Gene-targeted Mutant Mice Research Articles

Abstract 134: Role Of Epigenetic Mechanisms Regulating Renal Pathology And Disorders In Global Npr1 Gene-targeted Mutant Mice In Sex-dependent Manner

The objective of this study was to investigate the effect of class I-specific histone deacetylase inhibitor, mocetinostat (MGCD0103; MGCD) in the regulation of epigenetic mechanisms and renal pathology and disorders in Npr1 (coding for natriuretic peptide receptor A; NPRA) gene-disrupted haplotype mice. Male and female Npr1 gene-disrupted (1-copy; +/- ), wild-type (2-copy; +/+ ) and gene-duplicated (3-copy; ++/+) mice were administrated with MGCD (2 mg/kg) by intraperitoneal injection at alternate days for 2-weeks. The renal NPRA protein levels and guanylyl cyclase (GC) activity were significantly lower in 1-copy mice and higher in 3-copy than in 2-copy mice; however, MGCD increased NPRA protein levels and GC activity. The vehicle-treated females showed lower SBP than male mice, whereas both sexes of 1-copy mice had higher SBPs, and 3-copy mice had lower SBPs as compared with 2-copy mice. Treatment with MGCD distinctly reduced SBP in male (1-copy, Δ-21±2.0 mmHg, p<0.001; 2-copy, Δ-5±0.6 mmHg, p<0.01; and 3-copy, Δ-4±0.5 mmHg, p<0.05) and female mice (1-copy, Δ-17±2.1 mmHg, p<0.01; 2-copy, Δ-3±0.5 mmHg, p<0.01; and 3-copy, Δ-3±0.8 mmHg). Hematoxylin and eosin staining showed pronounced renal morphological damages in 1-copy male than female mice as compared to 2-copy and 3-copy mice. One-copy male mice exhibited a greater degree of mesangial matrix expansion and interstitial nephritis as compared with 2-copy and 3-copy mice, which was significantly (p<0.1) decreased with MGCD treatment as compared with vehicle-treated mice. Creatinine clearance was significantly reduced in 1-copy male mice (52%; p<0.05) than female mice (35%; p<0.05) compared with 2-copy mice. The renal HDAC activity and HDAC1 and 2 protein levels in 1-copy male mice were significantly (p<0.05) higher than female mice as compared with 2-copy mice. MGCD markedly increased acetylation of histone marks (H3-K9, H3-K27, and H3-K18) and greatly reduced the renal pathologies as compared with controls. The present results demonstrate that MGCD upregulates NPRA activity and repairs renal pathology epigenetically in 1-copy haplotype mice. These findings will have important implications for treatment of hypertension and renal injury in humans in both sexes.

Class I HDAC Differentially Regulates Blood Pressure and Renal Injury in Npr1 Gene‐targeted Male and Female Mutant Mice

Atrial and brain natriuretic peptides (ANP, BNP) activate guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), which plays a critical role in the regulation of blood pressure (BP) and fluid volume homeostasis, and inhibits the cell proliferation and fibrosis. Mice lacking functional Npr1 gene (coding for GC‐A/NPRA) exhibit pathophysiology of hypertension, kidney disease, and heart failure; however, the mechanisms regulating Npr1 expression are not well understood. The objective of the present study was to gain insight into the effect of class I‐specific histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103) on Npr1 gene expression and regulation of BP and renal injury in gene‐targeted mutant mice. We utilized 16‐ to 20‐weeks old male and female Npr1 gene‐knockout haplotype (1‐copy; Npr1+/−), wild‐type (2‐copy; Npr1+/+), and gene‐duplicated heterozygous (3‐copy; Npr1++/+) mice. All mice genotypes were injected intraperitoneally with MGCD0103 (2 mg/kg) at an alternate day for 2‐weeks. The Western blot results showed that MGCD0103 significantly increased the renal NPRA protein levels in male and female mice compared with vehicle‐treated (olive oil) controls. After MGCD treatment, the renal GC‐A activity was significantly increased in both male and female mice. Male mice exhibited significantly higher systolic BP (SBP) than female mice and treatment with MGCD0103 decreased SBP in both 1‐copy male (106 ± 0.6 vs. vehicle‐treated, 129 ± 1.9; mmHg) and female (97 ± 2.2 vs. vehicle‐treated, 110 ± 2.1 mmHg, p < 0.001) mice. Renal HDAC activity was considerably higher (p < 0.05) in male mice than female mice; however, treatment with MGCD0103 attenuated (p < 0.05) HDAC activity by 40% in male and ~50% in female animals. Significantly higher urinary albumin to creatinine ratio was detected in male mice compared with female mice, which was attenuated by MGCD0103 treatment in male mice. The present results demonstrate that class I HDAC inhibitor, MGCD0103 upregulates NPRA expression in vivo, lowers SBP, and repairs renal injury differentially in male vs. female mice. These findings will have important implications for treatment of hypertension and renal injury in humans in a gender‐related manner.Support or Funding InformationThis work was supported by NIH grant HL062147.

