Wild-type Transgenic Mice Research Articles

The mRNA of TCTP functions as a sponge to maintain homeostasis of TCTP protein levels in hepatocellular carcinoma

Translationally controlled tumor protein (TCTP) is a highly conserved protein that accumulated in the tumorigenesis of various malignancies. Despite the important role of TCTP protein in tumor progression, the precise function and underlying mechanistic regulation of TCTP mRNA in hepatocellular carcinoma (HCC) remain unclear. In this study, we found that TCTP protein was overexpressed in HCC patients but TCTP mRNA expression levels were reversed. TCTP knockout HCC cells exhibited attenuated abilities of proliferation, migration, and invasion. The knockdown of TCTP by siRNA effectively reduced TCTP mRNA levels but not protein levels in HCC cells. Moreover, although the constitutive knockdown of TCTP inhibited almost 80% of TCTP protein expression levels in tumors of wildtype transgenic mice (TCTP KD/WT), partial restoration of TCTP protein expression was observed in the tumors of heterozygous TCTP mice (TCTP KD/TCTP±). The blockage of mRNA synthesis with ActD stimulated TCTP protein expression in HCC cells. In contrast, combined treatment with ActD and CHX or MG132 treatment alone did not lead to the TCTP protein accumulation in cells. Furthermore, following the introduction of exogenous TCTP in cells and orthotopic HCC tumor models, the endogenous TCTP protein did not change with the recombinational TCTP expression and kept a rather stable level. Dual-luciferase assays revealed that the coding sequence of TCTP mRNA functions as a sponge to regulate the TCTP protein expression. Collectively, our results indicated that the TCTP mRNA and protein formed a closed regulatory circuit and works as a buffering system to keep the homeostasis of TCTP protein levels in HCC.

Involvement of CCR5 in bone resorption associated to apical periodontitis

Abstract The involvement of Cysteine-cysteine chemokine receptor 5 (CCR5) in the protection or stimulation of bone resorption associated with apical periodontitis that occur as consequence of endodontic infection has not yet been elucidated. The present study proposes to characterize the involvement of CCR5 in the genesis and progression of apical periodontitis, using animals deficient for this molecule. Experimental induction of periapical lesions was performed in CCR5 −/− (knockout) or wild type transgenic mice through the coronary opening of the lower 1st molars and exposure of the root canals to the oral environment for their contamination. After the death of the animals (at 0, 7, 21 and 42 days), the samples obtained in different experimental periods were evaluated by conventional microscopy and fluorescence (morphometry and characterization of the lesions), histoenzimology (TRAP for osteoclasts), immunohistochemistry, immunofluorescence and real-time PCR, to characterize the inflammatory cell in the presence or absence of CCR5 (neutrophils, macrophages and CD4 and CD8 lymphocytes), expression of cytokines (IL-1±, TNF-±, IFN-³, IL 4, IL-6 and IL-10), their ligands (RANTES and MIP- 1±) and factors involved in bone formation and resorption (RUNX2, NFATc1, ALP, RANK, RANKL, OPG and cathepsin K) and infection of tissues. Our results shows that CCR5 knockout mice developed more extense and severe inflammation and alveolar bone destruction at periapical region at 21 and 42 days after endodontic infection. It was concluded that CCR5 deficiency results in development of more severe periapical alveolar bone resorption associated to endodontic infection.

Presenilin Mutation Suppresses Lung Tumorigenesis via Inhibition of Peroxiredoxin 6 Activity and Expression.

Some epidemiological studies suggest an inverse correlation between cancer incidence and Alzheimer's disease (AD). In this study, we demonstrated experimental evidences for this inverse relationship. In the co-expression network analysis using the microarray data and GEO profile of gene expression omnibus data analysis, we showed that the expression of peroxiredoxin 6 (PRDX6), a tumor promoting protein was significantly increased in human squamous lung cancer, but decreased in mutant presenilin 2 (PS2) containing AD patient. We also found in animal model that mutant PS2 transgenic mice displayed a reduced incidence of spontaneous and carcinogen-induced lung tumor development compared to wildtype transgenic mice. Agreed with network and GEO profile study, we also revealed that significantly reduced expression of PRDX6 and activity of iPLA2 in these animal models. PS2 mutations increased their interaction with PRDX6, thereby increasing iPLA2 cleavage via increased γ-secretase leading to loss of PRDX6 activity. However, knockdown or inhibition of γ-secretase abolished the inhibitory effect of mutant PSs. Moreover, PS2 mutant skin fibroblasts derived from patients with AD showed diminished iPLA2 activity by the elevated γ-secretase activity. Thus, the present data suggest that PS2 mutations suppress lung tumor development by inhibiting the iPLA2 activity of PRDX6 via a γ-secretase cleavage mechanism and may explain the inverse relationship between cancer and AD incidence.

