Studies of Complex Biological Systems with Applications to Molecular Medicine: The Need to Integrate Transcriptomic and Proteomic Approaches
Table 3
Summary of the models used and of the major findings obtained by applying microarray technologies to the study of THs effects on relevant metabolically active tissues.
Hypothyroidism induced by low-iodine diet supplemented with 0.15% PTU for 4 weeks, then hyperthyroidism induced by single i.p. injection of L-T3 or T4 100 g/100 g body weight.
2225
Of 55 genes identified as target of T3, 41 were negatively regulated.
Glycogenolysis, gluconeogenesis, lipogenesis, proliferation, apoptosis, insulin signaling, immunogenity, and protein glycosylation.
2 to 3.5-months old (WT and TR) male mice.Studied tissue: liver.
At the onset of the experiment, all groups of animals were provided a low-iodine diet for 14 d to accustom them to the synthetic chow. Hypothyroidism was then induced by inclusion of 0.05% methimazole and 1% potassium perchlorate in the drinking water for 21 d while still on the low-iodine diet. From d 35, one group of animals was injected daily with 5 g T3 for an additional 5-d period to induce hyperthyroidism. Another group was injected with 5 g T3 and 5 g T4; on d 35, the animals in this group were killed 2 hours after the T3/T4 injection.
4000
T3 found to regulate more than 200 genes, more than 100 of which were not previously described. 60% of these genes showed dependence on the TR gene for T3 regulation.
Rapid or transient effects of T3 on lipogenic genes. Long-term effects of T3 on genes for the mitochondrial respiratory chain transcription factors and protein turnover.
8- to 10-week-old (TR and TR) male siblings (mice).Studied tissues: cerebellum, heart, and white adipose.
TR mice contain a cytosine insertion in exon 10 of the TR1 gene at nucleotide position 1,642 of the TR1 cDNA that leads to a frameshift of the carboxy- terminal 14 amino acids of TR1. For 7 days, T3 (5 g/mouse/day) was administered by i.p. injection.
11500
163 genes responsive to T3 treatment and 187 genes differentially expressed between TR mice and wild-type littermates.
T3 primarily acts to repress gene expression. TR has a powerful modulating effect in the heart. Novel physiologic candidates for T3 action are changes in immune-gene expression and in the induction of antiproliferative genes. The relative levels of TR isoforms lead to dramatic differential effects on gene expression.
Murine non-transfected hepatocyte cell line AML 12, expressing endogenous TRs.
RNA obtained from cells incubated for 3 hours or 24 hours 10 nM T3, in the presence of 10% stripped fetal bovine serum. Cells also incubated in the presence of cycloheximide (10 ìg/ml) 10 nM T3 for 3 hours to discriminate between primary and secondary responses.
15000
12 genes upregulated and 5 genes downregulated in the presence of T3.
Novel T3 responsive genes were identified. Insights were obtained into the role of T3 in processes such as cholesterol metabolism, bile acid secretion, and oncogenesis.
Gene expression analyzed in livers of mice rendered hypothyroid by treating pregnant mice from gestational d 13 to postnatal d 15 with 6-propyl-2-thiouracil in drinking water.
approximately 20000
96 differentially expressed genes were identified. Of these, 72 genes encode proteins of known function, 15 of which had previously been identified as regulated by TH.
Metabolism, development, cell proliferation, apoptosis, and signal transduction. A potential thyroid response element 1218 to 1188 bp upstream of the promoter region of Nr4a1 was identified and demonstrated to bind TH receptor TR and TR.
Hypothyroidism induced by i.p. injection of Na131I (250 Ci/100 g body weight) 28 days before the experiments. Hyperthyroidism provoked by i.p. injection of T3 (50 g/100 g body weight); repeated after 24 hours. Rats killed at 0, 6, 24, and 48 hours after thyroid hormone.
4608
Sixty-two of the genes were reproducibly T3-responsive.
Beside the “classical” pathway of T3-mediated gene regulation by TRs binding to TREs, an additional pathway appears to be mediated by transcription factors like NRF-1 and PPAR and coactivators (like the PGC-1 family of coactivators).
Cells were incubated without or with T3 (100 nM) for 1, 3, 6, 12, 24, or 72 hours. At each time-point, cells were harvested for total RNA preparation.
4400
358 responsive genes were identified. 88% had not previously been reported to be modulated by T3. A few genes showed biphasic expression patterns. In total, 203 genes were upregulated and the remainder were downregulated by T3.
Glucose metabolism, biosynthesis, transcriptional regulation, protein degradation, and detoxification in T3-induced cell proliferation.
Human adipose tissue obtained from the s.c. abdominal fat depots of Caucasian women for cDNA array and RT competitive PCR experiments.
Surgical adipose tissue samples were dissected from skin and vessels, and cultured adipocytes were obtained. Cultures were treated with T3 (100 nm) or vehicle for 24 hours. Medium-free T3 concentration was measured at 1 and 24 hours after addition of T3 (using RIA kits).
1 176
Among the statistically significant changes in mRNA levels of more than 1.3-fold, 13 and 6 genes were positively or negatively regulated, respectively.
Signal transduction, lipid metabolism, apoptosis, and inflammatory responses.
Human skin was obtained by punch biopsy from three normal individuals and two patients with RTH. Fibroblasts were grown in supplemented with 10% bovine calf serum (BCS). At confluency, the medium was replaced with one containing TH-depleted BCS (TxBCS), obtained from a thyroidectomized calf. For microarrays, incubation with T3 was for 24 hours.
more than 15000
Microarray analysis identified 148 genes induced by 1.4-fold or more and five genes repressed to 0.7 or less 24 hours after treatment with 2 10-9 M T3. Taking into account duplicate genes, these represented 91 up-regulated and five downregulated genes, respectively.
Aldo-keto reductase family 1 C1-3, collagen type VI 3, member RAS oncogene family brain antigen RAB3B, platelet phosphofructokinase, hypoxia-inducible factor-1, and enolase 1 genes, previously known to be induced by TH, were identified and validated. These genes have a variety of regulatory functions in development and metabolism.
Healthy male Caucasian volunteers (22–33 years old). The same investigations were performed on day 0 and day 14. Studied tissue: vastus lateralis muscle by percutaneous biopsies.
Participants took one tablet of 25 g of T3 three times a day (75 g per day) for 14 days.
24000
A transcriptional profile of 383 genes regulated by T3 was obtained (381 were upregulated and only two downregulated).
Novel target genes for T3 were identified. They belong to functional classes including transcriptional control, mRNA maturation, protein turnover, signal transduction, cellular trafficking, and energy metabolism.
Thyroidectomized patients treated for differentiated thyroid carcinoma (DTC) off and on L-thyroxine replacement.Studied tissue: skeletal muscle
Included were patients who had been diagnosed with DTC and had received initial therapy consisting of near-total thyroidectomy and radioiodine ablation therapy. Four weeks after L-thyroxine withdrawal and 8 wk after subsequent L-thyroxine replacement, patients were admitted to the clinical research unit. A catheter was inserted in a dorsal hand vein to collect blood samples for measurement of serum TSH, free T4 (fT4), and T3. Muscle biopsies were taken from the quadriceps muscle (vastus lateralis).
54674
607 differentially expressed genes on L-thyroxine treatment were identified, of which approximately 60% were positively and approximately 40% were negatively regulated.
New genes associated with thyroid state and involved in energy and fuel metabolism were overrepresented among the up-regulated genes. L-thyroxine therapy induced a large downregulation of the primary transcripts of the noncoding microRNA pair miR-206/miR-133b.
