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Angiotensin stimulates the expression of interferon-inducible genes in H295R cells
Molecular and Cellular Endocrinology 176 (2001) 21 – 27 www.elsevier.com/locate/mce
Angiotensin stimulates the expression of interferon-inducible genes in H295R cells Hisashi Daido a,b,e, Ming-Yi Zhou a, Celso E. Gomez-Sanchez a,c,d,* a
Di6ision of Endocrinology, Uni6ersity of Missouri, Columbia, MO 65212, USA Harry S. Truman Memorial Veterans Hospital, Columbia, MO 65201, USA c Di6ision of Endocrinology, Department of Medicine, The Uni6ersity of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216 -4505, USA d G.V. (Sonny) Montgomery VA Medical Center, Jackson, MS 39216, USA e Department of Medicine, Gifu Uni6ersity, Gifu, Japan b
Received 1 December 2000; accepted 12 March 2001
Abstract Angiotensin-II (A-II) induces proliferation of zona glomerulosa cells and stimulates expression of cytochrome P-450 aldosterone synthase. The genes activated during this adrenal remodeling are not well defined. To clarify this mechanism, we sought to identify the genes whose expression is stimulated by A-II in the H295R cell line. Using a subtractive hybridization technique, we identified one clone whose expression was stimulated by A-II. The sequence of this gene was homologous to the human interferon-inducible genes, 9-27, 1-8D and 1-8U. The 5% portion of the gene was identical to the 1-8D gene product and the 3% was identical to the 9-27 gene product, but the existence of a transcript was not demonstrated by RT-PCR. The expression of these three genes was stimulated by A-II, with the 9-27 gene being most abundant. Potassium and forskolin also stimulated the expression of the 9-27 gene in the H295R cells, but not as effectively as did A-II or interferon-g. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Angiotensin; Hybridization; Zona glomerulosa
1. Introduction Aldosterone (Aldo) synthesis in the zona glomerulosa of the adrenal gland is under the primary control of Angiotensin II (A-II). Physiological conditions like the adaptation to sodium depletion and diseases associated with chronic stimulation of the renin – angiotensin system result in the stimulation of aldosterone synthesis. A-II increases gene transcription and expression of cytochrome P-450 aldosterone synthase (CYP11B2), the final enzyme in the synthesis of aldosterone, and promotes adrenal zona glomerulosa remodeling (Tremblay et al., 1992; Mitani et al., 1994). Primary aldosteronism, a common cause of hypertension in humans, is caused by the excessive secretion of aldosterone by the adrenal zona glomerulosa (Gomez-Sanchez, 1998) and is caused * Corresponding author. Tel.: + 1-601-9845525; fax: +1-6019845769. E-mail address: [email protected] (C.E. Gomez-Sanchez).
by either the presence of an adrenal tumor or by bilateral zona glomerulosa hyperplasia. The cause and mechanisms of adrenal remodeling in bilateral zona glomerulosa hyperplasia is unknown. The H295R cell line is a pluripotential human adrenocortical carcinoma cell line (Gazdar et al., 1990; Bird et al., 1993; Rainey et al., 1993, 1994; Denner et al., 1996) shown to be a good model system to define mechanisms regulating human aldosterone production (Bird et al., 1993). A-II action in the adrenal not only involves an increase in the expression of the aldosterone synthase enzyme, but also an increase in cell proliferation (Vinson et al., 1998a,b). We attempted to identify genes that are stimulated by A-II in H295R cells using a subtractive hybridization technique (Diatchenko et al., 1996). This technique uses a powerful method to normalize transcript abundance, and enrich rare cDNAs that in this case were stimulated by A-II. We describe the identification of a gene product that is expressed after stimulation with A-II.
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H. Daido et al. / Molecular and Cellular Endocrinology 176 (2001) 21–27
2. Materials and methods
2.4. Screening of the subtraction library
2.1. Materials
A library of the subtracted cDNA was made by TA cloning (pGEM-T Vector system from Promega Corp., Madison, WI) and used to transform competent bacterial cells (One shot TOP10F% from Invitrogen Corp., Carlsbad, CA). About 784 bacterial colonies were picked at random and screened. For the first screening, we used the PCR-Select Differential Screening kit (Clontech Laboratories, Inc., Palo Alto, CA), with modifications. Two kinds of DNA probes were made. One of them, a subtracted probe, was made from cDNA from the above procedure. The other, a reverse-subtracted probe was made from reverse subtraction cDNA. This cDNA was constructed by the same method as subtractive hybridization. But in this case, tester cDNA was control cDNA, and driver cDNA was A-II-stimulated cells cDNA. Both probes were then labeled with biotin. The 784 clones were arrayed and grown in duplicate on nylon membranes and screened with two kinds of probes. The membranes were baked in a microwave oven and digested with proteinase K (200 mg/ml at 37°C for 1 h), followed by hybridization with the above probes. Hybridization solution consisted of 0.5 M Na2HPO4, 2 mM EDTA and 7% SDS, and 250 ng/ml of probe. After the overnight hybridization at 65°C, the membranes were washed three times. The first wash solution was 2× SSC and 1% SDS for 20 min, second was 1× SSC and 1% SDS for 20 min, final wash was 0.1× SSC and 1% SDS for 30 min at 50°C. Finally, the hybridized biotinilated probe was detected by a chemiluminescent detection system (BrightStar BioDetect from Ambion, Austin, TX). There were 159 positive colonies. Plasmids from the positive colonies were prepared by miniprep (Wizard Plus Minipreps from Promega Corp., Madison, WI), and screened by reverse dot blot technique. Denatured plasmids (2.5 mg) were placed on duplicate nylon membranes and crosslinked by UV light. The probes used were biotin-labeled cDNA made from mRNA of A-II stimulated cells and control cells using reverse transcriptase (Superscript-II form Life Technologies, Gaithersburg, MD). Hybridization, washing and detection methods were same as for the first screening. There were 40 clones that were positive only when using the probe generated from mRNA of A-II stimulated cells. These were assayed by conventional Northern blot technique with 32P-labeled probes using RNA from control and A-II stimulated cells. One clone was clearly differentially expressed in A-II-stimulated cells.