Preclinical Activity of Splicing Modulators in U2AF1 Mutant MDS/AML

Mutations in spliceosome genes are detectable in ~50% of patients with myelodysplastic syndromes (MDS), making this cellular pathway the most commonly mutated in MDS and providing a novel target for therapeutic intervention. Spliceosome gene mutations are mutually exclusive, implying they are either redundant in pathogenic function or are not tolerated in a cell when they co-occur. Our group and others identified recurrent heterozygous missense mutations in the splicing factor gene U2AF1 in 11% of MDS patient samples. The most common U2AF1 mutation results in a conversion of serine to phenylalanine at position 34 (S34F) of the U2AF1 protein. We previously reported that expression of mutant U2AF1(S34F) in vivo using doxycycline-inducible U2AF1(S34F) transgenic mice revealed an expansion of hematopoietic bone marrow progenitor cells and leukopenia following transgene induction; both phenotypes are seen in patients with MDS. We also identified mutant U2AF1-specific alterations in pre-mRNA splicing in transgenic mouse bone marrow progenitor cells, primary AML patient samples, and CD34+ cells by RNA sequencing. We hypothesize that cells harboring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome by splicing modulator drugs, providing a new treatment approach for patients with U2AF1 mutations. For our studies, we utilize sudemycins, which are compounds that bind the SF3B1 spliceosome protein and modulate pre-mRNA splicing in non-hematopoietic tissues. We examined the effects of sudemycin treatment on pre-mRNA splicing in primary hematopoietic cells by treating CD34+ cells isolated from human umbilical cord blood with sudemycin in vitro. We performed whole transcriptome (RNA-seq) analysis following 6 hours of sudemycin treatment (1µM) of CD34+ cells and identified robustly altered pre-mRNA splicing patterns that are sudemycin-specific (26,120 splice junctions by DEXSeq, FDR<0.05, n=3), thereby validating that splicing is altered in hematopoietic cells treated with sudemycin. We confirmed a subset of these altered pre-mRNA splicing changes by RT-PCR and gel electrophoresis, as well as by Nanostring assay of RNA. We performed in vitro studies to examine the sensitivity of cells expressing U2AF1(S34F) to sudemycin treatment. Primary human MDS/AML cells with U2AF1(S34F) mutations display increased sensitivity to sudemycin, compared to non-mutant controls in a cell cycle (EdU incorporation) assay (n=3), while treatment with daunorubicin showed no specificity for mutant U2AF1(S34F) samples compared to non-mutant controls. Primary mouse c-Kit+ bone marrow cells transduced with a retrovirus expressing U2AF1(S34F) display a marked increase in apoptosis (by flow cytometry for Annexin V+ staining) in response to increasing concentrations of sudemycin, compared to controls (p<0.001, n=3-5). In addition, in vivo treatment of U2AF1(S34F) transgenic mice with sudemycin resulted in attenuation of hematopoietic progenitor cell expansion by colony forming unit (CFU-C) assay (p<0.01, n=6-11) and by flow cytometry for lineage-, c-Kit+, Sca-1+ (KLS) cells (p<0.001, n=6-11). Ongoing studies are examining the splicing alterations in U2AF1 mutant and wild-type transgenic mouse bone marrow cells treated with vehicle versus sudemycin. Taken together, these data suggest that we may be able to specifically treat hematological cancers with U2AF1 mutations using small molecule pre-mRNA splicing modulators such as sudemycin. DisclosuresNo relevant conflicts of interest to declare.

Biogenesis and cytotoxicity of APOL1 renal risk variant proteins in hepatocytes and hepatoma cells

Two APOL1 gene variants, which likely evolved to protect individuals from African sleeping sickness, are strongly associated with nondiabetic kidney disease in individuals with recent African ancestry. Consistent with its role in trypanosome killing, the pro-death APOL1 protein is toxic to most cells, but its mechanism of cell death is poorly understood and little is known regarding its intracellular trafficking and secretion. Because the liver appears to be the main source of circulating APOL1, we examined its secretory behavior and mechanism of toxicity in hepatoma cells and primary human hepatocytes. APOL1 is poorly secreted in vitro, even in the presence of chemical chaper-ones; however, it is efficiently secreted in wild-type transgenic mice, suggesting that APOL1 secretion has specialized requirements that cultured cells fail to support. In hepatoma cells, inducible expression of APOL1 and its risk variants promoted cell death, with the G1 variant displaying the highest degree of toxicity. To explore the basis for APOL1-mediated cell toxicity, endoplasmic reticulum stress, pyroptosis, autophagy, and apoptosis were examined. Our results suggest that autophagy represents the predominant mechanism of APOL1-mediated cell death. Overall, these results increase our understanding of the basic biology and trafficking behavior of circulating APOL1 from the liver.

