Upstream Stimulatory Factor (USF) and CCAAT/Enhancer Binding Protein δ (C/EBPδ) Compete for overlapping Sites in the Negative Regulatory Region of the HIV-1 LTR

Human immunodeficiency virus type 1 (HIV-1) is a human retrovirus and the causative agent of the acquired immunodeficiency syndrome. Genetic analysis has revealed that the HIV-1 LTR contains a potential negative regulatory element (NRE) with an E box, the recognition sequence for the helix-loop-helix (HLH) family of transcription factors. Furthermore, the upstream stimulatory factor (USF) has been implicated as a negative regulator of HIV-1 expression. Here, we report that the NRE is a composite element and that both C/EBPδ and USF can specifically bind to the NRE. The recognition sequence for C/EBPδ overlaps with the E box in the NRE of HIV-1. Competition experiments showed that either USF or C/EBPδ binds to this NRE but not both together

Liver-Specific Inactivation of the Proprotein Convertase FURIN Leads to Increased Hepatocellular Carcinoma Growth

Proprotein convertases are subtilisin-like serine endoproteases that cleave and hence activate a variety of proproteins, including growth factors, receptors, metalloproteases, and extracellular matrix proteins. Therefore, it has been suggested that inhibition of the ubiquitously expressed proprotein convertase FURIN might be a good therapeutic strategy for several tumor types. Whether this is also the case for hepatocellular carcinoma (HCC) is currently not clear. In a mouse model for HCC expression of Furin was not altered in the tumors, while those of PC7, PC5/6, and PACE4 significantly decreased, at least at some time points. To investigate the impact of Furin inhibition on the development and progression of HCC in this model, Furin was genetically ablated in the liver. Furin inactivation resulted in an increased tumor mass after 5 weeks. This was not caused by decreased apoptosis, since no differences in the apoptosis index could be observed. However, it could at least partially be explained by increased hepatocyte proliferation at 5 weeks. The tumors of the Furin knockout mice were histologically similar to those in wild type mice. In conclusion, liver-specific Furin inhibition in HCC enhances the tumor formation and will not be a good therapeutic strategy for this tumor type

Metabolic and Behavioural Phenotypes in Nestin-Cre Mice Are Caused by Hypothalamic Expression of Human Growth Hormone.

The Nestin-Cre driver mouse line has mild hypopituitarism, reduced body weight, a metabolic phenotype and reduced anxiety. Although several causes have been suggested, a comprehensive explanation is still lacking. In this study we examined the molecular mechanisms leading to this compound phenotype. Upon generation of the Nestin-Cre mice, the human growth hormone (hGH) minigene was inserted downstream of the Cre recombinase to ensure efficient transgene expression. As a result, hGH is expressed in the hypothalamus. This results in the auto/paracrine activation of the GH receptor as demonstrated by the increased phosphorylation of signal transducer and activator of transcription 5 (STAT5) and reduced expression of growth hormone releasing hormone (Ghrh). Low Ghrh levels cause hypopituitarism consistent with the observed mouse growth hormone (mGH) deficiency. mGH deficiency caused reduced activation of the GH receptor and hence reduced phosphorylation of STAT5 in the liver. This led to decreased levels of hepatic Igf-1 mRNA and consequently postnatal growth retardation. Furthermore, genes involved in lipid uptake and synthesis, such as CD36 and very low-density lipoprotein receptor were upregulated, resulting in liver steatosis. In conclusion, this study demonstrates the unexpected expression of hGH in the hypothalamus of Nestin-Cre mice which is able to activate both the GH receptor and the prolactin receptor. Increased hypothalamic GH receptor signaling explains the observed hypopituitarism, reduced growth and metabolic phenotype of Nestin-Cre mice. Activation of either receptor is consistent with reduced anxiety

GHD in Nestin-Cre mice leads to a decrease in STAT5 phosphorylation, lower expression of <i>Igf1</i> and an increase in the expression of <i>CD36</i> and <i>Vldlr</i> in the liver.

