Abstract
Testosterone has been previously shown to induce persistent susceptibility to Plasmodium chabaudi malaria in otherwise resistant female C57BL/6 mice. Here, we investigate as to whether this conversion coincides with permanent changes of hepatic gene expression profiles. Female mice aged 10–12 weeks were treated with testosterone for 3 weeks; then, testosterone treatment was discontinued for 12 weeks before challenging with 106 P. chabaudi-infected erythrocytes. Hepatic gene expression was examined after 12 weeks of testosterone withdrawal and after subsequent infection with P. chabaudi at peak parasitemia, using Affymetrix microarrays with 22 690 probe sets representing 14 000 genes. The expression of 54 genes was found to be permanently changed by testosterone, which remained changed during malaria infection. Most genes were involved in liver metabolism: the female-prevalent genes Cyp2b9, Cyp2b13, Cyp3a41, Cyp3a44, Fmo3, Sult2a2, Sult3a1, and BC014805 were repressed, while the male-prevalent genes Cyp2d9, Cyp7b1, Cyp4a10, Ugt2b1, Ugt2b38, Hsd3b5, and Slco1a1 were upregulated. Genes encoding different nuclear receptors were not persistently changed. Moreover, testosterone induced persistent upregulation of genes involved in hepatocellular carcinoma such as Lama3 and Nox4, whereas genes involved in immune response such as Ifnγ and Igk-C were significantly decreased. Our data provide evidence that testosterone is able to induce specific and robust long-term changes of gene expression profiles in the female mouse liver. In particular, those changes, which presumably indicate masculinized liver metabolism and impaired immune response, may be critical for the testosterone-induced persistent susceptibility of mice to P. chabaudi malaria.
Introduction
Testosterone is known to increase susceptibility to a wide variety of infectious diseases (Müller 1992, Klein 2000, Roberts et al. 2001, Marriott & Huet-Hudson 2006), which also concerns human malaria (Müller 1992, Kurtis et al. 2001, Muehlenbein et al. 2005). In the experimental mouse malaria Plasmodium chabaudi, testosterone has been shown to induce a lethal outcome of otherwise self-healing infections (Wunderlich et al. 1988, 1991). Remarkably, this testosterone-induced conversion from resistance to susceptibility becomes somehow imprinted in female mice, i.e. it persists for rather a long time. Thus, when mice are pretreated with testosterone for 3 weeks, and then testosterone treatment is discontinued for 12 weeks, thereafter the mice are still susceptible to P. chabaudi infections (Benten et al. 1997). Obviously, this testosterone-induced susceptibility persists, even though the circulating testosterone levels have declined to those levels characteristic for female mice after withdrawal for 12 weeks (Benten et al. 1997). This indicates that testosterone is able to induce changes in mice, which continue to exist at low testosterone levels.
The liver is known to be a target organ for testosterone to mediate intrahepatic immune responses (Häussinger et al. 2004) and to play a central role in malaria. Indeed, the liver is not only that site in which the pre-erythrocytic development of malaria parasites takes place, but also it is an important effector against malarial blood stages (Balmer et al. 2000, Krücken et al. 2005), though largely neglected by current research. Moreover, specific populations of lymphocytes have been described to be generated in the liver, which mediate novel protective immune mechanisms against malaria blood stages in the mouse (Mannoor et al. 2001, 2002). Also, Kupffer cells are able to eliminate, via phagocytosis, parasite-derived material such as hemozoin and even Plasmodium-infected erythrocytes (Aikawa et al. 1980). Moreover, the liver is known for its sexual dimorphism, in particular for its sex- and testosterone-dependent pattern of phase I and phase II metabolism (Waxman & Holloway 2009). All this information led us to suppose that the liver may be one of those sites which is critically involved in mediating the suppressive testosterone effects on P. chabaudi malaria.
Testosterone acts on gene expression either directly through the androgen receptor (AR; Zhou et al. 1994, Quigley et al. 1995, Bennett et al. 2009) or indirectly by crosstalk with other signaling pathways (Guo et al. 2002, Rahman & Christian 2007, Wendler & Wehling 2009). Moreover, testosterone has been described to induce changes in gene expression, including those genes involved in liver metabolism (Kato & Onada 1970, Krücken et al. 2005, Waxman & Holloway 2009). However, there is no information available that testosterone is able to induce permanent changes in gene expression of the liver, i.e. changes that persist even after withdrawal of testosterone. This view prompted us to investigate possible permanent testosterone effects on hepatic gene expression using the Affymetrix microarray technology. At least, the present study provides evidence that testosterone is able to induce permanent changes in the expression of distinct genes in the liver, which, when once induced by treatment with testosterone for 3 weeks, remain existing even for at least 12 weeks after discontinuation of the testosterone treatment. In addition, the expression of such genes is rather robust upon infecting with P. chabaudi malaria, which is discussed with respect to relevance for the testosterone-induced persistent susceptibility of mice to P. chabaudi malaria.
Materials and methods
Mice
C57BL/6 mice were bred under specific pathogen-free conditions at the central animal facilities of our university. Experiments were performed with female mice. They were housed in plastic cages, and they received a standard diet (Wohrlin, Bad Salzuflen, Germany) and water ad libitum. The experiments were approved by the state authorities and followed German law on animal protection.
