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Departments of Medicine (L.C.M., S.R.), Pediatrics (S.R.), and Human Genetics (A.M.D.), Committees on Genetics and Molecular Medicine (S.R.), The University of Chicago, Chicago, Illinois 60637; and Cancer Genetics Branch (R.L.W., P.S.M.), National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892
Address correspondence and requests for reprints to: Samuel Refetoff, M.D., The University of Chicago, MC3090, 5841 South Maryland Avenue, Chicago, Illinois 60637. E-mail: refetoff{at}uchicago.edu.
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3, member RAS oncogene family brain antigen RAB3B, platelet phosphofructokinase, hypoxia-inducible factor-1, and enolase 1. These genes as well as three known to be TH regulated in other species and found in this study also in human cells (glucose transporter 1, solute carrier family 16 member 3, and basic transcription element-binding protein 1) have a variety of regulatory functions in development and metabolism. TH seems to induce these genes by initiating either genomic or nongenomic mechanisms. Surprisingly, TH-mediated down-regulation of fibroblast growth factor 7 and alcohol dehydrogenase 1B persisted in fibroblasts from patients with RTH. This first systematic study of TH-mediated gene expression in normal human cells identifies several new TH-responsive genes and demonstrates that skin fibroblasts are suitable for the study of TH action in health and disease.
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Introduction |
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and -ß. These TRs, best characterized for their ligand-dependent and gene-targeted transcription function (1, 2), recognize TH response elements (TREs) in the promoters of TH-responsive genes. In the case of positively regulated genes, ligand binding produces an exchange of the associated corepressors for coactivators resulting in the modification of chromatin structure and recruitment of the basal transcriptional machinery to increase the expression of target genes. cDNA microarray analysis has proven to be a powerful tool in determining the spectrum of hormonally responsive genes in a particular tissue or cell type (for review see Ref. 3). This is because of the capacity for simultaneous measurement of mRNA expression levels for thousands of genes. This technique has been used to study the effect of TH on gene expression in mouse liver (4, 5, 6, 7), brain (8), and osteoblasts (9) in rat liver (10) and the rat pituitary GC cell line (11).
Studies on the broad scale effect of TH on gene transcription in human cells are limited to tumor cell lines that, in addition, were stably transfected with TR1, such as HeLaTR (12) or HepG2-TR1 cells (13, 14). There is little knowledge about TH-mediated gene expression patterns in normal human cells. The aim of current study was to identify TH-responsive genes in human cells without the influence of artificial TR overexpression or neoplastic transformation. Information regarding the normal pattern of TH-regulated gene expression could serve as a tool for future studies of TH action and in the differential diagnosis of its defects.
We chose to study human skin fibroblasts because they are easily obtained from affected and unaffected members of families with inherited defects of TH action, thus allowing direct studies in these cells. Whereas skin fibroblasts are not as metabolically active as hepatic cells, they are TH responsive. Early studies have shown that T3 in physiological concentrations inhibited the accumulation of glycosaminoglycan and the synthesis of fibronectin in normal human fibroblasts (15, 16). These effects were attenuated in fibroblasts from patients with resistance to thyroid hormone (RTH), thus allowing their use for the tissue diagnosis of RTH (17, 18). Furthermore, ZAKI 4 had been found to be TH inducible in human skin fibroblasts (19), showing that fibroblasts are suitable for the study of TH-regulated gene expression.
We therefore set out to systematically examine the transcriptional effect of TH in human skin fibroblasts by quantitative fluorescent cDNA microarray, representing more than 15,000 genes. Confirmatory and more detailed studies were carried out with real-time PCR. To ascertain the specificity of the observed changes and test the ability of gene expression patterns to reflect a defect in TH action, we used in parallel fibroblasts from patients with RTH.
