Administration of growth hormone or IGF-I to pregnant rats on a reduced diet throughout pregnancy does not prevent fetal intrauterine growth retardation and elevated blood pressure in adult offspring

Increasing evidence from human epidemiological studies suggests that poor growth before birth is associated with postnatal growth retardation and the development of cardiovascular disease in adulthood. We have shown previously that nutritional deprivation in the pregnant rat leads to intrauterine growth retardation (IUGR), postnatal growth failure, changes in the endocrine parameters of the somatotrophic axis, and to increased blood pressure in later life. In the present study, we investigated whether administration of insulin-like growth factor-I (IGF-I) or bovine growth hormone (GH) during pregnancy could prevent IUGR and/or alter long-term outcome. Dams h-om day 1 of pregnancy throughout gestation received a diet of nd libitum available food or a restricted dietary intake of 30% of ad libitum fed dams. From day 10 of gestation, dams were treated for 10 days with three times daily subcutaneous injections of saline (100 mu l), IGF-I (2 mu g/g body weight) or GH (2 mu g/g body weight). Maternal weight gain was significantly increased (P

Sequential increase in Egr-1 and AP-1 DNA binding activity in the dentate gyrus following the induction of long-term potentiation

Establishment of long-term potentiation (LTP) at perforant path synapses is highly correlated with increased expression of Egr and AP-1 transcription factors in rat dentate gyrus granule cells. We have investigated whether increased transcription factor levels are reflected in increased transcription factor activity by assessing Egr and AP-I DNA binding activity using gel shift assays. LTP produced an increase in binding to the Egr element, which was NMDA receptor-dependent and correlated closely with our previously reported increase in Egr-1 (zif/268) protein levels. Supershift analysis confirmed involvement of Egr-1, but not Egr-2 in the DNA binding activity. AP-1 DNA binding was also rapidly elevated in parallel with protein levels, however, the peak increase in activity was delayed until 4 h, a time point when we have previously shown that only jun-D protein was elevated. These data indicate that binding of Egr-1 and AP-1 to their response elements is increased in two phases. This may result in activation of distinct banks of target genes which contribute to the establishment of persistent LTP. (C) 2000 Elsevier Science B.V. All rights reserved.

Placental growth hormone (GH), GH-binding protein, and insulin-like growth factor axis in normal, growth-retarded, and diabetic pregnancies: Correlations with fetal growth

We previously described significant changes in GH-binding protein (GHBP) in pathological human pregnancy. There was a substantial elevation of GHBP in cases of noninsulin-dependent diabetes mellitus and a reduction in insulin-dependent diabetes mellitus. GHBP has the potential to modulate the proportion of free placental GH (PGH) and hence the impact on the maternal GH/insulin-like growth factor I (IGF-I) axis, fetal growth, and maternal glycemic status. The present study was undertaken to investigate the relationship among glycemia, GHBP, and PGH during pregnancy and to assess the impact of GHBP on the concentration of free PGH. We have extended the analysis of specimens to include measurements of GHBP, PGH, IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFSP-2, and IGFBP-3 and have related these to maternal characteristics, fetal growth, and glycemia. The simultaneous measurement of GHBP and PGH has for the first time allowed calculation of the free component of PGH and correlation of the free component to indexes of fetal growth and other endocrine markers. PGH, free PGH, IGF-I, and IGF-II were substantially decreased in IUGR at 28-30 weeks gestation (K28) and 36-38 weeks gestation (K36). The mean concentration (+/-SEM) of total PGH increased significantly from K28 to K36 (30.0 +/- 2.2 to 50.7 +/- 6.2 ng/mL; n = 40), as did the concentration of free PGH (23.4 +/- 2.3 to 43.7 +/- 6.0 ng/mL; n = 38). The mean percentage of free PGH was significantly less in IUGR than in normal subjects (67% vs. 79%; P < 0.01). Macrosomia was associated with an increase in these parameters that did not reach statistical significance. Multiple regression analysis revealed that PGH/IGF-I and IGFBP-5 account for 40% of the variance in birth weight. IGFBP-3 showed a significant correlation with IGF-I, IGF-II, and free and total PGK at K28 and K36. Noninsulin-dependent diabetes mellitus patients had a lower mean percentage of free PGH (65%; P < 0.01), and insulin-dependent diabetics had a higher mean percentage of free PGH (87%; P < 0.01) than normal subjects. Mean postprandial glucose at K28 correlated positively with PGH and free PGH (consistent with the hyperglycemic action of GH). GHBP correlated negatively with both postprandial and fasting glucose. Although GHBP correlated negatively with PGH (r = -0.52; P