Testosterone treatment
Mice aged 10–12 weeks received s.c. injections of 100 μl sesame oil containing 0.9 mg testosterone (Testosterone-Depot-50, Schering, Berlin, Germany) twice a week for 3 weeks (Wunderlich et al. 1988, Benten et al. 1997). Controls were treated only with vehicle, i.e. sesame oil. Thereafter, they were kept under standard conditions for 12 weeks.
Infections
Blood stages of P. chabaudi were passaged weekly in NMRI mice (Wunderlich et al. 1982, Krücken et al. 2009). C57BL/6 mice were challenged with 106 P. chabaudi-parasitized erythrocytes. Parasitemia was determined in Giemsa-stained tail blood, and cell number was measured in a Neubauer chamber.
RNA isolation
Three mice per time point were killed by cervical dislocation, and livers were aseptically removed. Liver pieces were rapidly frozen in melting nitrogen and stored at −80 °C. For isolation of RNA, ∼250 mg frozen liver were homogenized with an ultra turrax in 5 ml Trizol (Peqlab Biotechnology, Erlangen, Germany) for 1 min, mixed with 1 ml chloroform for 15 s, incubated for 15 min at room temperature, and centrifugated at 3000 g for 45 min. The supernatant was treated with isopropanol and centrifugated, and the pellet was washed twice with 80% ethanol, air-dried, and dissolved in 200 μl RNase-free water. RNA concentrations were determined at 260 nm, and the quality of the RNA was examined with agarose gel electrophoresis.
Hybridization of microarrays
Quality control of RNA was performed with a Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany) on an RNA 6000 Nano chip. RNA was quantified using the RiboGreen RNA Quantitation kit (Molecular Probes, Leiden, The Netherlands). Biotin-labeled cRNA was synthesized from 5 μg total RNA using the One cycle kit (Affymetrix, Inc., Santa Clara, CA, USA) according to the manufacturer's protocol. Biotin-labeled cRNA (15 μg) was then hybridized to Affymetrix MOE430A Gene Chips for 16 h at 45 °C. The chips were stained and washed on an Affymetrix Fluidics Station 400, and the fluorescence of the hybridized cRNA was read with an Affymetrix 300 Scanner. The chips were quality controlled with the software ‘Expressionist Refiner’ (GeneData AG, Basel, Switzerland) detecting and correcting gradients, spots, and distortions. Each probe set is represented by 11 pairs of 25 mer perfect match and mismatch oligonucleotides. Using the MAS 5.0 statistical algorithms implemented in the Expressionist software, the intensities of all 11 probe pairs per probe were condensed to one intensity value. For comparability, the microarrays were scaled after condensing to an average signal intensity of 100.
Data analysis
Gene expression analysis was done using the software ‘Expression Analyst’ (GeneData AG). Gene expression profiles between individual mice were overall compared by principal component analysis using Genesis 1.7.2. (Sturn et al. 2002). To select testosterone-deregulated genes, we removed probe sets with an expression intensity <20 in each sample. In the next step, only those probe sets were selected, which were deregulated twofold by testosterone on day 0 post infectionem (p.i.) and on day 8 p.i., and these were subjected to two-way ANOVA (P<0.01). Genes were analyzed by the Database for Annotation, Visualization, and Integrated Discovery (Dennis et al. 2003) and categorized according to their major biological pathways involved. Genes with similar expression patterns were identified using Gene Cluster 3.0 (Eisen et al. 1998, de Hoon et al. 2004). Data were log2-transformed and normalized to the mean expression value for control mice. Hierachical clustering was done using uncentered correlation and average linkage mode.
Quantitative real-time PCR
All RNA samples were treated with DNase of the DNA-free kit (Applied Biosystems, Darmstadt, Germany) for 1 h and then converted into cDNA following the manufacturer's protocol using the QuantiTect Reverse Transcription (RT) kit (Qiagen). Amplifications were performed in the ABI Prism 7500HT Sequence Detection System (Applied Biosystems) using QuantiTect SYBR Green PCR kit (Qiagen) according to the manufacturer's instructions using gene-specific QuantiTect primer assays (Qiagen) for prominin 1 (Prom1), 5′-nucleotidase, ecto (Nt5e), sulfotransferase family 2A, dehydroepiandrosterone-preferring, member 2 (Sult2a2), 3 beta-hydroxysteroid dehydrogenase 5 (Hsd3b5), elongation of very long chain fatty acid-like 3 (Elovl3), interferon gamma (Ifnγ), peroxisome proliferator-activated receptor alpha (Pparα), liver X receptor (Lxr, listed as Nr1h3 in the MGI database), retinoid X receptor alpha (Rxrα), pregnane X receptor (Pxr, Nr1i2), farnesoid X receptor (Fxr, Nr1h4), Ar, estrogen receptor alpha (ERα, Esr1), estrogen receptor beta (ERβ, Esr2), aryl hydrocarbon receptor (Ahr), hepatic nuclear factor 4 alpha (Hnf4α), constitutive androstane receptor (Car, Nr1i3), and vitamin D receptor (VDR). PCRs were conducted as follows: 2 min at 50 °C to activate uracil-N-glycosylase (UNG), 95 °C for 10 min to deactivate UNG and to activate the PCR, 45 cycles at 94 °C for 15 s, at 60 °C for 30 s, and at 72 °C for 30 s. Reaction specificity was checked by performing dissociation curves after PCR. Relative quantitative evaluation of amplification data was done using Taqman7500 system software v.1.2.3f2 (Applied Biosystems), and the mRNA was normalized to 18S rRNA. The relative expression was measured using the 
Statistical analysis
Student's t-test was used for determination of significant differences (P<0.05).