DME/F12 medium was obtained from the CIC Core from the University of Missouri, Columbia. HyQCCM1 medium was obtained from Hyclone, (Logan, UT). Human interferon-g was purchased from PeproTech Inc. (Rocky Hill, NJ). Other reagents were obtained from Sigma Chemical Company (St. Louis, MO).
2.2. Incubation of H295R cells and RNA preparation H295R cells were grown in HyQ-CCM1 medium with 2% horse serum. The media was changed to serum-free DME/F12 medium and incubated for 24 h before the experiments because HyQ-CCM1 medium stimulates H295R cells to secrete aldosterone. Cells were incubated with or without stimulant in serum-free DME/F12 medium. [Sar1]-angiotensin-II and forskolin were purchased from Sigma (St. Louis, MO). After the incubation, aldosterone concentration in the medium was measured by ELISA (Gomez-Sanchez et al., 1987). Total RNA was prepared by a guanidine method using the ULTRASPEC RNA isolation system (Biotecx Laboratories, Inc., Houston, TX), and mRNA was prepared using oligo (dT) cellulose and a spin column (FastTrack 2.0 Kit from Invitrogen Corp., Carlsbad, CA).
2.3. Subtracti6e hybridization We used a PCR-Select cDNA Subtraction Kit for subtractive hybridization (Clontech Laboratories, Inc., Palo Alto, CA). Briefly, two kinds of cDNA were prepared, one from mRNA from cells incubated with 1 mM [Sar1]-angiotensin II for 48 h (A-II-cDNA), the other from mRNA of control cells. The cDNAs were digested with the restriction enzyme, Rsa I. The cDNA from the A-II stimulated cells was divided into two groups, and ligated with specially designed adapters for each group to serve as tester cDNAs. The digested cDNA from the control cells served as the driver cDNA. After denaturing, we added excess amount of driver cDNA to each of the two tester cDNAs and performed the first hybridization. A second hybridization was performed by mixing the two of the first hybridization solution with freshly denatured driver cDNA. After filling the ends of the cDNAs, polymerase chain reaction was done twice using this product as a template and primers with complementary sequences to a part of each adapter. This procedure lead to the normalization of transcript abundance and enrichment of rare cDNAs, in this case stimulated by A-II (Diatchenko et al., 1996).
2.5. Re6erse transcriptase-polymerase chain reaction (RT-PCR) cDNA was prepared from mRNA of A-II stimulated
H. Daido et al. / Molecular and Cellular Endocrinology 176 (2001) 21–27
cells and control cells by using reverse transcriptase (Superscript II form Life Technologies, Gaithersburg, MD). The total volume of PCR mixture was 50 ml, and it consisted of 5 ml of 10 × reaction buffer, 1 ml of dNTP mix (10 mM each), 0.4 mM of primers, 2 ml of RT reaction mixture and 1 ml of Advantage cDNA Polymerase Mix (from Clontech Laboratories, Inc., Palo Alto, CA). The PCR reactions were carried out under the following conditions, 94°C, 1 min; 60°C, 1 min; 72°C, 1 min for at most 35 cycles, followed by 72°C, 7 min. The sequence of primers are as follows; sense primer for the 9-27 gene; 5%-ACT TCC TTC CCC AAA GCC AGA A-3%, antisense primer for the 9-27 gene; 5%-ACG GAG TAG GCG AAT GCT ATG A-3%, sense primer for the 1-8D and 1-8U genes; 5%-ATG TCG TCT GGT CCC TGT TC-3%, antisense primer for the 1-8D and 1-8U genes; 5%-GCC ATT GTA GAA AAG CGT GT-3%, sense primer for No. 30 gene; 5%-CAG CCT CCC AAC TAC GAG AT-3%, anti sense primer for No. 30 gene; 5%-GTA ACC CCG TTT TTC CTG TA-3%, sense primer for human interferon (IFN)-b; 5%-TGC TAT CCT GTT GTG CTT CTC C-3%, antisense primer for IFN-b; 5%-CTG ATG ATA GAC ATT AGC CAG G-5%, sense primer for IFN-r; 5%-CAG ATG TAG CGG ATA ATG GAA C-3%, antisense primer for IFN-r; 5%-GGC GAC AGT TCA GCC ATC ACT T-3%. The sizes of PCR products were expected to be 204 bp for the 9-27 gene, 436 bp for the 1-8D gene, 368 bp for the 1-8U gene, 392 bp for No. 30 gene, 318 bp for IFN-ß, and 306 bp for IFN-g. The RT-PCR reactions were done twice with similar results.
2.6. Ribonuclease protection assay (RPA) A RNA probe of the 9-27 gene was prepared and labeled with biotin. The size of the fully protected fragment was 424 bp (Fig. 3). The probe for human a-actin was made from pTRI-a-actin-Human (Ambion, Austin, TX). RPA was performed with 40 mg total RNA by using a RPA III kit (Ambion, Austin, TX). After electrophoresis of the samples on a 5% denaturing polyacrylamide gel, RNA was transferred to a positively charged membrane by electroblotting, and crosslinked by UV. The biotinilated probe was detected by a chemiluminescent detection system described above.
