx, a Class 2 acute phase protein, rises dramatically within the first hour of treatment of rat hepatoma cells with either of two separate calcium ionophores or the phorbol ester, PMA. We have recently shown this acute phase protein to be GH-sensitive. The findings in the current study support the hypothesis that changes in intracellular calcium concentrations and/or activation of PKC can play a role in the regulation of the Hx gene.
Acknowledgements The authors would like to thank Dr Kevin Lynch and acknowledge the technical assistance of Cathy Saville, Melleny Hale, and Christopher Weirs. This project was supported by NIH DK02114 (SES), DK54440 (J.L. Messina), the Central New York Children’s Health Fund (S.E. Stred), the Department of Veterans Affairs (R.S. Weinstock), and the American Diabetes Association (J.L. Messina).
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