Results
Experimental design
This study is aimed at identifying genes in the female mouse liver, i) the expression of which is persistently deregulated by testosterone and ii) which even remains deregulated during malaria infection. To this end, we followed the experimental outline given in Fig. 1. Female C57BL/6 mice were treated with testosterone or vehicle twice a week for 3 weeks. The testosterone-treated mice were previously shown to loose their capability of self-healing malaria infections when challenged with 106 P. chabaudi-infected erythrocytes (Wunderlich et al. 1988, 1991). The treatment was discontinued for 12 weeks, before the mice were challenged with P. chabaudi malaria reaching peak parasitemia of ∼50% on day 8 p.i. in both testosterone- and vehicle-pretreated mice. Previously, we have shown that, though the circulating testosterone levels have declined to values characteristic for untreated females, from 3.79 to 0.21 ng/ml, by the end of the 12-week period of testosterone withdrawal, the mice still succumb to malaria (Benten et al. 1997). Livers were aseptically removed from three animals at each of the four time points on days 0 and 8 p.i., i.e. at Cd0, Cd8, Td0, and Td8 as outlined in Fig. 1. RNA was extracted from individual livers before subjecting to Affymetrix chip analysis. The used MOE430A array contains 22 690 oligo probe sets representing 14 000 different genes. Persistent testosterone effects were identified by two-way ANOVA test evaluating only those genes with at least twofold expression changes at a highly significant P value <0.01. Functional annotations of the genes were searched in several databases including SwissProt, Proteome, PubMed, and NetAffx from Affymetrix and were categorized into nine different groups. Quantitative RT-PCR (qRT-PCR) was used to verify expression profiles of several, arbitrarily selected genes from chips.
Experimental outline. Mice were treated with testosterone (T) or vehicle (C) twice a week for 3 weeks. The treatment was then discontinued for 12 weeks, before mice were challenged with 106 P. chabaudi-parasitized erythrocytes. Livers were removed from three mice just prior to infection at Cd0 and Td0 as well as at peak parasitemia on day 8 p.i. at Cd8 and Td8.
Citation: Journal of Molecular Endocrinology 45, 6; 10.1677/JME-10-0026
Overall expression
Principal component analysis revealed that the overall expression profiles were relatively similar among the three different biological replicates per time point, but differed among the four different time points (Fig. 2). The numbers of testosterone-deregulated genes identified on the microarrays of the four different groups are summarized in a Venn diagram (Fig. 3). In toto, the expression of 143 genes was still deregulated by testosterone after 12 weeks of testosterone withdrawal, especially 48 genes were upregulated and 95 genes were downregulated by testosterone (horizontal ellipses in Fig. 3). Upon infection with P. chabaudi malaria, 63 genes were deregulated, with 30 genes upregulated and 33 genes downregulated (vertical ellipses in Fig. 3). In particular, 24 genes out of the 48 upregulated genes on day 0 p.i. were still upregulated, and 29 genes out of the 95 genes downregulated by testosterone remained suppressed on day 8 p.i. (Fig. 3). Only one gene was upregulated by testosterone on day 0 p.i. and became relatively downregulated by malaria on day 8 p.i. (Fig. 3).
Principal component analysis represents the three major vectors contributing to variance between arrays. Variations exist in expression profiles within and between triplicates.
Citation: Journal of Molecular Endocrinology 45, 6; 10.1677/JME-10-0026
Venn diagram summarizing the numbers of testosterone-deregulated genes on day 0 p.i. (horizontal ellipses) and testosterone-deregulated genes on day 8 p.i. (vertical ellipses). The overlap of the ellipses represents the numbers of those genes persistently deregulated by testosterone.
Citation: Journal of Molecular Endocrinology 45, 6; 10.1677/JME-10-0026
Gene expression profiles
Out of the 143 genes persistently deregulated by testosterone at the end of the 12-week period of testosterone withdrawal, 54 genes remained significantly deregulated during infection with P. chabaudi malaria on day 8 p.i. (Table 1; cf. overlaps in Venn diagram of Fig. 3). The expression profile clustering of these genes is presented in Fig. 4.