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The sequence of this cDNA contained a full length transcript and was highly homologous to three kinds of human interferon-inducible genes 9-27, 1-8D and 1-8U (Hayzer et al., 1992; Deblandre et al., 1995; Lewin et al., 1991) (Fig. 2). Due to the high homology among these genes, we were unable to determine which gene contributed the most to the difference in the Northern blotting. RT-PCR revealed that all three genes, 9-27, 1-8D and 1-8U were expressed in H295R cells and their expression was stimulated by A-II. The transcript of 9-27 gene was most abundant (Fig. 3a). We could not detect a PCR product with the primers for No. 30 by RT-PCR, even at 35 cycles, whereas it was very easily and specifically detected using the No. 30 cDNA template. The No. 30 cDNA isolated consisted of sequence, which were identical to the 5% of gene 1-8D and the 3% of gene 9-27. The inability to detect mRNA for the isolated gene suggested that this cDNA was a PCR artifact. RPA showed that the 9-27 gene was stimulated by A-II in time dependent manner up to 48 h. Potassium 12 mM and forskolin 10 mM also stimulated the expression of this gene, but the effect of forskolin did not last as long (Fig. 3b). We also detected weak bands around 150 bp in the RPA. These are consistent with weak expression of the 1-8D and/or 1-8U genes, but we could not determine if they were differentially expressed by A-II. To determine if the stimulation of gene 9-27 was due to stimulation of interferon production, we performed RT-PCR for IFN-b or -g in H295R cells. Expression was not clearly detectable in either A-II stimulated cells or control cells (Fig. 4a). Incubation of H295R cells with interferon-g produced massive stimulation of the expression of the 9-27 gene compared with that produced by A-II (Fig. 4b). There was no significant simulation in aldosterone production between interferon-g stimulated cells and control cells (data not shown).
3. Results Using a Northern blot, only one of the 40 clones (No. 30) exhibited a significant differential expression in cells stimulated with A-II (Fig. 1). The others exhibited little or no difference between A-II stimulated cells and control cells.
Fig. 1. Northern blotting using No. 30 gene probe. There is a clear difference between mRNA from A-II stimulated cells and control cells. Each lane had 1.5 mg of mRNA.
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Fig. 2. Sequences of the clone of No. 30, 1-8D, 1-8U, and 1-8D genes. Numbering on the figure starts at the 5% end of No. 30, and other three genes. A point (.) indicates identity; (-) a gap in the sequence; (*) beginning and end of full protected fragment in RPA of 9-27 gene.
4. Discussion Using suppression subtractive hybridization and three steps of screening, we identified one cDNA clone
whose expression was stimulated by A-II in H295R cells. It was surprising that only one clone was identified using the technique, however, it is very likely that other genes are differentially expressed after A-II, but
H. Daido et al. / Molecular and Cellular Endocrinology 176 (2001) 21–27
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the technique used, Northern blot, was too insensitive to detect the transcripts. In addition, we only screened about 10 –20% of the clones (784 clones) that grew in the subtraction library. The gene was highly homologous with three interferon-inducible genes, 9-27, 1-8D, and 1-8U. The 5% region of the isolated clone was identical to that of the 1-8D gene; its 3% region was identical to that of the 9-27 gene. Studies using specific primers, which could easily amplify the isolated cDNA did not amplify it in tissues in which expression was indicated by Northern blot. This strongly suggests that this clone is a PCR artifact. However, this led to the study of other gene products from the family of Interferon-stimulated genes belonging to 1-8 gene family. This gene family is mainly expressed in leukocyte cell lines, endothelial cells and consists of at least these three genes and one or more pseudogenes (Lewin et al., 1991; Jaffe et al., 1989). These genes are highly inducible by both type I (a, ß) and type II (g) interferons (Lewin et al., 1991; Reid et al., 1989). Transcripts of these genes are thought to mediate interferon functions, including antiproliferative and differentiating effects
Fig. 4. Expression of interferons in H295R cells. We used RT (reverse transcription) reaction mixture (RT( +)) and mRNA without RT (RT(− )) as a template, and carried out PCR for 35 cycles. (b) Regulation of 9-27 gene by interferon-g (IFN-g) and angiotensin-II (A-II). About 40 mg of total RNA was used for RPA. 1. A-II 1 mM 48 h, 2. IFN-g 100 U/ml 24 h, 3. IFN-g 100 U/ml 48 h, 4. IFN-g 1000 U/ml 24 h, 5. IFN-g 1000 U/ml 48 h, 6. IFN-g 1000 U/ml+ A-II 1 mM 24 h, 7. IFN-g 1000 U/ml+A-II 1 mM 48 h, 8. Control 48 h.
Fig. 3. (a) Expression of interferon-inducible genes. We used undiluted RT product as a template for 1-8 D and U gene, and 1:10 diluted RT product for 9-27 gene or G3PDH. In these results of RT-PCR, 9-27 gene was at the end of the 24th cycle, 1-8D and U was at the end of the 26th cycle, G3PDH was at the end of 22nd cycle. (b) Regulation of 9-27 gene by angiotensin-II (A-II; 1 mM), potassium (12 mM), and forskolin (10 mM). We used 40 mg of total RNA for RPA.
(Deblandre et al., 1995; Sato et al., 1997). Of these, the 9-27 gene was most abundant in H295R cells. The transcript of 9-27 gene is also known as the Leu-13 antigen (Chen et al., 1984; Deblandre et al., 1995), which in the leukocyte is part of a membrane complex of proteins involved in the transduction of antiproliferative and homotypic adhesion signals (Evans et al., 1990; Deblandre et al., 1995). The functions of other genes in this family are still unknown.
