Growth hormone directly stimulates GATA2 expression.

OBJECTIVE: GATA2 is a key transcription factor involved in the differentiation and determination of thyrotrophs and gonadotrophs in pituitary and hematopoietic development. However, studies on the upstream ligands of the GATA2 signal transduction pathway have been limited. To identify upstream ligands, we examined growth hormone (GH) as a plausible stimulator. DESIGN: We evaluated GH-induced GATA2 expression in murine TtT/GF thyrotrophic pituitary tumor cells and its direct impact on the GHR/JAK/STAT5 pathway using a combination of a reporter assay, real-time quantitative polymerase chain reaction, and western blotting. RESULTS: GATA2 expression increased with activated STAT5B in a dose-dependent manner and was inhibited by a STAT5 specific inhibitor. Moreover, we found functional STAT5B binding site consensus sequences at -359 bp in the GATA2 promoter region. CONCLUSION: These findings suggest that GH directly stimulates GATA2 via the GHR/JAK/STAT pathway and participates in various developmental phenomena mediated by GATA2.

Cigarette Smoke Extract Modulates Functions of Peroxisome Proliferator-Activated Receptors.

Cigarette smoke extract (CSE) contains many toxicants and may derange the physiological processes, such as cholesterol metabolism. We examined the impact of CSE on transcriptional regulation mediated peroxisome proliferator-activated receptors (PPARs) and its interaction with cofactors to elucidate differences in the molecular mechanism between CSE and other agonists of PPARs. We constructed several mutant PPARs (mPPARs) with amino acid substitution in the ligand-binding domain, which according to the molecular modeling, may affect the binding of agonists. In transient expression assays, each wild-type peroxisome proliferator-activated receptor (PPAR) mediated transcription stimulated by CSE was faintly yet significantly elevated compared to the control. The CSE-induced transcriptional activation was abolished in the H323A, H323Y, S342A, and H449A mPPARγs, although the activation elevated by pioglitazone was reserved. In the mPPARγ with Y473A and mPPARß/δs with H286Y and Y436A, the pioglitazone-induced or L165041-activated transcriptional elevations were decreased and were lower than that of CSE-induced stimulation. These results suggested that CSE activated both mutant PPARs to be selectively different from those ligands. Mammalian two-hybrid assay illustrated that CSE could mildly recruit SRC1 or GRIP1 to the wild-type PPARγ. Representative ingredients, such as acrolein and crotonaldehyde present in CSE, could stimulate PPAR isoforms even at the toxicological concentrations and might possibly contribute to stimulatory effects. CSE mildly regulates the cholesterol metabolism-related genes, such as low density lipoprotein (LDL) receptor and Liver X receptor (LXR)ß. In conclusion, these CSE effects the nuclear hormone receptors and their cofactors thereby disturbing metabolic phenomena. Therefore, CSE might be involved in cholesterol metabolism.

Insulin-like growth factor-1 directly mediates expression of mitochondrial uncoupling protein 3 via forkhead box O4.

OBJECTIVE: The objective of our study was to examine the direct action of insulin-like growth factor-1(IGF-1) signaling on energy homeostasis in myocytes. DESIGN: We studied the IGF-1 stimulation of mitochondrial uncoupling protein 3 (UCP3) expression in the HEK 293 derived cell line TSA201, murine C2C12 skeletal muscle myoblasts, and rat L6 skeletal myoblasts. We also investigated the direct effect of IGF-1 on the Insulin/IGF-1 receptor (IGF-1R)/phosphatidylinositol 3 (PI3)-Akt/forkhead box O4 (FOXO4) pathway using a combination of a reporter assay, semi-quantitative polymerase chain reaction, western blotting, and animal experiments. RESULTS: We demonstrated that IGF-1 regulates UCP3 expression via phosphorylation of FOXO4, which is a downstream signal transducer of IGF-1. UCP3 expression increased with activated FOXO4 in a dose-dependent manner. We also examined the functional FOXO4 binding site consensus sequences and identified it as the -1922 bp site in the UCP3 promoter region. UCP3 was also found to be concomitantly expressed with IGF-1 during differentiation of C2C12 myoblasts. Our animal experiments showed that high fat diet induced IGF-1 levels which likely influenced UCP3 expression in the skeletal muscle. CONCLUSION: Our findings demonstrate that that IGF-1 directly stimulates UCP3 expression via the IGF-1/IGF-1R/PI3-Akt/FOXO4 pathway.

IGF-1 regulate the expression of uncoupling protein 2 via FOXO1.