Expression of genes persistently deregulated by testosterone and their functions annotated to date
| Gene symbol | Gene name | Td0/Cd0 | Td8/Cd8 | Affymetrix probe ID | Entrez gene | Representative public ID (A) | Functions | PMID |
|---|---|---|---|---|---|---|---|---|
| Phase I metabolism | ||||||||
| MGC25972 | Similar to cytochrome P450, 4a10 | 390.20 | 42.70 | 1424352_at | 277 753 | BC025936 | Decreased expression in HGF-knockout mice | 17241389 |
| Cyp2d9 | Cytochrome P450, family 2, subfamily d, polypeptide 9 | 22.58 | 4.38 | 1419349_a_at | 13 105 | BC010593 | Male-specific; regulated by GH; sterol 16α-hydroxylase | 16547391, 4074718 |
| Cyp7b1 | Cytochrome P450, family 7, subfamily b, polypeptide 1 | 5.37 | 3.59 | 1421075_s_at | 13 123 | NM_007825 | Oxysterol-7-α-hydroxylase involved in synthesis of 7-α-hydroxylated bile acids; male-specific expression in the liver | 9295351, 8530364, 11284740 |
| Cyp7b1 | Cytochrome P450, family 7, subfamily b, polypeptide 1 | 4.45 | 3.55 | 1421074_at | 13 123 | NM_007825 | ||
| Fmo2 | Flavin-containing monooxygenase 2 | 0.36 | 0.42 | 1422904_at | 55 990 | NM_018881 | Typically expressed at high levels in lung | 16872995 |
| Cyp3a44 | Cytochrome P450, family 3, subfamily a, polypeptide 44 | 0.10 | 0.20 | 1426064_at | 337 924 | AB039380 | Female-specific expression depends on GH | 12147261 |
| Cyp2b9 | Cytochrome P450, family 2, subfamily b, polypeptide 9 | 0.06 | 0.13 | 1419590_at | 13 094 | NM_010000 | Female-specific expression depends on GH | 15381067 |
| Cyp3a41 | Cytochrome P450, family 3, subfamily a, polypeptide 41 | 0.02 | 0.01 | 1419704_at | 53 973 | NM_017396 | Female-specific isoenzyme | 10775455 |
| Fmo3 | Flavin-containing monooxygenase 3 | 0.01 | 0.02 | 1449525_at | 14 262 | NM_008030 | Female-specific expression in the liver | 7473608 |
| Cyp2b13 | Cytochrome P450, family 2, subfamily b, polypeptide 13 | <0.01 | 0.01 | 1449479_at | 13 089 | NM_007813 | Female-specific expression | 15155787 |
| Phase II metabolism | ||||||||
| Cml5 | Camello-like 5 | 9.51 | 24.01 | 1424811_at | 69 049 | BC024605 | Putative N-acetyltransferase | |
| Ugt2b38 | UDP glucuronosyltransferase 2 family, polypeptide B38 | 9.47 | 3.06 | 1424934_at | 71 773 | BC027200 | Male-predominant expression in the liver; inductive effects by testosterone | 19131521 |
| Cml4 | Camello-like 4 | 6.30 | 8.91 | 1419520_at | 68 396 | NM_023455 | Putative N-acetyltransferase | |
| Ugt2b1 | UDP glucuronosyltransferase 2 family, polypeptide B1 | 1.76 | 2.08 | 1424934_at | 71 773 | BC027200 | Male-predominant expression in the liver; inductive effects by testosterone | 19131521 |
| Sult3a1 | Sulfotransferase family 3A, member 1 | 0.01 | 0.04 | 1421669_at | 57 430 | NM_020565 | Female-specific expression | 16807285 |
| Sult2a2 | Sulfotransferase family 2A, dehydroepiandrosterone (DHEA)-preferring, member 2 | <0.01 | <0.01 | 1419528_at | 20 865 | NM_009286 | Female-specific expression in the liver; involved in control of the amounts of active androgens in cells; protects against the toxic effects of lithocholic acid | 8570624, 9566751, 16864508 |
| Lipid metabolism | ||||||||
| Elovl3 | Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3 | 40.51 | 6.61 | 1420722_at | 12 686 | BC016468 | Circadian expression in the liver; cold induction in brown adipose tissue is controlled by PPARα and crucial for accumulation of very long chain | 17003504, 15855229 |
| Vldlr | Very low-density lipoprotein receptor | 0.21 | 0.30 | 1434465_x_at | 22 359 | AV333363 | Involved in uptake of triglycerides | 11453330 |
| Vldlr | Very low-density lipoprotein receptor | 0.17 | 0.22 | 1417900_a_at | 22 359 | NM_013703 | ||
| 1810022C23Rik | RIKEN cDNA 1810022C23 gene | 0.06 | 0.19 | 1451588_at | 69 123 | BC014724 | 78% identical and 85% similar to murine peroxisomal delta 3, delta 2-enoyl-coenzymeA isomerase involved in β-oxidation of fatty acids | 11781327 |
| Hao3 | Hydroxyacid oxidase (glycolate oxidase) 3 | 0.02 | 0.05 | 1418654_at | 56 185 | NM_019545 | Peroxisomal-2-hydroxy acid oxidase | 10777549 |
| General metabolism | ||||||||