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Materials and Methods |
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Human skin was obtained by punch biopsy from three normal individuals and two patients with RTH. One was heterozygous for single amino acid substitution in the T3-binding domain in the TRß gene (A317T) (20) and the other homozygous for a TRß gene deletion (21). The study was approved by the Institutional Review Board of the University of Chicago. Fibroblasts were grown in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% bovine calf serum (BCS) as previously described in detail (16). At confluency, the medium was replaced with one containing TH-depleted BCS (TxBCS), obtained from a thyroidectomized calf (15). For the time-course experiment, BCS was TH depleted by treatment with anion exchange resin (22). Forty-eight hours later, T3 was added to a final concentration of 0.1 x 10–9, 0.5 x 10–9, and 2 x 10–9 mol/liter. Based on the free T3 concentration in the medium containing 10% BCS, measured by equilibrium dialysis (14), these three T3 doses correspond to a third to half physiological, 1.5–2 times physiological, and 3–5 times physiological. For microarrays, incubation with T3 was for 24 h (4 x 10-cm dishes for each T3 concentration and eight dishes without addition of T3). Pools were made from two dishes for RNA extractions and a final pool for each individual and treatment was made for the microarray analyses. For additional real-time PCR studies, fibroblasts were incubated with 2 x 10–9 M T3 for 1, 3, 6, 12, and 24 h, and four 6-cm dishes were used for each time point. RNA extraction and real-time PCR were carried out separately for each culture dish.
The experiments were carried out on fibroblasts at comparable passages; the ages of the individuals at the time of biopsy are given in parentheses: normal 1 (5 yr), sixth and seventh passage; normal 2 (11 yr), 11th passage; normal 3 (3.5 yr) (time course), seventh passage; RTH/TRßmut (9 yr), seventh passage; RTH/TRß0 (10 yr), sixth passage.
Isolation of RNA
The medium was removed, and the dish was washed twice with Hank’s buffered saline solution. Total RNA was extracted using phenol/guanidine isothiocyanate (TRIZOL, Invitrogen). The supernatant, adjusted to 35% ethanol, was passed through a maxicolumn (Qiagen Inc., Valencia, CA). The yield after the final salt/ethanol precipitation was between 12 and 34 µg per 10-cm dish of good-quality RNA.
For experiments designed for real-time PCR analysis only, RNA extracted with TRIZOL was used without further purification.
Preparation of target cDNA, hybridization, and microarray
Cells were grown in TH-depleted medium, containing TxBCS, and those supplemented with T3, both derived from the same individual. Microarray analysis was performed according to standard protocols implemented at the National Human Genome Research Institute Microarray Core Facility (http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/protocol.html). Briefly, cDNA was synthesized from 15 µg total RNA using SuperScript II reverse transcriptase (Invitrogen) and an oligo-dT primer incorporating aminoallyl deoxyuridine 5-triphosphate (Sigma, St. Louis, MO) and coupled with Cy5 or Cy3 (Amersham, Piscataway, NJ) after synthesis. cDNA preparations containing distinct fluorescent labels allowing direct comparison between T3-treated cells and untreated cells were combined and hybridized to microarrays containing 15,360 cDNAs overnight under glass coverslips. A common reference probe from untreated cells was used to facilitate cross-comparison among the cell lines.
Fluorescence intensities detected by a laser scanner (Agilent Technologies, Palo Alto, CA) were processed digitally using DeArray software (23). Background was subtracted and data were normalized on the basis of a set of 88 stable genes. Data were managed with FileMaker Pro (FileMaker, Inc., Santa Clara, CA). Array features were required to have a fluorescence intensity greater than 750 in one channel and a DeArray-designated quality of 1.0 to enter the analysis. Positively (
1.4 ratio) or negatively (
0.7 ratio) regulated genes in normal hybridizations were then identified. TH-mediated changes of specific genes found on microarray analysis, according to criteria presented in Results, were analyzed by real-time PCR on RNA used for the microarray analysis and on separate experiments using fibroblasts from normal subjects. The microarray data set is available at http://research.nhgri.nih.gov/microarray/selected_publications.html.
Real-time PCR
For experiments designed for real-time PCR analysis, RNA was reverse transcribed with the Superscript II RNase H reverse transcriptase kit (Invitrogen) using 2 µg total RNA and 100 ng of random hexamers.