ASSA14-03-11 Role of GRK4γ variant 142V in the regulation of renal ETB receptor in hypertension

Background The endothelin receptor B (ETBR) regulates blood pressure and water and electrolyte balance by engendenring natriuresis. In hypertensive states, the ETBR mediated diureis and natriuresis is impaired. However, the underlying mechanisms are not clear. G protein-coupled receptor kinase 4 (GRK4), whose gene locus 4p16.3 is linked to essential hypertension, cause sodium retention and increase blood pressure via impairment of renal dopamine receptor and enhancement of renin-angiotensin system functions. Due to the higher activity of GRK4 in kidney from spontaneously hypertensive rats (SHRs) and hypertensive patients, we hypothesise that GRK4 might be the cause of ETBR impairment in hypertension. Method Experiments were carried out in male anaesthetic spontaneously hypertensive rats (SHR)and in normotensive Wistar-Kyoto (WKY) rats. The ETB receptor agonist, BQ-3020 (0.1, 0.5, 1.0 ug/kg/min) were infused via supra-renal artery at a rate of 0.04 ml/min for 40 min. The same experiments were conducted in GRK4 A142V and GRK4 Wild Type transgenic mice. The ETBR function were also checked in the wild-type and A142V transfected renal proximal tubule (RPT) cells from mice. Results We found that diuresis and natriuresis of ETBR agonist, BQ3020, in Wistar-Kyoto (WKY) rats, which was impaired in SHRs. The GRK4 expression was higher in renal cortex from SHRs as compared with WKY rats. In GRK4 A142V transgenic mice, it resulted that ETBR-mediated diuresis and natriuresis was impaired compared with Wild type. In wild-type transfected cells, activation of ETBR inhibited Na + -K + -ATPase activity; while in A142V transfected cells, the inhibitory effect was lost. There are co-localization and co-immunoprecipitation between ETBR and GRK4 in RPT cells. The linkage of ETBR/GRK4 was higher in wilde-type cells than in A142V cells. Similar phenomenon was found in the kidney from WKY and SHRs, SHRs had higher ETBR/GRK4 linkage, accompanied with higher ETBR phosphorylation, which might account for the impaired ETBR function in hypertension. Conclusion This study provides a mechanism by which GRK4, via regulation of renal ETBR function, participates in the pathogenesis of hypertension.

Role of GRK4 in the Regulation of Arterial AT 1 Receptor in Hypertension

G-protein-coupled receptor kinase 4 (GRK4) gene variants, via impairment of renal dopamine receptor and enhancement of renin-angiotensin system functions, cause sodium retention and increase blood pressure. Whether GRK4 and the angiotensin type 1 receptor (AT(1)R) interact in the aorta is not known. We report that GRK4 is expressed in vascular smooth muscle cells of the aorta. Heterologous expression of the GRK4γ variant 142V in A10 cells increased AT(1)R protein expression and AT(1)R-mediated increase in intracellular calcium concentration. The increase in AT(1)R expression was related to an increase in AT(1)R mRNA expression via the NF-κB pathway. As compared with control, cells expressing GRK4γ 142V had greater NF-κB activity with more NF-κB bound to the AT(1)R promoter. The increased AT(1)R expression in cells expressing GRK4γ 142V was also associated with decreased AT(1)R degradation, which may be ascribed to lower AT(1)R phosphorylation. There was a direct interaction between GRK4γ and AT(1)R that was decreased by GRK4γ 142V. The regulation of AT(1)R expression by GRK4γ 142V in A10 cells was confirmed in GRK4γ 142V transgenic mice; AT(1)R expression was higher in the aorta of GRK4γ 142V transgenic mice than control GRK4γ wild-type mice. Angiotensin II-mediated vasoconstriction of the aorta was also higher in GRK4γ 142V than in wild-type transgenic mice. This study provides a mechanism by which GRK4, via regulation of arterial AT(1)R expression and function, participates in the pathogenesis of conduit vessel abnormalities in hypertension.