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H. Daido et al. / Molecular and Cellular Endocrinology 176 (2001) 21–27
Beside interferons, it was reported that radiation stimulated expression of 1-8D and 9-27 genes in a leukemic cell line (Clave et al., 1997). This is the first report that this gene family is expressed in H295R cells, an adrenocortical carcinoma cell line, and that their expression could be stimulated by AII. Cytokines are mainly produced in lymphocytes or macrophages and are mediators of the immune system, though some other cells also produce them. The cytokines which are known to be produced in adrenal cells are interleukin-1 (IL-1), Interferon-g-inducing factor (IGIF), IL-6, and tissue necrosis factor ß (TNFa) (Ehrhart-Bornstein et al., 1998). Adrenal-produced cytokines are thought to work in a paracrine way. In this study, we could not clearly identify the expression of interferon-b or -g in H295R cells. Interferon-g is a much more potent inducer of the expression of these genes than is A-II. The significance of interferon-inducible genes in the adrenal is not certain, but the increase in their expression does not stimulate aldosterone secretion. Potassium also stimulated gene expression, though the effect was much weaker than A-II. Recently, it was reported that potassium stimulated local renin–angiotensin system inducing aldosterone secretion in NCIH295 cells (Hilbers et al., 1999). The stimulatory effect of potassium might be mediated by the local renin–angiotensin system. Forskolin also stimulated a transient expression; its effect was decreased after 48 h (Fig. 3b). Other peptides whose post-receptor signal is mediated by adenylate cyclase, for example ACTH, might also stimulate the expression of this gene. A-II stimulates the proliferation of adrenal glomerulosa cells. This reaction is mediated by angiotensin-1 (AT1) receptor; the PKC and lipoxygenase pathways have an important roles in this reaction (Natarajan et al., 1992). The transcript of 9-27 gene, whose production is also stimulated by A-II, has an opposite, antiproliferative effect in other cells. In this study, we identified the expression of interferon-inducible genes, 9-27, 1-8D and 1-8U in H295R cells. The expression of these genes was stimulated by A-II. The 9-27 gene transcript, which is known to have antiproliferative effect, was most abundant. It is possible that these genes might contribute to the regulation of adrenal glomerulosa cell proliferation and an abnormality of this gene might induce pathological proliferation of adrenocortical cells.
Acknowledgements These studies were supported by Medical Research Funds from the Department of Veterans Affairs and NHLBI/NIH grants HL 27255.
References Bird, I.M., Hanley, N.A., Word, R.A., Mathis, J.M., McCarthy, J.L., Mason, J.I., Rainey, W.E., 1993. Human NCI-H295 adrenocortical carcinoma cells: a model for angiotensin-II-responsive aldosterone secretion. Endocrinology 133, 1555 – 1561. Chen, Y.X., Welte, K., Gebhard, D.H., Evans, R.L., 1984. Induction of T cell aggregation by antibody to a 16 kDa human leukocyte surface antigen. J. Immunol. 133, 2496 – 2501. Clave, E., Carosella, E.D., Gluckman, E., Socie´ , G., 1997. Radiationenhanced expression of interferon-inducible genes in the KG1a primitive hematopoietic cell line. Leukemia 11, 114 – 119. Deblandre, G.A., Marinx, O.P., Evans, S.S., Majjaj, S., Leo, O., Caput, D., Huez, G.A., Wathelet, M.G., 1995. Expression cloning of an interferon-inducible 17-kDa membrane protein implicated in the control of cell growth. J. Biol. Chem. 270, 23860 – 23866. Denner, K., Rainey, W.E., Pezzi, V., Bird, I.M., Bernhardt, R., Mathis, J.M., 1996. Differential regulation of 11 beta-hydroxylase and aldosterone synthase in human adrenocortical H295R cells. Mol. Cell. Endocrinol. 121, 87 – 91. Diatchenko, L., Lau, Y.-F.C., Campbell, A.P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E., Siebert, P.D., 1996. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93, 6025 – 6030. Ehrhart-Bornstein, M., Hinson, J.P., Bornstein, S.R., Scherbaum, W.A., 1998. Intra adrenal interactions in the regulation of adrenocortical steroidogenesis. Endocr. Rev. 19, 101 – 143. Evans, S.S., Lee, D.B., Han, T., Tomasi, T.B., Evans, R.L., 1990. Monoclonal antibody to the interferon-inducible protein Leu-13 triggers aggregation and inhibits proliferation of leukemic B cells. Blood 76, 2583 – 2593. Gazdar, A.F., Oie, H.K., Shackleton, C.H., Chen, T.R., Triche, T.J., Myers, C.E., Brennan, M.F., Stein, C.A., La Rocca, R.V., 1990. Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Res. 50, 5488 – 5496. Gomez-Sanchez, C.E., 1998. Primary aldosteronism and its variants. Cardiovasc. Res. 37, 8 – 13. Gomez-Sanchez, C.E., Foecking, M.F., Ferris, M.W., Chavarri, M.R., Uribe, L., Gomez-Sanchez, E.P., 1987. The production of monoclonal antibodies against aldosterone. Steroids 49, 581 –587. Hayzer, D.J., Brison, E., Runge, M.S., 1992. A rat b-interferon-induced mRNA: sequence characterization. Gene 117, 277 –278. Hilbers, U., Peters, J., Bornstein, S.R., Correa, F.M.A., Jo¨ hren, O., Saavedra, J.M., Ehrhart-Bornstein, M., 1999. Local renin –angiotensin system is involved in K+-induced aldosterone secretion from human adrenocortical NCI-H295 cells. Hypertension 33, 1025 – 1030. Jaffe, E.A., Armellino, D., Lam, G., Cordon-Cardo, C., Murray, H.W., Evans, R.L., 1989. IFN-g and IFN-a induce the expression and synthesis of leu 13 antigen by cultured human endothelial cells. J. Immunol. 