Mitochondria uncoupling protein2 (UCP2) expressed ubiquitously is a key molecule of energy metabolism. Insulin-like growth factor-1 (IGF-1) is a hormone, a target molecule of growth hormone (GH) signal pathway, which is also known as the drug "mecasermin" for clinical usages. IGF-1 is seemed to be closely related to metabolic diseases, such as adult GH deficiency. However, there has not been reports depicted possible relationship with each other. So, we sought to elucidate the mechanisms by which expression of UCP2 is regulated by IGF-1 via FOXO1. The findings suggested that three sequences in the consensus UCP2 promoter play complementary functional roles in the functional expression of FOXO1. So, we found that FOXO1 is involved in IGF-1-mediated energy metabolism greater than that of direct action of GH via STAT5. Our findings suggested that IGF-1 was involved in energy metabolism by regulating the expression of UCP2 via the PI3K/Akt/FOXO1 pathway.

Cigarette Smoke Extract Disrupts Transcriptional Activities Mediated by Thyroid Hormones and Its Receptors.

Cigarette smoke contains over 4800 compounds, including at least 200 toxicants or endocrine disruptors. Currently, effects of cigarette smoke on thyroid hormone (TH) levels remains to be clarified. Here, we demonstrate that cigarette smoke extract (CSE) possesses thyroid hormone properties and acts synergistically as a partial agonist for thyroid hormone receptors (TRs) in the presence of TH. In transient gene expression experiments, CSE stimulated transcriptional activity with TH in a dose-dependent manner. Stimulatory effects were observed with physiological TH concentrations, although CSE did not activate TRs without TH. CSE (5%) dissolved in phosphate-buffered saline (PBS) supplemented with 1 nM TH was approximately comparable to 3.2±0.1 and 2.3±0.2 nM of TRα1 and TRß1, respectively. To illustrate probable mechanisms of the CSE agonistic activity, effects on TR mediated transcriptional functions with cofactors were investigated. With a mammalian two-hybrid assay, CSE recruited the nuclear coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC1) to the TR. Unsaturated carbonyl compounds, acrolein, crotonaldehyde, and methyl vinyl ketone, representative constituents of CSE, retained such agonistic properties and possibly contributed to stimulatory effects. The results suggest that CSE recruits a transcriptional activator and may reinforce TH binding to the TR additively, resulting in gene expression. CSE partially agonizes TH action and may disturb the function of various nuclear hormone receptor types and their cofactors to disrupt the physiological processes.

GH directly stimulates UCP3 expression.

OBJECTIVE: We evaluated the direct action of GH signaling in energy homeostasis in myocytes. DESIGN: We investigated the GH-induced expression of UCP3 in human embryonic kidney 293 cells, human H-EMC-SS chondrosarcoma cells, murine C2C12 skeletal muscle myoblasts, and rat L6 skeletal muscle cells, as well as its direct effect on the GHR/JAK/STAT5 pathway using a combination of a reporter assay, real-time quantitative polymerase chain reaction, and western blotting. RESULTS: We demonstrated that the regulation of energy metabolism by GH involves UCP3 via activated STAT5, a signal transducer downstream of GH. UCP3 expression increased with STAT5 in a dose-dependent manner and was higher than that of UCP2. We confirmed the functional STAT5 binding site consensus sequences at -861 and -507 bp in the UCP3 promoter region. CONCLUSION: The results suggest that GH stimulates UCP3 directly and that UCP2 and that UCP3 participate in the signal transduction pathway that functions downstream of the GHR/JAK/STAT.

Effects of growth hormone on uncoupling protein 1 in white adipose tissues in obese mice.

OBJECTIVE: The transition of white adipocytes to beige cells (a phenomenon referred to as browning or beigeing) during obesity has been previously reported. Our study aimed to examine the mechanisms through which obesity induced by a high fat diet (HFD) affects uncoupling protein 1 (UCP1) expression via signal transduction and activator of transcription 5 (STAT5s). DESIGN: Seven-week-old male C57BL/6J mice were fed a normal or HFD for 11weeks. Body weight, white adipose tissue weight, and blood lipid and glucose levels were measured. To unveil the molecular mechanisms of UCP1 expression in adipose tissue, we performed further studying 3T3-L1 cells using qRT-PCR. We also measured UCP1 promoter activity in the TSA201 cell line using a dual luciferase assay. In addition, we analyzed the predicted consensus sequences for STAT5 binding in the UCP1 promoter region. RESULTS: Mice fed an HFD had higher body weight and intra-abdominal adipose tissues weight and a higher expression of UCP1, GH receptor (GHR), STATs, suppressors of cytokine signaling (SOCSs), and cytokine-inducible SH2-containing protein (CISH) compared to control mice. In 3T3-L1 cell studies, GH induced phosphorylation of the STAT5, SOCSs, CISH and UCP1 expressions. UCP1 promoter activity was associated with constitutively active STAT5 in a dose-dependent manner. We confirmed functional STAT5 binding sites at -425, -279, and -178bp of the UCP1 promoter. CONCLUSION: We suggest that endogenous GH induces UCP1 expression in adipose tissue via STAT5.