Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells.

Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-ß-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single "all-in-one" Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities.

Mutation analysis of the LH receptor gene in Leydig cell adenoma and hyperplasia and functional and biochemical studies of activating mutations of the LH receptor gene.

CONTEXT: Germline and somatic activating mutations in the LH receptor (LHR) gene have been reported. OBJECTIVE: Our objective was to perform mutation analysis of the LHR gene of patients with Leydig cell adenoma or hyperplasia. Functional studies were conducted to compare the D578H-LHR mutant with the wild-type (WT)-LHR and the D578G-LHR mutant, a classic cause of testotoxicosis. The three main signal transduction pathways in which LHR is involved were studied. PATIENTS: We describe eight male patients with gonadotropin-independent precocious puberty due to Leydig cell adenoma or hyperplasia. RESULTS: The D578H-LHR mutation was found in the adenoma or nodule with hyperplasia in all but two patients. D578H-LHR displayed a constitutively increased but noninducible production of cAMP, led to a very high production of inositol phosphates, and induced a slight phosphorylation of p44/42 MAPK in the absence of human chorionic gonadotropin. The D578G-LHR showed a response intermediate between WT-LHR and the D578H-LHR. Subcellular localization studies showed that the WT-LHR was almost exclusively located at the cell membrane, whereas the D578H-LHR showed signs of internalization. D578H-LHR was the only receptor to colocalize with early endosomes in the absence of human chorionic gonadotropin. CONCLUSIONS: Although several LHR mutations have been reported in testotoxicosis, the D578H-LHR mutation, which has been found only as a somatic mutation, appears up until now to be specifically responsible for Leydig cell adenomas. This is reflected by the different activation of the signal transduction pathways, when compared with the WT-LHR or D578G-LHR, which may explain the tumorigenesis in the D578H mutant.

Improved lipid profile through liver-specific knockdown of liver X receptor alpha in KKAy diabetic mice.

Nuclear hormone receptors liver X receptor (LXRalpha and LXRbeta) ligands are attractive approaches for the treatment of dyslipidemia and atherosclerosis. To further elucidate the function of LXRalpha in liver lipid metabolism in a disease-relevant animal model, the KKAy mouse, we used adenoviral vectors to selectively knock down LXRalpha gene expression. Out of five different short hairpin RNAs (shRNAs) that were tested in vitro, one construct was selected for detailed analysis of LXRalpha knockdown in vivo. Reduction of LXRalpha transcript levels to 48 +/- 13% compared with control virus transduction resulted in a significant downregulation of the LXRalpha-regulated lipogenic genes sterol-regulatory element binding protein-1c (SREBP1c) and stearoyl CoA desaturase 1 in vivo. Interestingly, ABCA1 and phoshoenolpyruvate carboxykinase 1 expression was not affected, whereas lipoprotein lipase (LPL) expression was found to be increased. In addition, 8 days after virus transduction, both plasma and liver triglycerides (TGs) were reduced by about 50%. Changes in TG levels were not due to reduced food intake in virus-treated animals, because pair-fed mice showed unchanged TG levels. Taken together, liver-specific knockdown of LXRalpha in vivo by shRNA reduced expression of lipogenic master genes, like SREBP1c, and improved the lipid profile of hypertriglyceridemic KKAy mice.

Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor.

The H(4)R (histamine H(4) receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H(4)R is primarily expressed in eosinophils and mast cells and has the highest homology with the H(3)R. The occurrence of at least twenty different hH(3)R (human H(3)R) isoforms led us to investigate the possible existence of H(4)R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H(4)R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H(4)R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H(4)R-ligand induced signalling or constitutive activity for these H(4)R splice variants. However, when co-expressed with full-length H(4)R [H(4)R((390)) (H(4)R isoform of 390 amino acids)], the H(4)R splice variants have a dominant negative effect on the surface expression of H(4)R((390)). We detected H(4)R((390))-H(4)R splice variant hetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H(4)R splice variants were detected in various cell types and expressed at similar levels to the full-length H(4)R((390)) mRNA in, for example, pre-monocytes. We conclude that the H(4)R splice variants described here have a dominant negative effect on H(4)R((390)) functionality, as they are able to retain H(4)R((390)) intracellularly and inactivate a population of H(4)R((390)), presumably via hetero-oligomerization.

A flow-through fluorescence polarization detection system for measuring GPCR-mediated modulation of cAMP production.

A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z' factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H(3) G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC(50) values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements.

Normalization of prandial blood glucose and improvement of glucose tolerance by liver-specific inhibition of SH2 domain containing inositol phosphatase 2 (SHIP2) in diabetic KKAy mice: SHIP2 inhibition causes insulin-mimetic effects on glycogen metabolism, gluconeogenesis, and glycolysis.

Type 2 diabetes is characterized by a progressive resistance of peripheral tissues to insulin. Recent data have established the lipid phosphatase SH2 domain-containing inositol phosphatase 2 (SHIP2) as a critical negative regulator of insulin signal transduction. Mutations in the SHIP2 gene are associated with type 2 diabetes. Here, we used hyperglycemic and hyperinsulinemic KKA(y) mice to gain insight into the signaling events and metabolic changes triggered by SHIP2 inhibition in vivo. Liver-specific expression of a dominant-negative SHIP2 mutant in KKA(y) mice increased basal and insulin-stimulated Akt phosphorylation. Protein levels of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase were significantly reduced, and consequently the liver produced less glucose through gluconeogenesis. Furthermore, SHIP2 inhibition improved hepatic glycogen metabolism by modulating the phosphorylation states of glycogen phosphorylase and glycogen synthase, which ultimately increased hepatic glycogen content. Enhanced glucokinase and reduced pyruvate dehydrogenase kinase 4 expression, together with increased plasma triglycerides, indicate improved glycolysis. As a consequence of the insulin-mimetic effects on glycogen metabolism, gluconeogenesis, and glycolysis, the liver-specific inhibition of SHIP2 improved glucose tolerance and markedly reduced prandial blood glucose levels in KKA(y) mice. These results support the attractiveness of a specific inhibition of SHIP2 for the prevention and/or treatment of type 2 diabetes.