<p>A) Western blot analysis was performed for STAT5 and phospho-STAT5 on liver lysates from 1-month-old male Nestin-Cre mice and control littermates. B) Quantification of the phospho-STAT5/STAT5 ratio, **p<0.01. C) The expression of <i>Igf1</i>, <i>CD36</i> and <i>VLDLR</i> was investigated by RT-qPCR in the liver of 3-month-old male Nestin-Cre mice and control littermates (n = 3–4). Data are represented as mean ± SD. * p<0.05, **p<0.01, ***p<0.001.</p

<i>hGH</i> is expressed in the hypothalamus and to a lesser extent in the pituitary gland of Nestin-Cre mice.

<p>A) Schematic representation of the Cre-hGH transgene based on Tronche et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135502#pone.0135502.ref013" target="_blank">13</a>] B) The expression of <i>hGH</i> was investigated by RT-qPCR in the hypothalamus, the pituitary gland and the liver of 3-month-old male Nestin-Cre mice and control littermates (n = 4–5). C) Detection of a bicistronic Cre-hGH transcript by RT-qPCR, using a forward primer annealing to Cre and a reverse primer annealing to hGH. (n = 4–5) D) PCR was performed on hypothalamus cDNA from control and Nestin-Cre male mice (n = 4 per group), using primers in Cre and the last exon of hGH. E) hGH ELISA was performed on hypothalamus, pituitary and liver lysates from Nestin-Cre and control littermates (n = 6 for both groups). ND, not detected. Data are shown as mean ± SD, * p<0.05, **p<0.01, ***p<0.001.</p

Hypothalamic hGH expression increases STAT5 phosphorylation, induces <i>Cish</i> expression and leads to a reduction in the expression of <i>Ghrh</i>.

<p>A) Immunoblotting for STAT5 and phospho-STAT5 on hypothalamus lysates from Nestin-Cre versus control male mice. Three independent samples are shown per genotype. Actin was used as a loading control. B) Quantification of the phospho-STAT5/STAT5 ratio, **p<0.01. C) Hypothalamic <i>Cish</i> expression as quantified by RT-qPCR, n = 4–5, **p<0.01. D) The expression of <i>Ghrh</i> was investigated by RT-qPCR in the hypothalamus of 3-month-old male Nestin-Cre mice and control littermates, n = 4, *p<0.05.</p

The total body weight of Nestin-Cre mice is reduced.

<p>The total body weight of 3-month-old Nestin-Cre and wild type male mice (n = 4–5). Data are represented as mean ± SD. ***p<0.001.</p

Primer sequences used for transcript detection.

<p>Primer sequences used for transcript detection.</p

PREPL deficiency : Delineation of the phenotype and development of a functional blood assay

PurposePREPL deficiency causes neonatal hypotonia, ptosis, neonatal feeding difficulties, childhood obesity, xerostomia, and growth hormone deficiency. Different recessive contiguous gene deletion syndromes involving PREPL and a variable combination of SLC3A1 (hypotonia-cystinuria syndrome), CAMKMT (atypical hypotonia-cystinuria syndrome), and PPM1B (2p21 deletion syndrome) have been described. In isolated PREPL deficiency, previously described only once, the absence of cystinuria complicates the diagnosis. Therefore, we developed a PREPL blood assay and further delineated the phenotype.MethodsClinical features of new subjects with PREPL deficiency were recorded. The presence of PREPL in lymphocytes and its reactivity with an activity-based probe were evaluated by western blot.ResultsFive subjects with isolated PREPL deficiency, three with hypotonia-cystinuria syndrome, and two with atypical hypotonia-cystinuria syndrome had nine novel alleles. Their IQs ranged from 64 to 112. Adult neuromuscular signs included ptosis, nasal dysarthria, facial weakness, and variable proximal and neck flexor weakness. Autonomic features are prevalent. PREPL protein and reactivity were absent in lymphocytes from subjects with PREPL deficiency, but normal in the clinically similar Prader-Willi syndrome.ConclusionPREPL deficiency causes neuromuscular, autonomic, cognitive, endocrine, and dysmorphic clinical features. PREPL is not deficient in Prader-Willi syndrome. The novel blood test should facilitate the confirmation of PREPL deficiency