143, 3961 – 3966. Lewin, A.R., Reid, L.E., McMahon, M., Stark, G.R., Kerr, I.M., 1991. Molecular analysis of a human interferon-inducible gene family. Eur. J. Biochem. 199, 417 – 423. Mitani, F., Suzuki, H., Hata, J., Ogishima, T., Shimada, H., Ishimura, Y., 1994. A novel cell layer without corticosterone-synthesizing enzymes in rat adrenal cortex: histochemical detection and possible physiological role. Endocrinology 135, 431 –438. Natarajan, R., Gonzales, N., Hornsby, P.J., Nadler, J., 1992. Mechanism of angiotensin II-induced proliferation in bovine adrenocortical cells. Endocrinology 131, 1174 – 1180. Rainey, W.E., Bird, I.M., Sawetawan, C., Hanley, N.A., McCarthy, J.L., McGee, E.A., Mason, J.I., 1993. Regulation of human
H. Daido et al. / Molecular and Cellular Endocrinology 176 (2001) 21–27 adrenal carcinoma cell (NCI-H295) production of C19 steroids. J. Clin. Endocrinol. Metab. 77, 731 –737. Rainey, W.E., Bird, I.M., Mason, J.I., 1994. The NCI-H295 cell line: a pluripotent model for human adrenocortical studies. Mol. Cell. Endocrinol. 100, 45 – 50. Reid, L.R., Brasnett, A.H., Gilbert, C.S., Porter, A.C.G., Gewert, D.R., Stark, G.R., Kerr, I.M., 1989. A single DNA response element can confer inducibility by both a- and g-interferons. Proc. Natl. Acad. Sci. USA 86, 840 –844. Sato, S., Miller, A.S., Howard, M.C., Tedder, T.F., 1997. Regulation B lumphocyte development and activation by CD19/CD21/CD81/ Leu 13 complexes requires the cytoplasmic domain of CD19. J. Immunol. 159, 3278 –3287.
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Tremblay, A., Parker, K.L., Lehoux, J.G., 1992. Dietary potassium supplementation and sodium restriction stimulate aldosterone synthase but not 11b-hydroxylase P-450 messenger ribonucleic acid accumulation in rat adrenals and require angiotensin II production. Endocrinology 130, 3152 – 3158. Vinson, G.P., Ho, M.M., Puddefoot, J.R., 1998a. Adrenocortical zonation and the adrenal renin – angiotensin system. Endocr. Res. 24, 677 – 686. Vinson, G.P., Teja, R., Ho, M.M., Hinson, J.P., Puddefoot, J.R., 1998b. The role of the tissue renin – angiotensin system in the response of the rat adrenal to exogenous angiotensin II. J. Endocrinol. 158, 153 – 159.
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Angiotensin stimulates the expression of interferon-inducible genes in H295R cells Hisashi Daido a,b,e, Ming-Yi Zhou a, Celso E. Gomez-Sanchez a,c,d,* a
Di6ision of Endocrinology, Uni6ersity of Missouri, Columbia, MO 65212, USA Harry S. Truman Memorial Veterans Hospital, Columbia, MO 65201, USA c Di6ision of Endocrinology, Department of Medicine, The Uni6ersity of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216 -4505, USA d G.V. (Sonny) Montgomery VA Medical Center, Jackson, MS 39216, USA e Department of Medicine, Gifu Uni6ersity, Gifu, Japan b
Received 1 December 2000; accepted 12 March 2001
Abstract Angiotensin-II (A-II) induces proliferation of zona glomerulosa cells and stimulates expression of cytochrome P-450 aldosterone synthase. The genes activated during this adrenal remodeling are not well defined. To clarify this mechanism, we sought to identify the genes whose expression is stimulated by A-II in the H295R cell line. Using a subtractive hybridization technique, we identified one clone whose expression was stimulated by A-II. The sequence of this gene was homologous to the human interferon-inducible genes, 9-27, 1-8D and 1-8U. The 5% portion of the gene was identical to the 1-8D gene product and the 3% was identical to the 9-27 gene product, but the existence of a transcript was not demonstrated by RT-PCR. The expression of these three genes was stimulated by A-II, with the 9-27 gene being most abundant. Potassium and forskolin also stimulated the expression of the 9-27 gene in the H295R cells, but not as effectively as did A-II or interferon-g. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Angiotensin; Hybridization; Zona glomerulosa
1. Introduction Aldosterone (Aldo) synthesis in the zona glomerulosa of the adrenal gland is under the primary control of Angiotensin II (A-II). Physiological conditions like the adaptation to sodium depletion and diseases associated with chronic stimulation of the renin – angiotensin system result in the stimulation of aldosterone synthesis. A-II increases gene transcription and expression of cytochrome P-450 aldosterone synthase (CYP11B2), the final enzyme in the synthesis of aldosterone, and promotes adrenal zona glomerulosa remodeling (Tremblay et al., 1992; Mitani et al., 1994). Primary aldosteronism, a common cause of hypertension in humans, is caused by the excessive secretion of aldosterone by the adrenal zona glomerulosa (Gomez-Sanchez, 1998) and is caused * Corresponding author. Tel.: + 1-601-9845525; fax: +1-6019845769. E-mail address: [email protected] (C.E. Gomez-Sanchez).
