De Functionele Rol van Furine in de Lever tijdens Homeostase en Pathologische Condities: Een Reis van Uitdagingen en Valkuilen in Transgene Technologie

The biological role of hepatic Furin was investigated using conditional knock-out approaches in homeostatic and challenged conditions, including liver regeneration and HCC. On the one hand, the genetic inactivation of Furin in the liver under homeostatic conditions using AlfpCre Recombinase technology led to the characterization of an artifact related to the AlfpCre transgene explaining the phenotypes of post-natal growth retardation and liver steatosis. This type of artifact in Cre recombinase technology involves the use of the hGH minigene as a source of introns and a polyadenylation signal. In AlfpCre mice, this led to its expression in the hypothalamus and pituitary gland, causing GH deficiency and subsequently post-natal growth retardation and liver steatosis. On the other hand, liver regeneration and HCC challenged the liver in the setting of hepatic Furin deficiency. This led to a normal liver regenerative response, but an accelerated ASVB-induced tumor formation with significantly larger tumors due to an increased proliferation index in the early stage. The identification of FURIN substrates remains a subject of further research and may give more insights into why hepatic Furin deficiency led to a differential phenotype with regards to proliferation between liver regeneration and HCC.status: publishe

The AlfpCre mouse revisited, evidence for liver steatosis related to growth hormone deficiency

status: publishe

Inhibition of Sglt2-mediated streptozotocin uptake reduces direct renal toxicity in a mouse model for diabetic nephropathy

status: accepte

Endosome to trans-Golgi network transport of Proprotein Convertase 7 is mediated by a cluster of basic amino acids and palmitoylated cysteines

Proprotein Convertase 7 (PC7) is a Furin-like endoprotease that cleaves precursor proteins at basic amino acids. PC7 is concentrated in the trans-Golgi network (TGN) but it shuttles between the plasma membrane and the TGN depending on sequences in the cytoplasmic tail. A short region containing a three amino acids motif, P(724)-L(725)-C(726), is essential and sufficient for internalization of PC7 but not for TGN localization, which requires the additional presence of the juxtamembrane region. In this study we have investigated the contribution of a cluster of basic amino acids and two reversibly palmitoylated cysteine residues to endocytic trafficking. Stable cell lines overexpressing chimeric proteins (CD25 and CD46) containing the cytoplasmic domain of PC7 in which the basic cluster alone or together with both palmitoylated cysteines are mutated showed enhanced surface expression as demonstrated by immunofluorescence experiments and surface biotinylation. The mutant proteins no longer recycled to the TGN in antibody uptake experiments and accumulated in an endosomal compartment. Recycling of wild type PC7 to the TGN is blocked by nocodazole, suggesting that PC7 shuttles to the TGN via late endosomes, similar to Furin. Unlike furin, however, PC7 was found to recycle to a region within the TGN, which is deficient in sialyltransferase, as shown by resialylation experiments. In conclusion, a novel motif, composed of a basic amino acid cluster and two palmitoylated cysteines are essential for TGN localization and endocytic trafficking.status: publishe

The dwarf phenotype in GH240B mice, haploinsufficient for the autism candidate gene Neurobeachin, is caused by ectopic expression of recombinant human growth hormone.

Two knockout mouse models for the autism candidate gene Neurobeachin (Nbea) have been generated independently. Although both models have similar phenotypes, one striking difference is the dwarf phenotype observed in the heterozygous configuration of the GH240B model that is generated by the serendipitous insertion of a promoterless human growth hormone (hGH) genomic fragment in the Nbea gene. In order to elucidate this discrepancy, the dwarfism present in this Nbea mouse model was investigated in detail. The growth deficiency in Nbea+/- mice coincided with an increased percentage of fat mass and a decrease in bone mineral density. Low but detectable levels of hGH were detected in the pituitary and hypothalamus of Nbea+/- mice but not in liver, hippocampus nor in serum. As a consequence, several members of the mouse growth hormone (mGH) signaling cascade showed altered mRNA levels, including a reduction in growth hormone-releasing hormone mRNA in the hypothalamus. Moreover, somatotrope cells were less numerous in the pituitary of Nbea+/- mice and both contained and secreted significantly less mGH resulting in reduced levels of circulating insulin-like growth factor 1. These findings demonstrate that the random integration of the hGH transgene in this mouse model has not only inactivated Nbea but has also resulted in the tissue-specific expression of hGH causing a negative feedback loop, mGH hyposecretion and dwarfism

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.status: publishe

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.status: publishe

Alterations in the GH signaling cascade of <i>Nbea</i>^+/^<i>−</i> mice.