Expression of POU, Sox, and Pax genes in the brain ganglia of the tropical abalone Haliotis asinina

In gastropod mollusks, neuroendocrine cells in the anterior ganglia have been shown to regulate growth and reproduction. As a first step toward understanding the molecular mechanisms underlying the regulation of these physiological processes in the tropical abalone Haliotis asinina, ive have identified sets of POU, Sox, and Pax transcription factor genes that are expressed in these ganglia. Using highly degenerate oligonucleotide primers designed to anneal to conserved codons in each of these gene families, we have amplified by reverse transcriptase polymerase chain reaction 2 POU genes (HasPOU-III and HasPOU-IV), 2 Sox genes (HasSox-B and HasSox-C), and two Pax genes (HasPax-258 and HaxPax-6). Analyses with gene-specific primers indicated that the 6 genes are expressed in the cerebral and pleuropedal ganglia of both reproductively active and spent adults, in a number of sensory structures, and in a subset of other adult tissues.

Cited2 is required both for heart morphogenesis and establishment of the left-right axis in mouse development

Establishment of the left-right axis is a fundamental process of vertebrate embryogenesis. Failure to develop left-right asymmetry leads to incorrect positioning and morphogenesis of numerous internal organs, and is proposed to underlie the etiology of several common cardiac malformations. The transcriptional modulator Cited2 is essential for embryonic development: Cited2-null embryos die during gestation with profound developmental abnormalities, including cardiac malformations, exencephaly and adrenal agenesis. Cited2 is also required for normal establishment of the left-right axis; we demonstrate that abnormal heart looping and right atrial and pulmonary isomerism are consistent features of the left-right-patterning defect. We show by gene expression analysis that Cited2 acts upstream of Nodal, Lefty2 and Pitx2 in the lateral mesoderm, and of Lefty1 in the presumptive floor plate. Although abnormal left-right patterning has a major impact on the cardiac phenotype in Cited2-null embryos, laterality defects are only observed in a proportion of these embryos. We have therefore used a combination of high-resolution imaging and three-dimensional (3D) modeling to systematically document the full spectrum of Cited2-associated cardiac defects. Previous studies have focused on the role of Cited2 in cardiac neural crest cell development, as Cited2 can bind the transcription factor Tfap2, and thus affect the expression of Erbb3 in neural crest cells. However, we have identified Cited2-associated cardiac defects that cannot be explained by laterality or neural crest abnormalities. In particular, muscular ventricular septal defects and reduced cell density in the atrioventricular (AV) endocardial cushions are evident in Cited2-null embryos. As we found that Cited2 expression tightly correlated with these sites, we believe that Cited2 plays a direct role in development of the AV canal and cardiac septa. We therefore propose that, in addition to the previously described reduction of cardiac neural crest cells, two other distinct mechanisms contribute to the spectrum of complex cardiac defects in Cited2-null mice; disruption of normal left-right patterning and direct loss of Cited2 expression in cardiac tissues.

Phosphorus overload and PTH induce aortic expression of Runx2 in experimental uraemia

Background. Vascular calcification (VC) is commonly seen in patients with chronic kidney disease (CKD). Elevated levels of phosphate and parathormone (PTH) are considered nontraditional risk factors for VC. It has been shown that, in vitro, phosphate transforms vascular smooth muscle cells (VSMCs) into calcifying cells, evidenced by upregulated expression of runt-related transcription factor 2 (Runx2), whereas PTH is protective against VC. In addition, Runx2 has been detected in calcified arteries of CKD patients. However, the in vivo effect of phosphate and PTH on Runx2 expression remains unknown. Methods. Wistar rats were submitted to parathyroidectomy, 5/6 nephrectomy (Nx) and continuous infusion of 1-34 rat PTH (at physiological or supraphysiological rates) or were sham-operated. Diets varied only in phosphate content, which was low (0.2%) or high (1.2%). Biochemical, histological, immunohistochemistry and immunofluorescence analyses were performed. Results. Nephrectomized animals receiving high-PTH infusion presented VC, regardless of the phosphate intake level. However, phosphate overload and normal PTH infusion induced phenotypic changes in VSMCs, as evidenced by upregulated aortic expression of Runx2. High-PTH infusion promoted histological changes in the expression of osteoprotegerin and type I collagen in calcified arteries. Conclusions. Phosphate, by itself is a potential pathogenic factor for VC. It is of note that phosphate overload, even without VC, was associated with overexpression of Runx2 in VSMCs. The mineral imbalance often seen in patients with CKD should be corrected.

DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor. (c) 2006 Elsevier Inc. All rights reserved.

Regulation of CSF-1 receptor expression

Cells of the mononuclear phagocyte lineage possess receptors for macrophage colony-stimulating factor (CSF-1) encoded by the c-fms protooncogene and respond to CSF-1 with increased survival, growth, differentiation, and reversible changes in function. The c-fms gene is itself a macrophage differentiation marker. In whole mount analyses of mRNA expression in embryos, c-fms is expressed at very high levels on placental trophoblasts. It is detectable on individual cells in the yolk sac around 8.5 to 9 days postcoitus, appears on isolated cells in the head of the embryo around 9.5 dpc, and appears on numerous cells throughout the embryo by day 10.5. The extent of c-fms expression is much greater than for other macrophage-specific genes including lysozyme and a macrophage-specific protein tyrosine phosphatase. Our studies of the cis-acting elements of the c-fms promoter have indicated a key role for collaboration between the macrophage-specific transcription factor, Pu.1, which functions in determining the site of transcription initiation, and other members of the Ets transcription factor family. This is emerging as a common pattern in macrophage-specific promoters. We have shown that two PU box elements alone can function as a macrophage-specific promoter. The activity of both the artifical promoter and the c-fms promoter is activated synergistically by coexpression of Pu.1 and another Ets factor, c-Ets-2. A 3.5kb c-fms exon 2 promoter (but not the 300bp proximal promoter) is also active in a wide diversity of tumor cell lines. The interesting exception is the melanoma cell line K1735, in which the promoter is completely shut down and expression of c-fms causes growth arrest and cell death. The activity of the exon 2 promoter in these nonmacrophages is at least as serum responsive as the classic serum-responsive promoter of the c-fos gene. It is further inducible in nonmacrophages by coexpression of the c-fms product. Unlike other CSF-1/c-fms-responsive promoters, the c-fms promoter is not responsive to activated Ras even when c-Ets-2 is coexpressed. In most lines, production of full length c-fms is prevented by a downstream intronic terminator, but in Lewis lung carcinoma, read-through does occur, and expression of both c-fms and other macrophage-specific genes such as lysozyme and urokinase becomes detectable in conditions of serum deprivation. (C) 1997 Wiley-Liss, Inc.

Contractile activity per se induces transcriptional activation of SLC2A4 gene in soleus muscle: involvement of MEF2D, HIF-1a, and TR alpha transcriptional factors

Lima GA, Anhe GF, Giannocco G, Nunes MT, Correa-Giannella ML, Machado UF. Contractile activity per se induces transcriptional activation of SLC2A4 gene in soleus muscle: involvement of MEF2D, HIF-1a, and TR alpha transcriptional factors. Am J Physiol Endocrinol Metab 296: E132-E138, 2009. First published October 28, 2008; doi: 10.1152/ajpendo.90548.2008.-Skeletal muscle is a target tissue for approaches that can improve insulin sensitivity in insulin-resistant states. In muscles, glucose uptake is performed by the GLUT-4 protein, which is encoded by the SLC2A4 gene. SLC2A4 gene expression increases in response to conditions that improve insulin sensitivity, including chronic exercise. However, since chronic exercise improves insulin sensitivity, the increased SLC2A4 gene expression could not be clearly attributed to the muscle contractile activity per se and/or to the improved insulin sensitivity. The present study was designed to investigate the role of contractile activity per se in the regulation of SLC2A4 gene expression as well as in the participation of the transcriptional factors myocyte enhancer factor 2D (MEF2D), hypoxia inducible factor 1a (HIF-1a), and thyroid hormone receptor-alpha (TR alpha). The performed in vitro protocol excluded the interference of metabolic, hormonal, and neural effects. The results showed that, in response to 10 min of electrically induced contraction of soleus muscle, an early 40% increase in GLUT-4 mRNA (30 min) occurred, with a subsequent 65% increase (120 min) in GLUT-4 protein content. EMSA and supershift assays revealed that the stimulus rapidly increased the binding activity of MEF2D, HIF-1a, and TR alpha into the SLC2A4 gene promoter. Furthermore, chromatin immunoprecipitation assay confirmed, in native nucleosome, that contraction induced an approximate fourfold (P < 0.01) increase in MEF2D and HIF-1a-binding activity. In conclusion, muscle contraction per se enhances SLC2A4 gene expression and that involves MEF2D, HIF-1a, and TR alpha transcription factor activation. This finding reinforces the importance of physical activity to improve glycemic homeostasis independently of other additional insulin sensitizer approaches.