Reactions for the quantification of mRNAs were performed in an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA), using SYBR Green I as detector dye. The reaction mixtures contained 25 µl QuantiTect SYBR Green PCR kit (Qiagen), 0.3 µmol/liter of each primer, 10 ng template cDNA, and RNase-free water to a final volume of 50 µl. The oligonucleotide primers were designed to cross introns, and their sequences are available upon request. Whereas the cDNA microarray clones for aldo-keto reductase family 1 (AKR1) C1, C2, and C3 were located in their different 3' untranslated regions, it was not possible to design intron-crossing primers in the coding region due to extremely high homology between these members of the AKR1 family. We therefore chose to use a primer pair that amplified from all three members (AKR1C1–3).
The reaction conditions were 50 C for 2 min, 95 C for 10 min, followed by 40 cycles of 95 C for 15 sec and 60 C for 1 min. The expression of genes was calculated relative to that in untreated cells and normalized for 18S rRNA, measured under the same conditions with TaqMan ribosomal RNA control reagent (Applied Biosystems/Roche, Branchburg, NJ) primers, using the 2–
CT method (24).
Data analysis
Time-course real-time PCR results are expressed as mean ± SE, and statistical analysis was done by ANOVA. Time points were compared using ANOVA with Tukey’s adjustment for multiple pairwise comparisons.
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Results |
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For genes up-regulated by T3, we used as the cut-off point a T3-treated to untreated ratio or fold change (FC) of 1.4 or more, observed in all three microarrays of normal fibroblasts. Based on these criteria, 148 genes were identified. Taking into consideration duplicate genes and hypothetical genes, they contained 91 known genes induced by T3. These were arranged in descending order according to the average FC of the three normal fibroblast microarrays treated with 2 x 10–9 M T3 by hierarchical clustering using the average linkage method. The 24 genes with the highest FC are shown as a color plot in Fig. 1
. Genes from this list were used for further analysis.
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(ENO1)] and two represented hypothetical genes (DKFZp434P228 and KIAA1287). Finding duplicates of the same gene induced to the same extent by T3 supported the validity of the method in identifying TH-inducible genes. Further confirmatory evidence is in the observation that the T3 induction of these genes was attenuated in fibroblasts from the two patients with RTH (Fig. 1
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For genes down-regulated by T3, using a cut-off FC of 0.7 or less, five genes of interest were identified. These were fibroblast growth factor (FGF) 7, alcohol dehydrogenase 1B, ß-polypeptide (ADH1B), chondroitin sulfate proteoglycan (CSPG) 2, cytochrome P450 subfamily XIA, and Keratin16 (not shown in Fig. 1). In contrast to genes up-regulated by TH, down-regulation of the corresponding genes was not attenuated in fibroblasts from the patients with RTH.
Confirmation by real-time PCR
To verify the cDNA microarray data, we quantitated the mRNAs from the same experiment using real-time PCR. From the genes with the highest FC by microarray analysis, shown in Fig. 1, 10 were analyzed by real-time PCR: AKR1C1–3, member RAS oncogene family brain antigen RAB3B (RAB3B), PFKP, spermatid perinuclear RNA-binding protein (STRBP), collagen type (COL) VI 3 (COLVIA3), solute carrier family 16 member 3 (SLC16A3), collagen type VIII 1 (COLVIIIA1), and ENO1. The following three genes were from the list of five down-regulated genes: ADH1B, FGF7, and CSPG2. The hypoxia-inducible factor (HIF)-1, present in the 91 genes induced above the 1.4-FC threshold by microarray, had the highest normal to RTH expression ratio and was therefore also included in the real-time PCR analysis. In addition, mRNA measurement by real-time PCR was carried out for the following genes previously reported to be TH responsive in human cells: ZAKI 4, also known as Down syndrome critical region 1 L1, a calcineurin inhibitor (19, 25), and ATP-binding cassette transporter (ABC)A1 because an ABCA1-promoter plasmid was repressed by TH in transformed human embryonal kidney (293T) cells after cotransfection with TRß (26). We also examined expression of the human homologs of genes found to be TH responsive in other species: basic transcription element-binding protein 1 (BTEB1), TH inducible in rat (27) and tadpole (28); ß-amyloid precursor protein, repressed by TH in murine neuroblastoma cells (11); the glucose transporters (GLUT)1 and 4, increased in TH-treated rat 3T3 adipocytes (29); and SPOT14, TH induced in rat liver (30). These additional genes were either not included in the microarray chip or excluded from statistical analysis due to low fluorescence quality.