Abstract 601: Bcl10 Deficiency of Both Cardiac Cells and Bone Marrow Derived Cells Attenuates Angiotensin (Ang) II-induced Target Organ Damage

Background Bcl10 is a member of the CARMA3-Bcl10-MALT1 (CBM) signalosome that links angiotensin (Ang) II and NF-κB signaling. We have shown earlier that Ang II-infused Bcl10 knockout (KO) mice develop less inflammatory cell infiltration and less fibrosis coupled with improved arrhythmogenic electrical remodeling compared to wild-type (WT) littermates. In our present study we investigated the mechanism behind this phenotype. Methods and Results Bone marrow (BM) from WT and green fluorescence protein transgenic WT mice (GFP+ WT) was transferred into WT and Bcl10 KO mice. Conversely, Bcl10 KO BM was transferred into WT mice. After BM regeneration mice were uni-nephrectomized, 1% NaCl was given in the drinking water and received Ang II (1.44 mg/kg/d) for 14 days. Both Bcl10 BM transplanted WT mice and WT BM transplanted Bcl10 KO mice showed less immune cell infiltration compared to WT BM transplanted WT mice (macrophages: 44.8±2.7 and 46.8±1.8 vs. 62.0±1.5; CD4+ T-cells: 22.8±1.4 and 24.3±1.3 vs. 31.2±0.9; CD8+ T cells 24.2±1.2 and 28.0±1.1 vs. 39.5±1.1 cells/heart section, respectively) despite similar cardiac hypertrophy. In parallel with cell infiltration these mice developed less perivascular collagen 1 and interstitial fibronectin deposition, as well. Immunohistological and qPCR analysis revealed less VCAM-1, ICAM-1 and chemokines (MCP-1 and RANTES) expression in WT BM transplanted Bcl10 KO mice compared to Bcl10 BM transplanted or WT BM transplanted WT mice. Moreover, in vitro endothelial cells transfected with Bcl10 siRNA showed 5-fold less increase in monocyte attachment after Ang II treatment as endothelial cells transfected with control siRNA. Additionally, transplantation with GFP+ WT BM cells revealed that Bcl10 KO mice recruit less BM-derived GFP+/FSP1+ fibroblasts compared to GFP+ WT BM transplanted WT mice (51.3±1.3 vs. 23.5± 1.3 cells/heart section) suggesting that non-cardiac cells contribute to fibrosis too. Finally, intraperitoneal injection of MCP-1 into WT mice significantly increased the number of peritoneal CD11b+ cells, whereas Bcl10 KO mice showed no increase in response to MCP-1. Conclusion Our results demonstrate that cardiac fibrosis in Bcl10 KO mice depends on both immune/BM-derived cells and resident cardiac cells.

Abstract 100: Initial Characterization of Oxidant-Resistant Human ApoAI Transgenic Mice

Introduction HDL functions, such as its cholesterol acceptor and anti-inflammatory activities, are impaired by myeloperoxidase (MPO), which selectively targets and oxidizes HDL and apoAI. We previously found that the 4WF apoAI isoform, in which the 4 Trp residues are substituted with Phe, is equally competent as wild type (WT) human apoAI to interact with lipids and accept cholesterol in an ABCA1-dependent manner, but is resistant to MPO mediated loss of function. Hypothesis The oxidant resistant 4WF apoAI isoform will be superior in mediating reverse cholesterol transport than WT human apoAI in vivo under conditions of elevated oxidative stress. Methods and Results We generated transgenic mice expressing the 4WF human apoAI (h-apoAI) isoform. A genomic fragment containing the h-apoAI gene was subjected to site directed mutagenesis to convert the Trp to Phe residues, and microinjected into C57BL/6 fertilized oocytes. We selected the line with the highest h-apoAI expression to compare with existing WT h-apoAI transgenics. We assessed plasma apoAI and HDL-C levels in male and female mice. Male mice have significantly higher h-apoAI levels than females for both isoforms (p&lt;0.05). In general, the WT apoAI transgenics have higher plasma h-apoAI levels than the 4WF transgenics; however there is enough overlap that we were able to select male 4WF and WT h-apoAI transgenic mice having matched plasma h-apoAI levels of ~550 mg/dl, which are described below. HDL-C levels were slightly lower in 4WF vs. WT transgenics (157±4 vs 184±8 mg/dl, respectively, N=5 per group, p=0.03). We performed in vivo reverse cholesterol transport (RCT) assay by s.c. injection of cholesterol labeled and loaded bone-marrow macrophages. RCT to the plasma reflected the HDL-C levels and was lower in the 4WF vs. WT transgenic mice (4.5% vs 6.6%, respectively p=0.007). However, RCT to the liver and feces was equivalent in the 4WF and WT transgenics (NS). This suggests that the flux of HDL-C in the 4WF mice may be higher than in the WT apoAI transgenics, leading to equal RCT to the feces and liver despite lower HDL-C levels. Conclusion 4WF transgenic mice are now breeding with MPO transgenic mice in order to test our hypothesis that this isoform retains more HDL function under oxidative stress conditions.