PPAR gamma expression is increased in systemic lupus erythematosus patients and represses CD40/CD40L signaling pathway

Systemic lupus erythematosus (SLE) is a heterogeneous disease involving several immune cell types and pro-inflammatory signals, including the one triggered by binding of CD40L to the receptor CD40. Peroxisome-proliferator activated receptor gamma (PPAR gamma) is a transcription factor with anti-inflammatory properties. Here we investigated whether CD40 and PPAR gamma could exert opposite effects in the immune response and the possible implications for SLE. Increased PPAR gamma mRNA levels were detected by real-time PCR in patients with active SLE, compared to patients with inactive SLE PPAR gamma/GAPDH mRNA = 2.21 +/- 0.49 vs. 0.57 +/- 0.14, respectively (p < 0.05) or patients with infectious diseases and healthy subjects (p < 0.05). This finding was independent of the corticosteroid therapy. We further explored these observations in human THP1 and in SLE patient-derived macrophages, where activation of CD40 by CD40L promoted augmented PPAR gamma gene transcription compared to non-stimulated cells (PPAR gamma/GAPDH mRNA = 1.14 +/- 0.38 vs. 0.14 +/- 0.01, respectively; p < 0.05). This phenomenon occurred specifically upon CD40 activation, since lipopolysaccharide treatment did not induce a similar response. In addition, increased activity of PPAR gamma was also detected after CD40 activation, since higher PPAR gamma-dependent transcription of CD36 transcription was observed. Furthermore, CD40L-stimulated transcription of CD80 gene was elevated in cells treated with PPAR gamma-specific small interfering RNA (small interfering RNA, siRNA) compared to cells treated with CD40L alone (CD80/GAPDH mRNA = 0.11 +/- 0.04 vs. 0.05 +/- 0.02, respectively; p < 0.05), suggesting a regulatory role for PPAR gamma on the CD40/CD40L pathway. Altogether, our findings outline a novel mechanism through which PPAR gamma regulates the inflammatory signal initiated by activation of CD40, with important implications for the understanding of immunological mechanisms underlying SLE and the development of new treatment strategies. Lupus (2011) 20, 575-587.

AP-1 and Egr DNA-binding activities are increased in rat brain during ethanol withdrawal

The DNA-binding activities of AP-1 and Egr proteins were investigated in nuclear extracts of rat brain regions during ethanol withdrawal. Both DNA-binding activities were transiently elevated in the hippocampus and cerebellum 16 h after withdrawal. In the cerebral cortex, AP-1 and Egr DNA-binding activities increased at 16 h and persisted until 32 and 72 h, respectively. The AP-1 DNA-binding activities in all regions at all times after withdrawal were composed of FosB, c-Jun, JunB, and JunD. c-Fos was detected at all times in the cerebral cortex, at 16 h only in the hippocampus, and from 16 to 72 h in the cerebellum. Withdrawal severity did not affect the composition of the AP-1 DNA-binding activities. Two Egr DNA-binding activities were present in the cortex and hippocampus. The faster-migrating complex predominated in hippocampus, and only the slower-migrating complex (identified as Egr-1) was present in the cerebellum. The increase in DNA-binding activity of immediate early gene-encoded transcription factors supports their proposed role in initiating a cascade of altered gene expression underlying the long-term neuronal response to ethanol withdrawal.

Evaluation of the insulin-like growth factors (IGF) IGF-I and IGF binding protein 3 in patients at high risk for breast cancer

Our data suggest that serum concentrations of insulin-like growth factor I and insulin-like growth factor binding protein 3 do not correlate with breast cancer development. (Fertil Steril (R) 2011;95:2753-5. (C)2011 by American Society for Reproductive Medicine.)

A new FOXL2 gene mutation in a woman with premature ovarian failure and sporadic blepharophimosis-ptosis-epicanthus inversus syndrome