<p>(<b>A</b>) Schematic representation including the members of the GH pathway of which the mRNA level was measured by qRT-PCR. Black arrows indicate receptor binding, a + or – added to the arrow implicates a stimulation or inhibition effect, respectively. (<b>B</b>) In the hypothalamus of <i>Nbea</i>^+/<i>−</i> mice, the expression level of <i>Ghrh</i> was significantly reduced. The mRNA level of <i>Ghrelin</i> was too low to generate conclusive data (n = 4 litters/genotype; 12 weeks old). (<b>C</b>) In the anterior pituitary, the mRNA level of <i>Gh</i> was significantly decreased in <i>Nbea</i>^+/<i>−</i> mice. Moreover, the expression level of <i>Ghr</i> and <i>Sstr2</i> was significantly increased in <i>Nbea</i>^+/<i>−</i> mice (n = 4 litters/genotype; 12 weeks old). (<b>D</b>) The mRNA level of <i>Igf-1</i> was significantly reduced in the liver of <i>Nbea</i>^+/<i>−</i> mice (n  =  3/genotype; 12 weeks old). (<b>E</b>) The reduced mRNA level is reflected in a decrease of plasma IGF-1 in <i>Nbea</i>^+/<i>−</i> mice (n = 3/genotype; 12 weeks old). <i>Gh: growth hormone; Ghr: Gh receptor; Ghrh: Gh-releasing hormone; Ghrhr: Ghrh receptor; Sst: somatostatin; Sstr: Sst receptor; Ghrlr: Ghrelin receptor; Igf-1: insulin-like growth factor 1;</i> *: <i>P</i><0.05, **: <i>P</i><0.01; ***: <i>P</i><0.001.</p

Somatotrope cells in <i>Nbea</i>^+/^<i>−</i> mice are less numerous and contain less GH than <b>Nbea</b>^+/+ mice.

<p>(<b>A</b>) The pituitary of <i>Nbea^+/−</i> mice is smaller. More specifically, the anterior pituitary is affected since the size of the posterior pituitary (dashed line) appears to be unaffected. (<b>B</b>) Percent change in absolute number of growth hormone (GH) and prolactin (PRL) immunopositive cells in the anterior pituitary of <i>Nbea^+/−</i> compared to control mice, shown for 2 experiments separately (1,000–2,000 cells per hormone were counted per genotype; the experiment was conducted twice on 12 weeks old male mice). (<b>C</b>) In addition to the reduced presence of somatotrope cells in the anterior pituitary of <i>Nbea</i>^+/<i>−</i> mice, they also contained significantly less GH (n = 3/genotype; 12 weeks old). (<b>D</b>) Primary anterior pituitary cell cultures of <i>Nbea^+/−</i> mice secrete similar to <i>Nbea^+/+</i> mice under basal conditions and upon stimulation with IBMX and Forskolin (Stim.), after we normalized the GH content in the medium for the total GH level in somatotropes (n = 4/genotype). *: <i>P</i><0.05, **: <i>P</i><0.01; ***: <i>P</i><0.001.</p

Normal morphology of somatotrope cells of <i>Nbea</i>^+/^<i>−</i> mice.

<p>Electron microscopic views show representative somatotrope cells of 12 weeks old <i>Nbea</i>^+/+ and <i>Nbea</i>^+/<i>−</i> mice with no apparent ultrastructural differences. Nu: nucleus of a somatotrope cell.</p