Table 1 shows the real-time PCR results for genes whose TH responsiveness, as identified by microarray analysis (see Fig. 1), could be confirmed as well as three additional, previously reported genes. No increase in expression after T3 could be observed for STRBP, COLVIIIA1, ABCA1, ß-amyloid precursor protein, SPOT14, and GLUT4. Down-regulation of CSPG could not confirmed by real-time PCR.
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ZAKI 4 had the highest magnitude of TH-mediated induction, a FC of 4–6. The AKR1 gene family 1 members, C1, C2, and C3, showed very similar levels of stimulation (average FCs 3.5–3.1) by microarray. Although the TH response was attenuated in fibroblasts from both patients with RTH, the FC of those heterozygous for a single amino acid substitution (RTH/TRßmut) was higher than those with a deletion of both TRß alleles (RTH/TRß0). This observation suggests that the hormonal resistance could be overcome more easily with higher doses of T3 in the case of a receptor mutation than in its complete absence. ENO1 might represent a gene at the threshold of detectable TH effect. The FC by T3, measured by real-time PCR, was only 1.4 and 1.2 in normal fibroblasts, whereas fibroblasts from the two individuals with RTH showed no stimulation whatsoever (FC of 0.9).
For two genes repressed by TH, ADH1B and FGF7, real-time PCR could reproduce the T3-induced decrease in expression observed by microarrays. A change of similar magnitude, as in fibroblasts from normal individuals, was also found in fibroblasts from the two patients with RTH.
Dose response
To reduce the likelihood of nonspecific effect of TH treatment, the hormonal effect was tested by adding three concentrations of T3 (0.1 x 10–9, 0.5 x 10–9, and 2 x 10–9 M) to medium containing 10% TH-depleted BCS, ranging from below physiological to 5-fold above physiological levels. Microarray data for the 24 clones, including 14 known genes, with the highest FC demonstrate an enhancement in red intensity with increasing T3 doses (Fig. 1). Figure 2 shows the level of mRNA expression after T3 treatments relative to untreated fibroblasts measured by real-time PCR for the four genes with the highest up-regulation (Fig. 2A) and two down-regulated genes (Fig. 2B).
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, BTEB1, AKR1C1, and COLVIA3, the mRNA increase is correlated with the increasing T3 treatment, whereas in the fibroblasts from patients with RTH, this correlation is either absent or the response is markedly reduced (Fig. 2A). This suggests that the effect of TH on the induction of these genes is TRß mediated. For both down-regulated genes, FGF7 and ADH1B, repression shows a dose-dependent pattern that is preserved in the fibroblasts of the patients with RTH, suggesting that TRß may not be required for this apparent TH-mediated effect (Fig. 2B). Time course
To explore the time course of TH effect on gene expression, real-time PCR measurements were performed for five different time points in an independent series of experiments. Fibroblasts from a third normal individual were grown for 48 h in TH-depleted medium that was then supplemented to 2 x 10–9 M T3 for 1, 3, 6, 12, and 24 h. Five genes, three up-regulated (ZAKI 4, BTEB1, AKR1C1–3) and two down-regulated (ADH1B and FGF7) were examined (Fig. 3).
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mRNA induction, in contrast, was slower, with a 2-fold (2.1 ± 0.3) increase at 6 h and further increase to 3-fold after 12 (3.1 ± 0.4) and 24 h (3.1 ± 0.3). AKR1C1–3 mRNA stimulation was observed at 3 h (1.8 ± 0.8) but reached significance at 6 h with a 3-fold induction (3.1 ± 0.6), which was maintained for 24 h. Down-regulation of genes could be more difficult to detect because for mRNA reduction to be observed due to decrease of new transcription, accumulated mRNA has first to be degraded. Significantly lower mRNA levels could be detected for ADH1B 6 h after T3 treatment (33 ± 5%), with further decrease reaching 21 ± 5%. The FGF7 mRNA decrease was significant from 6 h on (70 ± 2%), reaching 60 ± 8% after 24 h.