Abstract P255: Differential Regulation of Blood Pressure and Renal Injury by HDAC Inhibitor Mocetinostat in Npr1 Gene-targeted Male and Female Mutant Mice

The objective of the present study was to elucidate the systolic blood pressure (SBP) lowering effect of class I histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103) by enhanced expression of natriuretic peptide receptor-A (NPRA) in gene ( Npr1 ). Adult male and female Npr1 heterozygous (1-copy; Npr1 +/- ), wild-type (2-copy; Npr1 +/+ ), and gene-duplicated (3-copy; Npr1 ++/+ ) mice were intraperitonealy injected with MGCD (2 mg/kg) for 2-weeks. The renal NPRA protein levels were significantly increased in the treated male (1-copy, 2.1-fold; 2-copy, 2.3-fold; and 3-copy, 1.5-fold; p < 0.05) and female (1-copy, 3.6-fold; 2-copy, 2.1-fold; and 3-copy, 1.7-fold; p < 0.05) mice compared with untreated animals. HDAC activity was significantly higher in male (1-copy, 24.4 ± 2.8; 2-copy, 15.9 ± 1.2; and 3-copy, 6.4 ± 0.7; ng/min/mg protein) than female (1-copy, 13.7 ± 1.4; 2-copy, 8.5 ± 0.9; and 3-copy, 4.7 ± 0.9, p < 0.05) mice; however, treatment with MGCD significantly attenuated HDAC activity in both male (~40%, p < 0.05) and female (~50%, p < 0.05) animals. Male mice exhibited significantly higher SBP than female mice and treatment with MGCD decreased SBP in both 1-copy male (106 ± 0.6 vs. control, 129 ± 1.9; mm Hg) and female (97 ± 2.2 vs. control, 110 ± 2.1, p < 0.001) mice. Significantly high urinary protein to creatinine ratio was detected in male compared with female mice which was attenuated by MGCD treatment in male mice (1-copy, 3.2 ± 0.4 vs. untreated 5.5 ± 04; 2-copy 2.5 ± 0.4 vs. untreated 4.6 ± 0.2; and 3-copy, 1.6 ± 0.3 vs. untreated 3.1 ± 0.4; p < 0.05). Lower creatinine clearance (μl/min) was observed in both male (1-copy, 51 ± 11 vs. 2-copy, 130 ± 14.0; p < 0.05) and female (1-copy, 55 ± 2.7 vs. 2-copy, 129 ± 6.4; p < 0.01) mice. However, treatment with MGCD increased creatinine clearance in male (105 ± 13; p < 0.05) and female (97 ± 8.4; p < 0.05) vs. control mice. The present results demonstrate that class I HDAC inhibitor, MGCD upregulates NPRA expression in vivo , lowers SBP, and repairs renal injury differentially in male vs. female mice. These findings will have important implications for treatment of hypertension and renal injury in humans in a sex-related manner.

Soluble Fas Ligand Is Essential for Blister Formation in Pemphigus.