by either the presence of an adrenal tumor or by bilateral zona glomerulosa hyperplasia. The cause and mechanisms of adrenal remodeling in bilateral zona glomerulosa hyperplasia is unknown. The H295R cell line is a pluripotential human adrenocortical carcinoma cell line (Gazdar et al., 1990; Bird et al., 1993; Rainey et al., 1993, 1994; Denner et al., 1996) shown to be a good model system to define mechanisms regulating human aldosterone production (Bird et al., 1993). A-II action in the adrenal not only involves an increase in the expression of the aldosterone synthase enzyme, but also an increase in cell proliferation (Vinson et al., 1998a,b). We attempted to identify genes that are stimulated by A-II in H295R cells using a subtractive hybridization technique (Diatchenko et al., 1996). This technique uses a powerful method to normalize transcript abundance, and enrich rare cDNAs that in this case were stimulated by A-II. We describe the identification of a gene product that is expressed after stimulation with A-II.
0303-7207/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 1 ) 0 0 4 7 8 - 6
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H. Daido et al. / Molecular and Cellular Endocrinology 176 (2001) 21–27
2. Materials and methods
2.4. Screening of the subtraction library
2.1. Materials
A library of the subtracted cDNA was made by TA cloning (pGEM-T Vector system from Promega Corp., Madison, WI) and used to transform competent bacterial cells (One shot TOP10F% from Invitrogen Corp., Carlsbad, CA). About 784 bacterial colonies were picked at random and screened. For the first screening, we used the PCR-Select Differential Screening kit (Clontech Laboratories, Inc., Palo Alto, CA), with modifications. Two kinds of DNA probes were made. One of them, a subtracted probe, was made from cDNA from the above procedure. The other, a reverse-subtracted probe was made from reverse subtraction cDNA. This cDNA was constructed by the same method as subtractive hybridization. But in this case, tester cDNA was control cDNA, and driver cDNA was A-II-stimulated cells cDNA. Both probes were then labeled with biotin. The 784 clones were arrayed and grown in duplicate on nylon membranes and screened with two kinds of probes. The membranes were baked in a microwave oven and digested with proteinase K (200 mg/ml at 37°C for 1 h), followed by hybridization with the above probes. Hybridization solution consisted of 0.5 M Na2HPO4, 2 mM EDTA and 7% SDS, and 250 ng/ml of probe. After the overnight hybridization at 65°C, the membranes were washed three times. The first wash solution was 2× SSC and 1% SDS for 20 min, second was 1× SSC and 1% SDS for 20 min, final wash was 0.1× SSC and 1% SDS for 30 min at 50°C. Finally, the hybridized biotinilated probe was detected by a chemiluminescent detection system (BrightStar BioDetect from Ambion, Austin, TX). There were 159 positive colonies. Plasmids from the positive colonies were prepared by miniprep (Wizard Plus Minipreps from Promega Corp., Madison, WI), and screened by reverse dot blot technique. Denatured plasmids (2.5 mg) were placed on duplicate nylon membranes and crosslinked by UV light. The probes used were biotin-labeled cDNA made from mRNA of A-II stimulated cells and control cells using reverse transcriptase (Superscript-II form Life Technologies, Gaithersburg, MD). Hybridization, washing and detection methods were same as for the first screening. There were 40 clones that were positive only when using the probe generated from mRNA of A-II stimulated cells. These were assayed by conventional Northern blot technique with 32P-labeled probes using RNA from control and A-II stimulated cells. One clone was clearly differentially expressed in A-II-stimulated cells.
DME/F12 medium was obtained from the CIC Core from the University of Missouri, Columbia. HyQCCM1 medium was obtained from Hyclone, (Logan, UT). Human interferon-g was purchased from PeproTech Inc. (Rocky Hill, NJ). Other reagents were obtained from Sigma Chemical Company (St. Louis, MO).
2.2. Incubation of H295R cells and RNA preparation H295R cells were grown in HyQ-CCM1 medium with 2% horse serum. The media was changed to serum-free DME/F12 medium and incubated for 24 h before the experiments because HyQ-CCM1 medium stimulates H295R cells to secrete aldosterone. Cells were incubated with or without stimulant in serum-free DME/F12 medium. [Sar1]-angiotensin-II and forskolin were purchased from Sigma (St. Louis, MO). After the incubation, aldosterone concentration in the medium was measured by ELISA (Gomez-Sanchez et al., 1987). Total RNA was prepared by a guanidine method using the ULTRASPEC RNA isolation system (Biotecx Laboratories, Inc., Houston, TX), and mRNA was prepared using oligo (dT) cellulose and a spin column (FastTrack 2.0 Kit from Invitrogen Corp., Carlsbad, CA).
2.3. Subtracti6e hybridization We used a PCR-Select cDNA Subtraction Kit for subtractive hybridization (Clontech Laboratories, Inc., Palo Alto, CA). Briefly, two kinds of cDNA were prepared, one from mRNA from cells incubated with 1 mM [Sar1]-angiotensin II for 48 h (A-II-cDNA), the other from mRNA of control cells. The cDNAs were digested with the restriction enzyme, Rsa I. The cDNA from the A-II stimulated cells was divided into two groups, and ligated with specially designed adapters for each group to serve as tester cDNAs. The digested cDNA from the control cells served as the driver cDNA. After denaturing, we added excess amount of driver cDNA to each of the two tester cDNAs and performed the first hybridization. A second hybridization was performed by mixing the two of the first hybridization solution with freshly denatured driver cDNA. After filling the ends of the cDNAs, polymerase chain reaction was done twice using this product as a template and primers with complementary sequences to a part of each adapter. This procedure lead to the normalization of transcript abundance and enrichment of rare cDNAs, in this case stimulated by A-II (Diatchenko et al., 1996).