Effects of Cardiomyopathy-Related Troponin T Mutations on the Ubiquitin-Proteasome System

Familial hypertrophic cardiomyopathy (FHC) is a disease of the myocardium that can be caused by a mutation in a sarcomeric gene. TNNT2 encodes cardiac troponin T (cTnT), a sarcomeric protein important for cardiac muscle contraction. Mutations in TNNT2 are frequently linked to sudden cardiac death (SCD); however their role in heart failure and cardiomyopathy is unclear. We hypothesize that changes in the ubiquitin-proteasome system (UPS) are related to increased risk of cardiac death, and that changes in the UPS may be related to calcium sensitization caused by TNNT2 mutations. We examined two mutations in TNNT2, R278C, which is associated with mild effects and late-onset heart disease, and the calcium-sensitizing mutation I79N, which is early-onset and has severe cardiac effects, including SCD. In 3-month-old transgenic mice expressing the I79N mutant form of troponin T, expression of the proteasome subunit PSMA6 was decreased by approximately 35% in I79N mice when compared to hearts from wild-type transgenic mice. Protein levels of the 19S regulator subunit Rpt1 were increased in the hearts of 3-month-old I79N mice and decreased in the hearts of 1-year-old R278C mice. Expression of PKC alpha, an important regulator of cardiac contractility, was decreased in R278C mice at 1 year. Activity of the lysosomal protease cathepsin L was increased in 1-year-old I79N mice, indicating disruption of another major proteolytic system in these hearts. Quantitative real-time PCR of the genes involved in the ubiquitination pathway (E1, E2, and E3 enzymes) showed that transcript levels were increased or decreased for 25 genes in I79N mice and three genes in R278C mice. These data demonstrate that the UPS is affected at the gene expression and protein level in R278C and I79N mice, indicating that changes in the UPS may be involved in FHC.

Effect of the Troponin I Restrictive Cardiomyopathy Mutation R145W on Protein Expression in Murine Murine Hearts

Restrictive cardiomyopathy (RCM) is a relatively rare type of cardiomyopathy but is assocated with a high incidence of sudden cardiac death (SCD). The R145W mutation in troponin I have been shown to be associated with RCM and in patients with this mutation SCD has been found to occur. A transgenic mouse model of RCM expressing troponin I R145W showed increased calcium sensitivity of force development and impaired muscle relaxation. Isobaric tags for relative and absolute quantitation (iTRAQ), a non-gel-based technique used to quantify proteins from different sources in a single experiment, was used to determine the differences between hearts from 3 month old transgenic control mice (mice expressing human cardiac troponin I) and R145W mice (mice expressing human troponin I with the R145W mutation). Four hearts each from R145W and wild-type transgenic mice were proteomically investigated and several signaling pathways were found to be affected by the R145W mutation. Increases in enzymes involved in ATP production (such as NAD(P) transhydrogenase), kinases (calmodulin dependent protein kinase II beta), intermediate filament proteins (desmin), de-ribosylating enzyme (ADP-ribosylarginine hydrolase), proteolytic enzymes (tripeptidy peptides II), glycolytic enzymes (glyceraldehyde-3-phosphate dehydrogenase), and stress related proteins (inducible heat shock protein 90aa). At the proteolytic level the activity of the proteasome was decreased in hearts from R145W transgenic mice. No change in the protein expression levels of the 20S protein PSMA6 or the 19S protein Rpt1 was observed in these hearts. These results suggest that the RCM mutation R145W is associated with significant and complex changes in cardiac protein expression.

Taurocholic acid-induced secretion in normal and cystic fibrosis mouse ileum.