Pemphigus is a blistering disease characterized by pemphigus autoantibodies (PVIgG) directed mostly against desmogleins (Dsgs), resulting in the loss of keratinocyte adhesion (acantholysis). Yet, the mechanisms underlying blister formation remain to be clarified. We have shown previously that anti-Fas ligand (FasL) antibody (Ab) prevents PVIgG-induced caspase-8 activation and Dsg cleavage in human keratinocytes, and that sera from pemphigus patients contain abnormally increased levels of FasL. Here, we demonstrate that recombinant FasL induces the activation of caspases prior to Dsg degradation, and anti-FasL Ab prevents acantholysis in cultured keratinocytes. Moreover, the silencing of FasL reduces PVIgG-induced caspase-8 activation and Dsg3 cleavage. Following injection of PVIgG into mice, FasL is upregulated at 1–3 h and is followed by caspase-8-mediated keratinocyte apoptosis, before blister formation. The administration of anti-FasL Ab after PVIgG injection blocks blister formation in mice. Furthermore, we injected PVIgG into two different gene-targeted mutant mice that selectively lack either secreted soluble FasL (sFasL), FasLΔs/Δs mice, or the membrane-bound form of FasL (mFasL), FasLΔm/Δm mice. After PVIgG treatment, blisters are only visible in FasLΔm/Δm animals, lacking mFasL, but still producing sFasL, similar to wild-type (C57BL/6) animals. By contrast, a significant decrease in the relative acantholytic area is observed in the FasLΔs/Δs animals. These results demonstrate that soluble FasL plays a crucial role in the mechanisms of blister formation, and blockade of FasL could be an effective therapeutic approach for pemphigus.

Death Domain Signaling by Disulfide-Linked Dimers of the p75 Neurotrophin Receptor Mediates Neuronal Death in the CNS

The p75 neurotrophin receptor (p75(NTR)) mediates neuronal death in response to neural insults by activating a caspase apoptotic pathway. The oligomeric state and activation mechanism that enable p75(NTR) to mediate these effects have recently been called into question. Here, we have investigated mutant mice lacking the p75(NTR) death domain (DD) or a highly conserved transmembrane (TM) cysteine residue (Cys(259)) implicated in receptor dimerization and activation. Neuronal death induced by proneurotrophins or epileptic seizures was assessed and compared with responses in p75(NTR) knock-out mice and wild-type animals. Proneurotrophins induced apoptosis of cultured hippocampal and cortical neurons from wild-type mice, but mutant neurons lacking p75(NTR), only the p75(NTR) DD, or just Cys(259) were all equally resistant to proneurotrophin-induced neuronal death. Homo-FRET anisotropy experiments demonstrated that both NGF and proNGF induce conformational changes in p75(NTR) that are dependent on the TM cysteine. In vivo, neuronal death induced by pilocarpine-mediated seizures was significantly reduced in the hippocampus and somatosensory, piriform, and entorhinal cortices of all three strains of p75(NTR) mutant mice. Interestingly, the levels of protection observed in mice lacking the DD or only Cys(259) were identical to those of p75(NTR) knock-out mice even though the Cys(259) mutant differed from the wild-type receptor in only one amino acid residue. We conclude that, both in vitro and in vivo, neuronal death induced by p75(NTR) requires the DD and TM Cys(259), supporting the physiological relevance of DD signaling by disulfide-linked dimers of p75(NTR) in the CNS. A detailed understanding of the physiological significance of distinct structural determinants in the p75 neurotrophin receptor (p75(NTR)) is crucial for the identification of suitable drug targets in this receptor. We have tested the relevance of the p75(NTR) death domain (DD) and the highly conserved transmembrane residue Cys(259) for the ability of p75(NTR) to induce apoptosis in neurons of the CNS using gene-targeted mutant mice. The physiological importance of these determinants had been contested in some recent in vitro studies. Our results indicate a requirement for DD signaling by disulfide-linked dimers of p75(NTR) for neuronal death induced by proneurotrophins and epileptic seizures. These new mouse models will be useful for clarifying different aspects of p75(NTR) physiology.

Abstract 453: Ablation of TRPV1 Impairs Exendin-4 Induced Suppression in Renal Cytokine Release