| Hsd3b5 | Hydroxysteroid dehydrogenase-5, delta<5>-3-beta | 1203.67 | 119.72 | 1420531_at | 15 496 | NM_008295 | Involved in steroid synthesis with both dehydrogenase and isomerase activity; male-specific | 8319586, 8477648, 8647315 |
| Ela3b | Elastase 3B, pancreatic | 4.19 | 3.82 | 1415884_at | 67 868 | NM_026419 | Protease of the exocrine pancreas | 2826474 |
| Serpine2 | Serine (or cysteine) proteinase inhibitor, clade E, member 2 | 2.91 | 2.78 | 1416666_at | 20 720 | NM_009255 | Plasminogen activator inhibitor (PAI1); protective against malaria, since disruption of the Pai1 gene results in partial loss of the ability to control the course of P. chabaudi infections | 15618182 |
| 4833442J19Rik | RIKEN cDNA 4833442J19 gene | 2.80 | 2.29 | 1427202_at | 320 204 | AV002340 | Highly conserved protein containing putative methyltransferase domain | |
| Csad | Cysteine sulfinic acid decarboxylase | 2.37 | 2.74 | 1427981_a_at | 246 277 | AY033912 | Rate-limiting enzyme in taurine biosynthesis | 11997111 |
| Mcm10 | Minichromosome maintenance deficient 10 (S. cerevisiae) | 2.20 | 2.16 | 1433408_a_at | 70 024 | AK010648 | DNA replication protein required for assembly and progression of the replication fork | 19081065 |
| Serpinb1a | Serine (or cysteine) proteinase inhibitor, clade B, member 1a | 0.42 | 0.46 | 1416318_at | 66 222 | AF426024 | Inhibitor of neutrophil serine proteases (elastase, cathepsin G and proteinase 3) | 17664292 |
| Serpinb1a | Serine (or cysteine) proteinase inhibitor, clade B, member 1a | 0.36 | 0.41 | 1448301_s_at | 66 222 | AF426024 | ||
| Nt5e | 5′ nucleotidase, ecto | 0.18 | 0.22 | 1422974_at | 23 959 | NM_011851 | Reduced activity by ischemia | 9306922 |
| Carcinogenesis | ||||||||
| Lama3 | Laminin, alpha 3 | 3.93 | 2.64 | 1427512_a_at | 16 774 | X84014 | Expression correlated with dedifferentiation of hepatocellular tumors | 12079511 |
| Nox4 | NADPH oxidase 4 | 3.88 | 2.52 | 1419161_a_at | 50 490 | AB041034 | NADPH oxidase generating ROS; Nox4 is most frequently expressed in tumor cells | 11376945, 15155719 |
| Nox4 | NADPH oxidase 4 | 3.29 | 2.44 | 1451827_a_at | 50 490 | BC021378 | ||
| Rad51l1 | RAD51-like 1 (S. cerevisiae) | 0.24 | 0.55 | 1421430_at | 19 363 | NM_009014 | Involved in genetic instability | 15723711 |
| Prom1 | Prominin 1 | 0.06 | 0.51 | 1419700_a_at | 19 126 | NM_008935 | Marker for hematopoietic and endothelial progenitor cells | 1569483 |
| Immune response | ||||||||
| Ifnγ | Interferon gamma | 11.72 | 0.58 | 1425947_at | 15 978 | K00083 | Plays a protective role during cerebral malaria | 16088835 |
| C6 | Complement component 6 | 8.66 | 6.37 | 1449308_at | 12 274 | NM_016704 | Complement component produced in the liver; higher expression in males than in females | 4007963 |
| Cklf | Chemokine-like factor | 0.43 | 0.49 | 1436242_a_at | 75 458 | BE852312 | Chemoattractant for leukocytes | 11415443 |
| Tox | Thymocyte selection-associated HMG box gene | 0.38 | 0.55 | 1425484_at | 252 838 | BB547854 | Upregulated in immature thymocytes but not in matured naive T-cells upon stimulation of the T-cell receptors | 11850626 |
| Lag3 | Lymphocyte activation gene 3 | 0.36 | 0.54 | 1449911_at | 16 768 | NM_008479 | Negatively regulates T-cell proliferation | 12421911 |
| Igk-C | Immunoglobulin kappa chain variable 8 (V8) | 0.22 | 0.52 | 1426200_at | 16 071 | AY058910 | ||
| Signal transduction | ||||||||
| Prlr | Prolactin receptor | 0.50 | 0.48 | 1425853_s_at | 19 116 | M22958 | Treatment of female mice with testosterone reduces Prlr levels | 6329976 |
| Prlr | Prolactin receptor | 0.45 | 0.52 | 1448556_at | 19 116 | BC005555 | ||
| Dscr1l1 | Down syndrome critical region gene 1-like 1 | 0.25 | 0.31 | 1421425_a_at | 53 901 | NM_030598 | Mimics the inhibitory effects of DSCR1 on calcineurin signaling pathways in endothelial cells and inhibits angiogenesis | 10756093 |
| Mmd2 | Monocyte to macrophage differentiation-associated 2 | 0.23 | 0.47 | 1424534_at | 75 104 | BC025064 | Member of the PAQR seven transmembrane protein family that also encompasses non-genomic progesterone receptors | 16044242, 12617826 |
| Mmd2 | Monocyte to macrophage differentiation-associated 2 | 0.23 | 0.47 | 1438654_x_at | 75 104 | AV269411 | ||
| Transport | ||||||||