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, COLVIA3) and negatively (FGF7, ADH1B) regulated. Furthermore, our results show TH responsiveness in normal human cells for genes that had been found in other species (BTEB1, GLUT1, SLC16A3). The congruent finding of TH responsiveness for the described genes in microarray and real-time PCR and the progressive changes in mRNA concentrations with graded doses of TH support the validity of the results. This is further strengthened because the transcriptional responses were not found in a single isogenic cell line but in primary culture cells from two unrelated normal individuals and reliably so again, in normal fibroblasts from yet another individual, in a separate time-course experiment.
The level of induction or repression in general was much lower than previously described in TR-overexpressing HeLa or HepG2 cells or mouse livers, in which up to 20-fold induction was found for some genes. This was to be expected because fibroblasts are not as metabolically active as hepatic or neoplastic cells and have lower levels of TRs. In addition, the T3 treatments with concentrations from 0.1 to 2 nM were lower and closer to physiological concentrations than in most studies. In fact, high T3 doses can overcome the relative resistance in RTH, as shown in vivo in patients with RTH (31) and can abolish gene expression differences between normal and RTH fibroblasts (our unpublished data). This was not the case here because even for our highest dose of 2 nM T3, no overlap in FC could be observed between normal and resistant fibroblasts. The reduced or absent response in the TH-resistant fibroblasts confirms the role of the TRß and the TH specificity of our results.
T3 is known to play a major role in the regulation of development, growth, and metabolism in most tissues. How do our findings of TH-responsive genes in human fibroblasts contribute to defining their role in humans? TH is essential for the development of the central nervous system, and TH deficiency in neonatal and early postnatal life results in irreversible mental retardation. In rat brain, TH influences neural migration, axonal maturation, and dendritic outgrowth (32). Neurite branching is regulated by BTEB1, a zinc-finger transcription factor, which itself is regulated by TH. BTEB1 induction was rapidly up-regulated in rat neuro-2a cells expressing the TRß1 isoform but not in those expressing TR, suggesting TRß-specific induction (33), which is compatible with the markedly reduced response for BTEB1 in fibroblasts with TRß mutation or deletion. Significant 3.6-fold induction of BTEB1 mRNA in primary rat embryonic neurons was detectable already after 1 h (27), as it was here in human fibroblasts, too. BTEB1 is a rapidly and strongly TH-regulated gene in tadpole metamorphosis, in which TH controls tissue remodeling and limb growth (28). Rapid TH responsiveness in human cells suggests that BTEB1 also may be an important mediator of TH action in human brain and body development and growth.
ZAKI 4, a calcineurin (Cn) inhibitor, is expressed predominantly in rat and human brain and is believed to also play a role in mediating TH effect on brain development and maturation. Its expression overlaps with that of Cn. How TH-dependent Cn inhibition through ZAKI 4 might affect central nervous system development and function is unknown, though (34, 35).
HIF-1 is responsible for elevated expression of glycolytic enzymes and glucose transporters, especially in tumors, leading to increased metabolism (36). Our results of HIF-1 mRNA increase in response to 2 nM T3 in normal cells suggest that HIF-1 induction by TH has a physiological role. Furthermore, we also found four of its known target genes to be induced by TH, namely ENO1, triose phosphate isomerase, phosphoglycerate kinase 1, and GLUT1.
GLUT1, a glucose transporter, has been described to be induced by T3 in murine-derived 3T3-L1 fibroblasts after treatment with 50 nM T3 25 times higher than our highest T3 treatment dose (29). SLC16A3, a widely expressed lactate transporter, had been shown to be TH responsive in rat muscle (37). The induction of the glucose transporter GLUT1, several enzymes of glycolysis, and the lactate exporter SLC16A3 strongly suggests a role for TH in glycolysis regulation. Because GLUT1 and the glycolytic enzymes are target genes of the transcription factor HIF-1, the effect of TH on the induction of these genes seems to be indirect and HIF-1 mediated.