Impairment of function and expression of the transient receptor potential vanilloid type 1 (TRPV1) occurs in obesity and diabetes, which may aggravate inflammation and tissue injury given that the TRPV1 mediated pathway has been shown to be anti-inflammatory. Glucagon-like peptide-1 (GLP-1), an incretin hormone released in response to food intake, is capable of protecting tissues from injury, but its mechanism largely unknown. We tested the hypothesis that exendin-4 (Ex4), a GLP-1 receptor agonist, suppresses hypoxia-induced cytokine release via a TRPV1 mediated pathway. Using gene-targeted TRPV1-null mutant (TRPV1 -/- ) or wild-type (WT, C57BL/6) mice, plasma creatinine or urine lactate dehydrogenase (LDH) levels were measured 40- min after ischemia plus 3-hr reperfusion (I/R). Renal I/R increased plasma creatinine and urine LDH levels with a greater magnitude in TRPV1-/- than WT mice (creatinine, uM: WT: 99.4 ± 4.4 to TRPV1: 129.7 ± 11.9, p < 0.05; LDH, mU: WT: 132.3± 39.8 to TRPV1-/-: 178.5 ± 44.4, p < 0.05). In vitro, cytokine or chemokine secretion from renal tissues incubated either with 95% O 2 -5% CO 2 or 99% nitrogen (hypoxia) was measured. TNFα, IL-6, or MCP-1 release in response to nitrogen (1 hr) was increased in both strains with a bigger magnitude in TRPV1-/- than WT mice (p<0.05). Blockade of receptors for calcitonin gene-related peptide (CGRP) with CGRP8-38 (10 -7 M) further increased hypoxia-induced MCP-1 release in WT mice (p<0.05). Ex4 suppressed nitrogen (0.5hr)-induced cytokine/chemokine release from the kidneys in WT but not TRPV1-/-mice (WT: IL-6: 0.74 ± 0.08 to 0.66 ± 0.03; TNF-a: 0.73 ± 0.07 to 0.66 ± 0.03; MCP-1: 16.1± 3.5 to 12.7 ± 1.3 pg/g/min, p < 0.05. TRPV1-/-: p>0.05 for all). Moreover, Ex4 increased CGRP release from the renal pelvis in WT ( p < 0.05) but not TRPV1 -/- mice, which can be inhibited by Ex9-39, an Ex4 antagonist. Thus, ablation of TRPV1 or blockade of CGRP receptors enhances hypoxia-induced inflammation and renal injury following I/R. Ex4 suppresses hypoxia-induced cytokine/chemokine release and enhances CGRP release from the kidney only when TRPV1 function and expression are intact, suggesting that TRPV1/CGRP may play a key role mediating Ex4-induced anti-inflammatory effects.

Abstract 462: Gene-targeting of Npr1 Reveals the Retinoic Acid-mediated Enhanced Expression of Guanylyl Cyclase-A/Natriuretic Peptide Receptor-A and Attenuation of Renal Fibrosis in Mutant Mice

Mice lacking functional Npr1 gene (coding for guanylyl cyclase/natriuretic peptide receptor-A, GC-A/NPRA) exhibit hypertension and kidney disease, however, the mechanisms regulating Npr1 expression are not well understood. The objective of the present study was to determine the effect of all- trans retinoic acid (ATRA) on Npr1 gene expression and reduction of renal fibrosis in gene-targeted mutant mice. Adult (24-week old) male Npr1 gene-disrupted heterozygous (1-copy; +/-), wild-type (2-copy; +/+), and gene-duplicated (3-copy; ++/+) mice were treated by injecting ATRA (0.5 mg/Kg/day) intraperitoneally for 2-weeks. After treatment with ATRA, the renal Npr1 mRNA levels was increased in 1-copy mice by 4.6-fold, (1.4 ± 0.05 vs. control, 0.3 ± 0.02; p < 0.05), 2-copy mice by 3-fold, (3.7 ± 0.2 vs. control 1.2 ± 0.1; p < 0.05), and 3-copy mice by 2-fold (3.2 ± 0.3 vs. control 1.6 ± 0.2; p < 0.05). The retinoic acid increased renal cGMP levels (pmol/mg protein) in 1-copy (28.9 ± 1.1 vs. control, 7.1 ± 0.4; p < 0.05), 2-copy (55.8 ± 1.9 vs. control 22.6 ± 1.8; p < 0.05), and 3-copy (85.8 ± 4.3 vs. control 47.8 ± 3.2; p < 0.05) mice. A significant decrease in systolic blood pressure (SBP) was observed in ATRA-treated 1-copy mice (114.6 ± 2.2, p < 0.01) vs. 1-copy control mice (128.2 ± 2.1). Higher tubulo-interstitial fibrosis present in 1-copy mice was attenuated after treatment with ATRA (35%, p < 0.05). Immunohistochemistry and Western blot analyses exhibited the reduction in renal expression of α-SMA (25%, p < 0.05 and 35%, p < 0.05, respectively) in ATRA-treated 1-copy mice compared with 1-copy control mice. Interestingly, ATRA significantly enhanced the levels of immunosuppressive cytokine IL-10 (pg/mg protein) in the kidneys of 1-copy (43.3 ± 3.1 vs. control 23.4 ± 1.8; p < 0.05), 2-copy (67.1 ± 5.5 vs. control 56.5 ± 3.7; p < 0.05), and 3-copy (102.7 ± 6.7 vs. control 81.4 ± 3.2; p < 0.05) mice. The results show that ATRA induces Npr1 gene expression and function, lowers SBP, and attenuates renal fibrosis in 1-copy Npr1 mice. The findings of this study indicate that ATRA can serve as a possible therapeutic agent in attenuation of renal fibrosis under reduced Npr1 gene copy number, which will have important implications in prevention of hypertension and renal remodeling process.