| Slco1a1 | Solute carrier organic anion transporter family, member 1a1 | 25.65 | 22.65 | 1420379_at | 28 248 | AB031813 | Organic anion transporter with male preponderance in the liver; regulated by androgens and GH secretion pattern | 16807376, 12399219 |
| Slco1a1 | Solute carrier organic anion transporter family, member 1a1 | 22.05 | 41.28 | 1449844_at | 28 248 | AB031813 | ||
| Slc1a4 | Solute carrier family 1 (glutamate/neutral amino acid transporter), member 4 | 0.37 | 0.44 | 1423549_at | 55 963 | BB277461 | Neutral amino acid transporter | 12533615 |
| D630002G06|C730048C13Rik | Hypothetical protein D630002G06|RIKEN cDNA C730048C13 gene | 0.17 | 0.31 | 1451635_at | 236 293|319 800 | AB056443 | Cation transport protein | |
| D630002G06 | Hypothetical protein D630002G06 | 0.15 | 0.31 | 1425222_x_at | 236 293 | AB056443 | Cation transport protein | |
| AB056442 | cDNA sequence AB056442 | 0.05 | 0.11 | 1419751_x_at | 171 405 | NM_134256 | Organic anion transporter 6 | |
| BC014805 | cDNA sequence BC014805 | 0.05 | 0.15 | 1425751_at | 236 149 | AJ132857 | 64% identical and 79% similar to Slc22a9, an organic anion transporter in transepithelial transport of steroid sulfates | 16150593 |
| BC014805 | cDNA sequence BC014805 | 0.04 | 0.13 | 1425752_at | 236 149 | AJ132857 | ||
| Miscellaneous | ||||||||
| Omd | Osteomodulin | 17.26 | 2.55 | 1418745_at | 27 047 | NM_012050 | Bone-specific extracellular matrix protein | 10607915 |
| Susd4 | Sushi domain-containing protein 4 | 8.14 | 2.49 | 1424221_at | 96 935 | BC021842 | Unknown | |
| 2810439F02Rik | RIKEN cDNA 2810439F02 gene | 3.49 | 3.04 | 1426223_at | 72 747 | BC020021 | Unknown | |
| Clec2h | C-type lectin domain family 2, member h | 2.88 | 3.16 | 1451438_s_at | 94 071 | AF350410 | Unknown | |
| Arrdc4 | Arrestin domain-containing protein 4 | 0.42 | 0.54 | 1426818_at | 66 412 | BC025091 | Unknown | |
| Pcp4l1 | Purkinje cell protein 4-like 1 | 0.41 | 0.36 | 1452913_at | 66 425 | AV337888 | Reported to be expressed only in the central nervous system | 15053978 |
| 2610528H13Rik | Coiled-coil domain containing 25 | 0.37 | 0.55 | 1451799_at | 67 179 | BC025545 | Unknown |
Hierarchical cluster analysis of expression levels of persistently deregulated genes by testosterone, with the upregulated genes on the left and the downregulated genes on the right. Analysis was performed using Gene Cluster 3. All expression levels were normalized to the mean signal intensity of control mice (Cd0; cf. outline in Fig. 1), and data are log2-transformed. Green and red colors represent down- and upregulation respectively as indicated by the logarithmic color scale bar.
Citation: Journal of Molecular Endocrinology 45, 6; 10.1677/JME-10-0026
Testosterone persistently upregulated the gene expression of well-known male-prevalent enzymes such as CYP2D9, CYP7B1, CYP4A10, UGT2B1, UGT2B38, and HSD3B5 as well as the male-prevalent transporter SLCO1A1 (Table 1). By contrast, there also remained a persistent downregulation of gene expression of female-prevalent enzymes such as CYP2B9, CYP2B13, CYP3A41, CYP3A44, FMO3, SULT2A2, and SULT3A1 as well as the female-prevalent transporter BC014805. Remarkably, 27 genes, i.e. 50% of the genes, which were deregulated by testosterone, belonged to the categories phase I and phase II metabolism, lipid, and general metabolism. Phase I expression of genes encoding CYP2B13, CYP3A41, and FMO3 and phase II expression of genes encoding SULT2A2 and SULT3A1 were persistently suppressed by testosterone (>100-fold), whereas HSD3B5 showed the highest persistent testosterone-upregulated expression (>100-fold; Table 1).
Remarkably, the expression of four genes, which were involved in cancerogenic processes, was persistently deregulated by testosterone. These genes encode LAMA3, NOX4, RAD51L1, and PROM1 (Table 1). LAMA3 is an indicator of hepatocellular carcinoma dedifferentiation, NOX4 is normally expressed in cancerous tissues, RAD51L1 is involved in genetic instability, and PROM1 is a marker for hematopoietic and endothelial progenitor cells. Furthermore, persistent deregulation also occurred with four genes involved in signal transduction and genes encoding diverse transporters and seven genes of miscellaneous function.