Bile acids cause secretion throughout the intestinal tract and this process contributes to maintaining the fluidity of intestinal contents. In cystic fibrosis (CF) defective intestinal secretion can lead to excessive dehydration of the luminal contents and the development of clinical symptoms. This study was designed to investigate bile acid-induced secretion in mouse ileum and to determine whether this process was defective in CF. Taurocholic acid-induced secretion was monitored as a rise in short-circuit current (SCC) in ileal sheets from normal (Swiss MF1) and transgenic CF mice. Taurocholic acid increased the SCC in both intact and stripped ileal sheets from Swiss MF1 mice. This effect was due to a stimulation of electrogenic Cl- secretion as it was inhibited by Cl(-)-free conditions, serosal furosemide (frusemide), mucosal diphenylamine-2-carboxylic acid (DPC) and increased serosal K+ concentration, without being affected by reduced mucosal Na+ concentration. Taurocholic acid-induced secretion was inhibited by tetrodotoxin, indicating the involvement of a neural pathway, but this did not include capsaicin-sensitive afferent neurons or muscarinic cholinoreceptors. Mucosal mast cells also contributed to the response. Responses in tissues from transgenic wild-type mice were similar to those obtained with Swiss MF1 animals, but ilea from CF mice exhibited a lower basal SCC with significantly reduced secretory responses to acetylcholine and taurocholic acid. We concluded that taurocholic acid induces ileal secretion by a mechanism that entails activation of enteric nerves and degranulation of mucosal mast cells. Impaired bile acid-induced secretion in CF may contribute to luminal dehydration.

Ursodeoxycholic acid action on the transport function of the small intestine in normal and cystic fibrosis mice.

Ursodeoxycholic acid possesses choleretic and cytoprotective properties and in cystic fibrosis (CF) it is used to treat the hepatobiliary symptoms of the disease. This study investigated the effects of this bile acid on the transport function of the small intestine in normal and CF mice. The effects of ursodeoxycholic acid were monitored as changes in short-circuit current (SCC) in stripped sheets of small intestine from normal (Swiss MF1) and transgenic CF (Cftr(tm2Cam)) mice. In ileal sheets from Swiss MF1 mice, mucosal ursodeoxycholic acid caused a biphasic increase in SCC. The first phase was reduced by lowering the mucosal Na+ concentration, while the second phase was inhibited by (Cl-)-free conditions, serosal furosemide or mucosal diphenylamine-2-carboxylic acid (DPC), suggesting an initial Na+-dependent bile acid absorption followed by a stimulation of electrogenic Cl- secretion. Serosal application of ursodeoxycholic acid to the ileum and mucosal or serosal application to the mid-intestine and jejunum elicited a secretory response only. Secretion was Ca2+-dependent, but did not involve neural mechanisms. Mucosal mast cells, histamine and serotonin (5-HT) were implicated in the secretory response. Responses in tissues from transgenic wild-type mice were similar to those obtained with Swiss MF1 mice, but the secretory response to mucosal or serosal application of the bile acid was impaired in CF tissues. In ilea from CF mice the initial absorptive phase of the response to mucosal ursodeoxycholic acid was still observed. It is concluded that ursodeoxycholic acid induces secretion throughout the murine small intestine by a mechanism that involves degranulation of mucosal mast cells. In the ileum Na+-dependent absorption can also be detected. The secretory response is defective in CF intestine, but the absorptive effect is still present.

All of the human β‐type globin genes compete for LCR enhancer activity in embryonic erythroid cells of yeast artificial chromosome transgenic mice

In primitive erythroid cells of human beta-globin locus transgenic mice (TgM), the locus control region (LCR)-proximal epsilon- and gamma-globin genes are transcribed, whereas the distal delta- and beta-globin genes are silent. It is generally accepted that the beta-globin gene is competitively suppressed by gamma-globin gene expression at this developmental stage. Previously, however, we observed that epsilon-globin gene expression was severely attenuated when its distance from the LCR was extended, implying that beta-globin gene might also be silenced because of its great distance from the LCR. Here, to clarify the beta-globin gene silencing mechanism, we established TgM lines carrying either gamma- or epsilon- plus gamma-globin promoter deletions, without significantly altering the distance between the beta-globin gene and the LCR. Precocious expression of delta- and beta-globin genes was observed in primitive erythroid cells of mutant, but not wild-type TgM, which was most evident when both the epsilon and gamma promoters were deleted. Thus, we clearly demonstrated that the repression of the delta- and beta-globin genes in primitive erythroid cells is dominated by competitive silencing by the epsilon- and gamma-globin gene promoters, and that epsilon- and the other beta-like globin genes might be activated by two distinct mechanisms by the LCR.