2.5. Re6erse transcriptase-polymerase chain reaction (RT-PCR) cDNA was prepared from mRNA of A-II stimulated
H. Daido et al. / Molecular and Cellular Endocrinology 176 (2001) 21–27
cells and control cells by using reverse transcriptase (Superscript II form Life Technologies, Gaithersburg, MD). The total volume of PCR mixture was 50 ml, and it consisted of 5 ml of 10 × reaction buffer, 1 ml of dNTP mix (10 mM each), 0.4 mM of primers, 2 ml of RT reaction mixture and 1 ml of Advantage cDNA Polymerase Mix (from Clontech Laboratories, Inc., Palo Alto, CA). The PCR reactions were carried out under the following conditions, 94°C, 1 min; 60°C, 1 min; 72°C, 1 min for at most 35 cycles, followed by 72°C, 7 min. The sequence of primers are as follows; sense primer for the 9-27 gene; 5%-ACT TCC TTC CCC AAA GCC AGA A-3%, antisense primer for the 9-27 gene; 5%-ACG GAG TAG GCG AAT GCT ATG A-3%, sense primer for the 1-8D and 1-8U genes; 5%-ATG TCG TCT GGT CCC TGT TC-3%, antisense primer for the 1-8D and 1-8U genes; 5%-GCC ATT GTA GAA AAG CGT GT-3%, sense primer for No. 30 gene; 5%-CAG CCT CCC AAC TAC GAG AT-3%, anti sense primer for No. 30 gene; 5%-GTA ACC CCG TTT TTC CTG TA-3%, sense primer for human interferon (IFN)-b; 5%-TGC TAT CCT GTT GTG CTT CTC C-3%, antisense primer for IFN-b; 5%-CTG ATG ATA GAC ATT AGC CAG G-5%, sense primer for IFN-r; 5%-CAG ATG TAG CGG ATA ATG GAA C-3%, antisense primer for IFN-r; 5%-GGC GAC AGT TCA GCC ATC ACT T-3%. The sizes of PCR products were expected to be 204 bp for the 9-27 gene, 436 bp for the 1-8D gene, 368 bp for the 1-8U gene, 392 bp for No. 30 gene, 318 bp for IFN-ß, and 306 bp for IFN-g. The RT-PCR reactions were done twice with similar results.
2.6. Ribonuclease protection assay (RPA) A RNA probe of the 9-27 gene was prepared and labeled with biotin. The size of the fully protected fragment was 424 bp (Fig. 3). The probe for human a-actin was made from pTRI-a-actin-Human (Ambion, Austin, TX). RPA was performed with 40 mg total RNA by using a RPA III kit (Ambion, Austin, TX). After electrophoresis of the samples on a 5% denaturing polyacrylamide gel, RNA was transferred to a positively charged membrane by electroblotting, and crosslinked by UV. The biotinilated probe was detected by a chemiluminescent detection system described above.
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The sequence of this cDNA contained a full length transcript and was highly homologous to three kinds of human interferon-inducible genes 9-27, 1-8D and 1-8U (Hayzer et al., 1992; Deblandre et al., 1995; Lewin et al., 1991) (Fig. 2). Due to the high homology among these genes, we were unable to determine which gene contributed the most to the difference in the Northern blotting. RT-PCR revealed that all three genes, 9-27, 1-8D and 1-8U were expressed in H295R cells and their expression was stimulated by A-II. The transcript of 9-27 gene was most abundant (Fig. 3a). We could not detect a PCR product with the primers for No. 30 by RT-PCR, even at 35 cycles, whereas it was very easily and specifically detected using the No. 30 cDNA template. The No. 30 cDNA isolated consisted of sequence, which were identical to the 5% of gene 1-8D and the 3% of gene 9-27. The inability to detect mRNA for the isolated gene suggested that this cDNA was a PCR artifact. RPA showed that the 9-27 gene was stimulated by A-II in time dependent manner up to 48 h. Potassium 12 mM and forskolin 10 mM also stimulated the expression of this gene, but the effect of forskolin did not last as long (Fig. 3b). We also detected weak bands around 150 bp in the RPA. These are consistent with weak expression of the 1-8D and/or 1-8U genes, but we could not determine if they were differentially expressed by A-II. To determine if the stimulation of gene 9-27 was due to stimulation of interferon production, we performed RT-PCR for IFN-b or -g in H295R cells. Expression was not clearly detectable in either A-II stimulated cells or control cells (Fig. 4a). Incubation of H295R cells with interferon-g produced massive stimulation of the expression of the 9-27 gene compared with that produced by A-II (Fig. 4b). There was no significant simulation in aldosterone production between interferon-g stimulated cells and control cells (data not shown).
3. Results Using a Northern blot, only one of the 40 clones (No. 30) exhibited a significant differential expression in cells stimulated with A-II (Fig. 1). The others exhibited little or no difference between A-II stimulated cells and control cells.
Fig. 1. Northern blotting using No. 30 gene probe. There is a clear difference between mRNA from A-II stimulated cells and control cells. Each lane had 1.5 mg of mRNA.
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H. Daido et al. / Molecular and Cellular Endocrinology 176 (2001) 21–27
Fig. 2. Sequences of the clone of No. 30, 1-8D, 1-8U, and 1-8D genes. Numbering on the figure starts at the 5% end of No. 30, and other three genes. A point (.) indicates identity; (-) a gap in the sequence; (*) beginning and end of full protected fragment in RPA of 9-27 gene.