Long-term testosterone-induced deregulation also occurred in expression of genes involved in the immune response, in particular those encoding IFNγ and IGK-C (Table 1). Out of the 24 upregulated genes, 3 genes encoding CYP2D9, ELOVL3, and SUSD4 remained upregulated, but at significantly lower levels after infection on day 8 p.i. By contrast, only the expression of Ifnγ was increased by testosterone on day 0 p.i. (Td0/Cd0=11.78), but its upregulation appeared relatively suppressed on day 8 p.i. (Td8/Cd8=0.58), i.e. it was strongly induced by infection at Cd8 than at Td8 (Table 1). Finally, Fig. 5 shows that the expression profiles of arbitrarily selected genes from microarrays, i.e. testosterone-upregulated Hsd3b5, Elovl3, and Ifnγ and testosterone-downregulated Sult2a2, Nt5e, and Prom1, could be verified by real-time PCR analysis.
Expression of testosterone-deregulated genes analyzed by quantitative real-time PCR. Gene expression profiles of testosterone-upregulated Hsd3b5, Ifnγ, and Elovl3 on the left and testosterone-downregulated Nt5e, Prom1, and Sult2a2 on the right, as revealed by microarrays, can be verified by real-time PCR analysis. Relative mRNA expression was normalized to the mean expression of control mice (Cd0). For definition of Cd0, Cd8, Td0, and Td8, see outline in Fig. 1. Means±s.d. from three different mice are indicated for each group. Significant differences between Td0 and Cd0 are indicated by*, and between Td8 and Cd8 by § using t-test (P<0.05).
Citation: Journal of Molecular Endocrinology 45, 6; 10.1677/JME-10-0026
Nuclear receptors
Nuclear receptors play a central role in liver metabolism of endo- and xenobiotics (Tirona & Kim 2005, Plant & Aouabdi 2009). However, some nuclear receptors such as RXRα, LXRα, and PPARα are not represented on the Affymetrix chip, and others may be removed according to the criteria we applied for analysis. We therefore decided to re-examine a possible persistent testosterone effect on expression of genes encoding some known nuclear receptors using qRT-PCR. Table 2 summarizes data obtained for 12 different receptors. It is conspicuous that after testosterone withdrawal for 12 weeks, only the expression of Car was more than twofold downregulated. However, expression of Car did not remain deregulated by testosterone after infection with P. chabaudi on day 8 p.i.
Effects of testosterone pretreatment and Plasmodium chabaudi malaria on gene expression of different nuclear receptors in the female mouse liver as revealed by quantitative reverse transcription-PCR
| Gene symbol | Gene name | Td0/Cd0 | Td8/Cd8 |
|---|---|---|---|
| Pparα | Peroxisome proliferator-activated receptor alpha | 0.76±0.07 | 1.25±0.18 |
| Lxrα (Nr1h3) | Liver X receptor alpha | 0.76±0.06 | 0.72±0.19 |
| Rxrα | Retinoid X receptor alpha | 0.53±0.33 | 0.92±0.33 |
| Pxr (Nr1i2) | Pregnane X receptor | 1.13±0.12 | 1.01±0.57 |
| Fxr (Nr1h4) | Farnesoid X receptor | 0.56±0.42 | 0.98±0.09 |
| Ar | Androgen receptor | 0.91±0.22 | 1.15±0.27 |
| ERα (Esr1) | Estrogen receptor alpha | 0.88±0.22 | 1.19±0.21 |
| ERβ (Esr2) | Estrogen receptor beta | 1.46±0.33 | 0.95±0.14 |
| Ahr | Aryl hydrocarbon receptor | 1.16±0.16 | 1.09±0.17 |
| Hnf4α | Hepatic nuclear factor 4 alpha | 1.28±0.33 | 1.13±0.38 |
| Car (Nr1i3) | Constitutive androstane receptor | 0.31±0.06 | 0.99±0.26 |
| Vdr | Vitamin D receptor | ND | 0.31±0.17 |
Discussion
Using Affymetrix microarray technology, the present study has revealed that, among 14 000 genes examined, only 54 genes were persistently deregulated by testosterone in livers of female mice and remained deregulated after subsequent infection with P. chabaudi for 8 days. The fact that only 54 genes out of 14 000 are persistently deregulated by testosterone indicates that this deregulation reflects specific testosterone effects rather than long-term toxic testosterone effects. The genes permanently changed by testosterone can be summarized into three groups: 1) genes involved in liver metabolism, 2) genes involved in hepatocellular carcinogenesis, and 3) genes involved in immune response.