Functional Effects of a Restrictive-Cardiomyopathy-Linked Cardiac Troponin I Mutation (R145W) in Transgenic Mice

The human cardiac troponin I (hcTnI) mutation R145W has been associated with restrictive cardiomyopathy. In this study, simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) were explored. Tg-R145W fibers showed an ∼ 13–16% increase in maximal Ca 2+-activated force and ATPase activity compared to hcTnI wild-type transgenic mice. The force-generating cross-bridge turnover rate ( g) and the energy cost (ATPase/force) were the same in all groups of fibers. Also, the Tg-R145W fibers showed a large increase in the Ca 2+ sensitivity of both force development and ATPase. In intact fibers, the mutation caused prolonged force and intracellular [Ca 2+] transients and increased time to peak force. Analysis of force and Ca 2+ transients showed that there was a 40% increase in peak force in Tg-R145W muscles, which was likely due to the increased Ca 2+ transient duration. The above cited results suggest that: (1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca 2+ transients that would result in a decrease in ventricular filling (diastolic dysfunction); and (2) there would be a large (approximately 53%) increase in force during systole, which may help to partly compensate for diastolic dysfunction. These functional results help to explain the mechanisms by which these mutations give rise to a restrictive phenotype.

Proteasome Dysfunction in Troponin Related Cardiomyopathies

Hypertrophic cardiomyopathy (HCM) is a disease characterized by small to significant increases in calcium sensitivity of force development. Our results suggest that patients with mutations which are associated with large increases in calcium sensitivity of force development show poor prognosis. Investigation of two cardiac troponin T (cTnT) transgenic mice, I79N and F110I, which are associated with HCM using a functional proteomics approach showed that several key proteins which are known to be degraded by the proteasome are altered when compared to wild-type cTnT transgenic mice. Skinned fiber studies from I79N and F110I transgenic mice showed that these mutations both caused similar increases in calcium sensitivity of force development (change in pCa50 = approx. 0.22) when compared to wild-type transgenic mice. The 179N and F110I mice both showed significant decreases in 20S proteasome activities related to wild-type cTnT transgenic mice suggesting that proteasomal dysfunction may be a contributing factor to the pathogenesis of HCM. The levels of polyubiquitinated proteins present in the I79N and F110I were also increased relative to wild-type cTnT transgenic mice. Transgenic mice expressing cTnT I79N or cTnT F110I do not develop significant hypertrophy or ventricular fibrosis even after chronic exercise challenge. This suggests that increased heart size may not the critical factor for causing proteasome dysfunction in some cardiomyopathies. These results suggest that the UPS is an important system involved in the pathogenesis of troponin related HCM which is initially caused by significant increases in calcium sensitivity of force development.

P3-211: Overexpression of the C-terminal fragment of APP altered gene expression profiles in APPV717I-CT100 transgenic mice

Many carboxy-terminal (C-terminal) fragments of the human amyloid precursor protein (APP) are neurotoxic, including β amyloid (Aβ) and APP-CT100. We used a transgenic mouse model expressing the familial Alzheimer's disease (AD) ‘London mutation' with the V717I (valine to isoleucine) substitution within APP-CT100, which includes the Aβ sequence. APPV717I is associated with aggressive early onset dementia and a characteristic increase in the long amyloidogenic form of Aβ (Aβ1–42) in AD brain. β amyloid, in the form of diffuse non-congophilic extracellular plaques, deposits in the brain of transgenic mice expressing APPV717I-CT100 from 6 months of age. To elucidate the neurodegenerative mechanisms of AD, we evaluated several genes that might be involved in APP pathology in 11-month-old APPV717I-CT100 transgenic mice using microarray analysis. We compared the profiles of the altered genes in the hippocampal region of transgenic mice with that of age-matched wild-type transgenic mice. We also clustered the genes associated with apoptosis, cell cycle, cell death, cellular physiology, immune response, signal transduction, and transcription. We selected several genes that might have essential roles in AD pathology and their altered expression in the cortical and hippocampal brain tissues used in the microarray anaysis was confirmed with RT-PCR and Western blot analysis. In addition, the expression pattern of the selected genes was examined in human AD brain by immunohistochemistry and Western blot, and their pathogenic mechanisms were studied in an APP intracellular domain -overexpressing cell culture system. These findings suggest that APPV717I-CT100 overexpression selectively alters the levels of specific transcripts in the brains of transgenic mice. This study provides valuable information about several AD-related genes and their pathogenic roles.