Objective: To describe a new FOXL2 gene mutation in a woman with sporadic blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and hypergonadotropic hypogonadism. Design: Case report. Setting: University medical center. Patient(s): A 28-year-old woman. Intervention(s): Clinical evaluation, hormone assays, gene mutation research. Main Outcome Measure(s): FOXL2 gene mutation. Result(s): The patient with hypergonadotropic hypogonadism was diagnosed with BPES due to a new FOXL2 gene mutation. Conclusion(s): Blepharophimosis-ptosis-epicanthus inversus syndrome is a rare disorder associated with premature ovarian failure (POF). The syndrome is an autosomal dominant trait that causes eyelid malformations and POF in affected women. Mutations in FOXL2 gene, located in chromosome 3, are related to the development of BPES with POF (BPES type I) or without POF (BPES type II). This report demonstrates a previously undescribed de novo mutation in the FOXL2 gene-a thymidine deletion, c. 627delT (g. 864delT)-in a woman with a sporadic case of BPES and POF. This mutation leads to truncated protein production that is related to a BPES type I phenotype. This report shows the importance of family history and genetic analysis in the evaluation of patients with POF and corroborates the relationship between mutations on the FOXL2 gene and ovarian insufficiency. (Fertil Steril (R) 2010; 93: 1006.e3-e6. (C) 2010 by American Society for Reproductive Medicine.)

Prognostic relevance of TTF-1 and MMP-9 expression in advanced lung adenocarcinoma

Background: The thyroid transcription factor-1 (TTF-1) is a tissue-specific transcription factor that Could playan important role in cell differentiation and morphogenesis of lung tumors. Matrix metalloproteinase-9 (MMP-9) is a protease commonly expressed in non-small cell lung cancer, conferring angiogenic and metastatic potential. Methods: We assessed TTF-1 and MMP-9 tumor expression by immunohistochemistry in 51 patients with lung adenocarcinoma, stage 11113 or IV, treated with platinum regimens. A bicategorical prognostic model was obtained using the Kaplan-Meier method, COX regression, and conjunctive consolidation. Results: The median expression of TTF-1 was 30.0% (range: 0-85.9%). All tumors expressed MMP-9 (median: 78.7%: range: 15.2-96.1%). Median survival was 41.6 weeks, with estimated 1- and 2-year survival rates of 45.0% and 22.0%, respectively. Poor performance status (Karnofsky scale) - hazards ratio(HR): 1.03. 95% confidence interval (CI): 1.01-1.06: low TTF-1 expression (<40%) - FIR: 4.00, 95% CI: 1.75-9.09: and high MMP-9 expression (>= 80%) - HR: 2.82, 95% CI: 1.30-6.08 were independent prognostic factors. Patients could be stratified in three death risk groups according to markers expression: low risk (high TTF-1 and low MMP-9; median survival: 127.6 weeks), intermediate risk (low TTF-1 OF high MMP-9; median survival: 39.0 weeks): and high risk (low TTF-1 and high MMP-9: median survival: 16.4 weeks). Conclusion: TTF-1 and MMP-9 tumor expression as detected by immunohistochemistry may allow identification of different, clinically meaningful, prognostic groups of advanced lung adenocarcinoma patients treated with platinum regimens. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Steroidogenic Factor 1 Overexpression and Gene Amplification Are More Frequent in Adrenocortical Tumors from Children than from Adults

Background: Steroidogenic factor 1 (SF-1) is a key determinant of endocrine development and function of adrenal cortex. SF-1 overexpression and gene amplification were previously demonstrated in a small group of pediatric adrenocortical tumors. Objective: Our objective was to determine the frequency of SF-1 protein expression and gene amplification in a large cohort of pediatric and adult adrenocortical tumors. Patients: SF-1 protein expression was assessed in a cohort of 103 adrenocortical tumors from 36 children and 67 adults, whereas gene amplification was studied in 38 adrenocortical tumors ( 17 from children). Methods: Tissue microarray, multiplex ligation-dependent probe amplification, and quantitative real-time PCR were used. Results: Astrong nuclear SF-1 expression was detected by tissue microarray in 56% (20 of 36) and 19% (13 of 67) of the pediatric and adult adrenocortical tumors, respectively (P = 0.0004). Increased SF-1 copy number was identified in 47% (eight of 17) and 10% (two of 21) of the pediatric and adult adrenocortical tumors, respectively (P = 0.02). All adrenocortical tumors with SF-1 gene amplification showed a strong SF-1 staining, whereas most of the tumors (61%) without SF-1 amplification displayed a weak or negative staining (P = 0.0008). Interestingly, a strong SF-1 staining was identified in five (29%) pediatric adrenocortical tumors without SF-1 amplification. The frequency of SF-1 overexpression and gene amplification was similar in adrenocortical adenomas and carcinomas. Conclusion: We demonstrated a higher frequency of SF-1 overexpression and gene amplification in pediatric than in adult adrenocortical tumors, suggesting an important role of SF-1 in pediatric adrenocortical tumorigenesis. (J Clin Endocrinol Metab 95: 1458-1462, 2010)