Most of the genes permanently changed by testosterone belong to the first group, i.e. genes encoding enzymes involved in phase I–III liver metabolism. Conspicuously, there is a persistent downregulation of some female-prevalent genes such as Cyp2b9, Cyp2b13, Cyp3a41, Cyp3a44, Fmo3, Sult2a2, Sult3a1, and BC014805, and an upregulation of some male-prevalent genes such as Cyp2d9, Cyp7b1, Hsd3b5, Ugt2b1, Ugt2b38, and Slco1a1 (cf. Table 1). The testosterone-induced permanent changes of these genes contribute to an at least partial masculinization of the metabolism of the female mouse liver. Such a masculinization is ultimately also in accordance with the testosterone-induced permanent changes of those genes listed in the second group, which are involved in and contribute to hepatocellular carcinogenesis, such as Lama3 and Nox4. Indeed, hepatocellular carcinoma is known for a long time to be much more prevalent in male mice than in female mice (Drinkwater et al. 1990, Kemp & Drinkwater 1990, Nagasue & Kohno 1992). The changes of Igk-C and Ifnγ in the third group of testosterone-deregulated genes indicate that testosterone also affects genes involved in the immune response, though our data cannot discriminate as to whether testosterone affects only genes of intrahepatic T- and B-cells and/or also circulatory B- and T-cells. At least, the persistent downregulation of Igk-C by testosterone indicates that testosterone ultimately leads to a partially reduced production of antibodies. Indeed, this confirms previous studies that testosterone lowers the production of antibodies (Fujii et al. 1975, Hirota et al. 1980, Morton et al. 1981, Kincade et al. 1994, Benten et al. 1997). More complicated is the situation with Ifnγ which is upregulated by the end of the testosterone withdrawal period, but the subsequent malaria infection induces expression of Ifnγ in both the vehicle- and the testosterone-pretreated mice, however, at a higher extent in control than in testosterone-pretreated mice.
The mechanisms by which testosterone exerts reprograming of gene expression, evidenced as permanent changes in the expression of distinct genes in the female mouse liver, are unknown, but are expected to be rather complex and, possibly, even to be different for different genes, involving both direct and indirect actions of testosterone on gene expression (Wunderlich et al. 2002, Centenera et al. 2008, Bennett et al. 2009). An indirect action of testosterone may also take place through the hypothalamus–pituitary gland–liver axis, which is imprinted neonatally by androgens and which operates through pulsatile versus continuous patterns of GH secretion in males versus females respectively (Colby et al. 1973, Einarson et al. 1973, Sakuma et al. 2002, Gustafsson 2005, Waxman & Holloway 2009). Indeed, this axis is currently envisaged as to maintain the sexual dimorphism of hepatic gene expression, especially expression of cytochrome P450 genes involved in liver metabolism. GH in turn activates the GH-responsive transcription factor STAT5b, which is, besides HNF4α, essential for establishment and maintenance of sexually dimorphic gene expression in the liver of male mice (Clodfelter et al. 2007, Holloway et al. 2007, 2008). In female mice, however, STAT5b plays only – if at all – a minor role in sexually dimorphic pattern of hepatic gene expression (Holloway et al. 2007). Remarkably, our microarray and qRT-PCR data indicate that testosterone affects expression of neither Stat5b nor Hnf4α in the female mouse liver. Also, any other transcription factors including common nuclear factors are apparently not affected by testosterone. Moreover, testosterone-induced reprograming of liver gene expression may involve changes in the epigenome. Indeed, steroid hormones are known to induce long-lasting chromatin remodeling through epigenetic mechanisms, as e.g. DNA methylation and/or covalent modifications of histones (Grunstein 1997, Goldberg et al. 2007, Murray et al. 2009, Waxman & Holloway 2009).
Some of those genes that may be important for the testosterone-induced persistent susceptibility to P. chabaudi malaria have been possibly not detected due to masking since our experiments have been conducted with livers in toto. Also, it is not unlikely that genes may be critical in other organs, as e.g. in the spleen representing the other major effector site against blood stage malaria (Wunderlich et al. 2005). Nevertheless, it appears as if the testosterone-induced permanent changes we have found in hepatic expression of those genes involved in liver metabolism and in particular those involved in immune response are presumably relevant for the testosterone-induced persistent susceptibility to P. chabaudi malaria. For instance, masculinization of liver metabolism possibly reflects a decreased hepatic capacity to detoxify substances, which are derived in abundance from destroyed parasites and host cells during malaria infection. At least, the testosterone-induced decrease in production of antibodies and IFNγ indicates an association with the testosterone-induced persistent susceptibility to P. chabaudi malaria. Indeed, the current view predominates that protective immunity to P. chabaudi malaria is eventually mediated through antibodies (Achtman et al. 2005, 2007) requiring activation by the TH2 response. However, this activation has to be preceded by an IFNγ-dependent activation of the TH1 response (Taylor-Robinson & Phillips 1998, Balmer et al. 2000, Su & Stevenson 2000, Batchelder et al. 2003, Cernetich et al. 2006). The specific upregulation of Ifnγ we observe after testosterone withdrawal could reflect an overactivation of the TH1 response; the delicately balanced switching to the TH2 response may be then perturbed (Zhang et al. 2000), thus delaying the maturation and secretion of the antibodies from plasma B cells. In addition, the testosterone-increased production of IFNγ may perturb the proper activation of those genes, which encode, e.g. other effectors directed against infections such as diverse GTPases (Boehm et al. 1998, Klamp et al. 2003, Degrandi et al. 2007).
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by Deutsche Forschungsgemeinschaft through GRK1427 and the Centre of Excellence for Biodiversity Research, College of Science, King Saud University, Riyadh, Saudi Arabia.
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