The TH-dependent gene expression described herein seems to be the result of very different mechanisms of TH action through TRß. This is probably also reflected in the different time-course patterns of gene expression. BTEB1, TH responsive in tadpoles (28) and rats (27), has a well-studied direct repeat 4-TRE. BTEB1 is therefore most likely induced by classical genomic action involving TRß binding to TRE in the promoter region of TH regulated genes, which, after hormone binding, recruits cofactors leading to the assembly of the basic transcription machinery. Consistent with this classical model of TH/TRß action, BTEB1 induction was abrogated in the fibroblasts from patients with RTH. The rapid nearly 4-fold increase of BTEB1 mRNA within 1 h of T3 treatment is compatible with this direct TH action.
Induction of ZAKI 4, on the other hand, has recently been shown to be secondary to nongenomic action of TH (38). Instead of binding to a TRE, liganded TRß activates phosphatidylinositol-3OH-kinase, which initiates a signaling pathway of several kinases including mammalian target of rapamycin, leading to the putative induction of a transcription factor and ZAKI4 mRNA increase. The slower increase of ZAKI 4 mRNA over time in comparison with BTEB1 seems to reflect this indirect mode of induction with the requirement of preceding protein synthesis (Fig. 3). The absence of a dose response to TH in the RTH fibroblasts (Fig. 2A) demonstrates the necessary involvement of TRß in this cytosolic, nongenomic action of TH, which ultimately does lead to changes in gene expression. HIF-1 is also induced via the phosphatidylinositol-3OH-kinase pathway (39). The mechanism of TH-mediated induction of HIF-1 is probably very similar to ZAKI 4.
We conclude that the genes responding to TH treatment can do so directly, as BTEB1, or indirectly, as GLUT1, located downstream of the TH-responsive transcription factor HIF-1. Induction of TH-responsive genes can be initiated by either nuclear (classical) or cytosolic (nongenomic or nontranslational) action of TH.
The response of two genes negatively regulated by TH, FGF7 and ADH1B, was not attenuated by RTH. The reason for this observation is not clear, and selective repression by TR is one possible explanation. It is also possible that the effect of TH on these genes is TR independent and initiated by other receptors, such as the suggested membrane receptor (40).
We examined the effect of TH on gene expression in individuals with RTH, either due to a TRß mutation or a homozygous TRß deletion. The clear separation between normal and TH-resistant cells for all genes positively regulated by TH indicates that measurement of gene expression patterns can be diagnostic in the study of individuals with defects of TH action. Whereas RTH in most cases is caused by a TRß mutation, in 16% of the patients, no TRß mutation could be found, defining the subtype of non-TR-RTH (41). So far, no differences in phenotype, such as hyposensitivity to T3-induced changes in serum cholesterol, SHBG, creatinine kinase, ferritin, or TSH, could be found between patients with RTH due to TRß gene mutations and those without (42). It will be interesting to study gene expression in those fibroblasts in comparison with normal and RTH/TRßmut fibroblasts.
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Footnotes |
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First Published Online October 26, 2004
Abbreviations: ABC, ATP-binding cassette transporter; ADH1B, alcohol dehydrogenase 1B; AKR1, aldo-keto reductase family 1; BCS, bovine calf serum; BTEB1, basic transcription element-binding protein 1; Cn, calcineurin; COLVIA3, collagen type VI 3; COLVIIIA1, collagen type VIII 1; CSPG, chondroitin sulfate proteoglycan; ENO1, enolase 1; FC, fold change; FGF, fibroblast growth factor; GLUT, glucose transporter; HIF, hypoxia-inducible factor; PFKP, platelet phosphofructokinase; RAB3B, member RAS oncogene family brain antigen RAB3B; RTH, resistance to thyroid hormone; SLC16A3, solute carrier family 16 member 3; STRBP, spermatid perinuclear RNA-binding protein; TH, thyroid hormone; TR, TH receptor; TRE, TH response element; TxBCS, medium containing TH-depleted BCS.
Received September 4, 2004.
Accepted October 15, 2004.
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C(T)] method. Methods 25:402–408[CrossRef][Medline]
or ß genes may be due to a defective cofactor. J Clin Endocrinol Metab 81:4196–4203[Abstract]
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