Myosin regulatory light chain E22K mutation results in decreased cardiac intracellular calcium and force transients

The glutamic acid to lysine mutation at the 22nd amino acid residue (E22K) in the human cardiac myosin regulatory light chain (RLC) gene causes familial hypertrophic cardiomyopathy (FHC) with a phenotype of midventricular obstruction and septal hypertrophy. Our recent histopathology results have shown that the hearts of transgenic E22K mice (Tg-E22K) resemble those of human patients, demonstrating enlarged interventricular septa and papillary muscles. In this study, we show no effect of the E22K mutation on the kinetics of mutated myosin in its ATP-powered interaction with fluorescently labeled single actin filaments compared to nontransgenic or transgenic wild-type (Tg-WT) control mice. Likewise, no change in cross-bridge dissociation rates (g(app)) was observed in freshly skinned papillary muscle fibers. In contrast, maximal force and ATPase were decreased approximately 20% in Tg-E22K skinned papillary muscle fibers and intracellular [Ca2+] and force transients were significantly decreased in intact papillary muscle fibers from Tg-E22K compared to Tg-WT mice. Moreover, energy metabolism measured in isolated working Tg-E22K mouse hearts perfused under conditions of physiologically relevant levels of metabolic demand was similar in Tg-E22K and control hearts before and after 20 min of no-flow ischemia. Our results suggest that the pathological response observed in the E22K myocardium might be triggered by mutation induced changes in the properties of the RLC Ca2+-Mg2+ site, the state of the Ca2+/Mg2+ occupancy and consequently the Ca2+ buffering ability of the RLC. By decreasing the affinity of the RLC for Ca2+, the E22K mutation most likely promotes a Mg2+-saturated RLC producing less force and ATPase than the Ca2+-saturated RLC of WT fibers. Decreased Ca2+ binding may also lead to faster Ca2+ dissociation kinetics in Tg-E22K intact fibers resulting in decreased duration and amplitude of [Ca2+] and force transients. These changes when placed in vivo would result in higher workloads and consequently cardiac hypertrophy.

The elevated blood pressure of humanGRK4γA142Vtransgenic mice is not associated with increased ROS production

G protein-coupled receptor (GPCR) kinases (GRKs) regulate the sensitivity of GPCRs, including dopamine receptors. The GRK4 locus is linked to, and some of its polymorphisms are associated with, human essential hypertension. Transgenic mice overexpressing human (h) GRK4gamma A142V on a mixed genetic background (C57BL/6J and SJL/J) have impaired renal D(1)-dopamine receptor (D(1)R) function and increased blood pressure. We now report that hGRK4gamma A142V transgenic mice, in C57BL/6J background, are hypertensive and have higher blood pressures than hGRK4gamma wild-type transgenic and nontransgenic mice. The hypertensive phenotype is stable because blood pressures in transgenic founders and F6 offspring are similarly increased. To determine whether the hypertension is associated with increased production of reactive oxygen species (ROS), we measured renal NADPH oxidase (Nox2 and Nox4) and heme oxygenase (HO-1 and HO-2) protein expressions and urinary excretion of 8-isoprostane and compared the effect of Tempol on blood pressure in hGRK4gamma A142V transgenic mice and D(5)R knockout (D(5)(-/-)) mice in which hypertension is mediated by increased ROS. The expressions of Nox isoforms and HO-2 and the urinary excretion of 8-isoprostane were similar in hGRK4gamma A142V transgenic mice and their controls. HO-1 expression was increased in hGRK4gamma A142V relative to hGRK4gamma wild-type transgenic mice. In contrast with the hypotensive effect of Tempol in D(5)(-/-) mice, it had no effect in hGRK4gamma A142V transgenic mice. We conclude that the elevated blood pressure of hGRK4gamma A142V transgenic mice is due mainly to the effect of hGRK4gamma A142V transgene acting via D(1)R and increased ROS production is not a contributor.

Humanized β-Thalassemia Mouse Model Containing the Common IVSI-110 Splicing Mutation

Splicing mutations are common causes of beta-thalassemia. Some splicing mutations permit normal splicing as well as aberrant splicing, which can give a reduced level of normal beta-globin synthesis causing mild disease (thalassemia intermedia). For other mutations, normal splicing is reduced to low levels, and patients are transfusion-dependent when homozygous for the disease. The development of therapies for beta-thalassemia will require suitable mouse models for preclinical studies. In this study, we report the generation of a humanized mouse model carrying the common IVSI-110 splicing mutation on a BAC including the human beta-globin ((hu)beta-globin) locus. We examined heterozygous murine beta-globin knock-out mice ((mu)beta(th-3/+)) carrying either the IVSI-110 or the normal (hu)beta-globin locus. Our results show a 90% decrease in (hu)beta-globin chain synthesis in the IVSI-110 mouse model compared with the mouse model carrying the normal (hu)beta-globin locus. This notable difference is attributed to aberrant splicing. The humanized IVSI-110 mouse model accurately recapitulates the splicing defect found in comparable beta-thalassemia patients. This mouse model is available as a platform for testing strategies for the restoration of normal splicing.