4. Discussion Using suppression subtractive hybridization and three steps of screening, we identified one cDNA clone
whose expression was stimulated by A-II in H295R cells. It was surprising that only one clone was identified using the technique, however, it is very likely that other genes are differentially expressed after A-II, but
H. Daido et al. / Molecular and Cellular Endocrinology 176 (2001) 21–27
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the technique used, Northern blot, was too insensitive to detect the transcripts. In addition, we only screened about 10 –20% of the clones (784 clones) that grew in the subtraction library. The gene was highly homologous with three interferon-inducible genes, 9-27, 1-8D, and 1-8U. The 5% region of the isolated clone was identical to that of the 1-8D gene; its 3% region was identical to that of the 9-27 gene. Studies using specific primers, which could easily amplify the isolated cDNA did not amplify it in tissues in which expression was indicated by Northern blot. This strongly suggests that this clone is a PCR artifact. However, this led to the study of other gene products from the family of Interferon-stimulated genes belonging to 1-8 gene family. This gene family is mainly expressed in leukocyte cell lines, endothelial cells and consists of at least these three genes and one or more pseudogenes (Lewin et al., 1991; Jaffe et al., 1989). These genes are highly inducible by both type I (a, ß) and type II (g) interferons (Lewin et al., 1991; Reid et al., 1989). Transcripts of these genes are thought to mediate interferon functions, including antiproliferative and differentiating effects
Fig. 4. Expression of interferons in H295R cells. We used RT (reverse transcription) reaction mixture (RT( +)) and mRNA without RT (RT(− )) as a template, and carried out PCR for 35 cycles. (b) Regulation of 9-27 gene by interferon-g (IFN-g) and angiotensin-II (A-II). About 40 mg of total RNA was used for RPA. 1. A-II 1 mM 48 h, 2. IFN-g 100 U/ml 24 h, 3. IFN-g 100 U/ml 48 h, 4. IFN-g 1000 U/ml 24 h, 5. IFN-g 1000 U/ml 48 h, 6. IFN-g 1000 U/ml+ A-II 1 mM 24 h, 7. IFN-g 1000 U/ml+A-II 1 mM 48 h, 8. Control 48 h.
Fig. 3. (a) Expression of interferon-inducible genes. We used undiluted RT product as a template for 1-8 D and U gene, and 1:10 diluted RT product for 9-27 gene or G3PDH. In these results of RT-PCR, 9-27 gene was at the end of the 24th cycle, 1-8D and U was at the end of the 26th cycle, G3PDH was at the end of 22nd cycle. (b) Regulation of 9-27 gene by angiotensin-II (A-II; 1 mM), potassium (12 mM), and forskolin (10 mM). We used 40 mg of total RNA for RPA.
(Deblandre et al., 1995; Sato et al., 1997). Of these, the 9-27 gene was most abundant in H295R cells. The transcript of 9-27 gene is also known as the Leu-13 antigen (Chen et al., 1984; Deblandre et al., 1995), which in the leukocyte is part of a membrane complex of proteins involved in the transduction of antiproliferative and homotypic adhesion signals (Evans et al., 1990; Deblandre et al., 1995). The functions of other genes in this family are still unknown.
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Beside interferons, it was reported that radiation stimulated expression of 1-8D and 9-27 genes in a leukemic cell line (Clave et al., 1997). This is the first report that this gene family is expressed in H295R cells, an adrenocortical carcinoma cell line, and that their expression could be stimulated by AII. Cytokines are mainly produced in lymphocytes or macrophages and are mediators of the immune system, though some other cells also produce them. The cytokines which are known to be produced in adrenal cells are interleukin-1 (IL-1), Interferon-g-inducing factor (IGIF), IL-6, and tissue necrosis factor ß (TNFa) (Ehrhart-Bornstein et al., 1998). Adrenal-produced cytokines are thought to work in a paracrine way. In this study, we could not clearly identify the expression of interferon-b or -g in H295R cells. Interferon-g is a much more potent inducer of the expression of these genes than is A-II. The significance of interferon-inducible genes in the adrenal is not certain, but the increase in their expression does not stimulate aldosterone secretion. Potassium also stimulated gene expression, though the effect was much weaker than A-II. Recently, it was reported that potassium stimulated local renin–angiotensin system inducing aldosterone secretion in NCIH295 cells (Hilbers et al., 1999). The stimulatory effect of potassium might be mediated by the local renin–angiotensin system. Forskolin also stimulated a transient expression; its effect was decreased after 48 h (Fig. 3b). Other peptides whose post-receptor signal is mediated by adenylate cyclase, for example ACTH, might also stimulate the expression of this gene. A-II stimulates the proliferation of adrenal glomerulosa cells. This reaction is mediated by angiotensin-1 (AT1) receptor; the PKC and lipoxygenase pathways have an important roles in this reaction (Natarajan et al., 1992). The transcript of 9-27 gene, whose production is also stimulated by A-II, has an opposite, antiproliferative effect in other cells. In this study, we identified the expression of interferon-inducible genes, 9-27, 1-8D and 1-8U in H295R cells. The expression of these genes was stimulated by A-II. The 9-27 gene transcript, which is known to have antiproliferative effect, was most abundant. It is possible that these genes might contribute to the regulation of adrenal glomerulosa cell proliferation and an abnormality of this gene might induce pathological proliferation of adrenocortical cells.
Acknowledgements These studies were supported by Medical Research Funds from the Department of Veterans Affairs and NHLBI/NIH grants HL 27255.
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