Abstract 535: Impaired Natriuresis and Increased Salt Sensitivity in Response to Acute Salt Load During Western Diet-intake: Role of Trpv1

Western diet (WD) intake increases morbidity of obesity, diabetes, and salt sensitive hypertension albeit mechanisms are largely unknown. We tested the hypothesis that the transient receptor potential vanilloid type 1 (TRPV1) channels are activated during WD intake to protect against salt-induced increases in blood pressure possibly via enhancing natriuresis. Wild-type (WT, C57BL/6) or gene-targeted TRPV1-null mutant (TRPV1 -/- ) mice were fed a normal (CON) or Western (WD) diet for 16-18 weeks. Mean arterial pressure (MAP), determined by radiotelemetry after acute sodium loading (NS, 0.9% NaCl) or vehicle (C) by gavage with or without treatment with Nω-nitro-l-arginine methyl ester ( L - NAME, 10mg/kg, ip. ) or a TRPV1 agonist, N-oleoyldopamine ( OLDA, 0.1mg/kg, ip ), was not different between two strains fed a CON diet. WT or TRPV1-/- mice fed a WD diet had increased MAP after NS, with a greater magnitude in TRPV1-/- mice (p<0.05). OLDA decreased while L-NAME increased MAP in WT-WD-NS but not TRPV1-/-WD-NS mice (p<0.05). The urinary nitrates plus nitrites excretion (UNOx, umol*4h urine/kg BW), an indicator of renal nitric oxide (NO) production, was increased in both strains fed a CON diet after NS (p<0.05). OLDA further increased while L-NAME prevented NS-induced increases in UNOx in WT-CON-NS only or in both strains, respectively. TRPV1 ablation with WD intake decreased UNOx levels before NS and abolished NS-induced increment in UNOx (WT-WD-C: 0.3 ±0.06 vs. TRPV1-/-WD-C: 0.2 ±0.05; and WT-WD-NS: 0.7 ±0.1 vs. TRPV1-/-WD-NS: 0.3 ±0.1; p<0.05). OLDA further increased while L-NAME prevented NS-induced increment in UNOx in WT-WD-NS mice only (p<0.05). Urinary sodium excretion was increased in both strains fed a CON diet after NS (p<0.05), which was unaffected by OLDA or L-NAME. UNa was increased in WT-WD-NS but not TRPV1-/-WD-NS after NS. OLDA further increased while L-NAME prevented NS-induced increases in UNa in WT-WD-NS only. Thus, TRPV1 ablation increases salt sensitivity during WD intake possibly via impaired NS-induced renal NO production and sodium excretion. Activation of TRPV1 with OLDA enhances renal NO production and sodium excretion, resulting in prevention of increased salt sensitivity during WD intake.

Abstract 94: High Sucrose Intake-induced Endoplasmic Reticulum Stress, Inflammation and Fibrosis in hte Pancreas: Role of Trpv1

Excessive consumption of sugar may serve as a primary and independent factor for increased morbidity of diabetes. We test the hypothesis that the transient receptor potential vanilloid type 1 (TRPV1) channels play a protective role against sugar-induced pancreatic endoplasmic reticulum (ER) stress, inflammatory cell infiltration, and tissue fibrosis. Wild-type (WT, C57BL/6) or gene-targeted TRPV1-null mutant (TRPV1 -/- ) mice were fed a normal (CON) or high sucrose diet for 22-23 weeks. TRPV1 ablation impaired glucose tolerance and suppressed glucose-induced insulin release when fed a CON diet (p<0.05). Sucrose intake further impaired glucose tolerance and insulin release in TRPV1 -/- compared to WT mice (p<0.05). Sucrose intake increased pancreatic ER stress, showing elevated levels of chop protein content in both strains and GRP78, XBP-1, and CREB-2 in TRPV1 -/- mice only (p<0.05). TRPV1 ablation increased levels of phosphorylated JNK (WT, 0.12±0.14; TRPV1-/-, 0.31±0.13) and decreased glut2 contents (WT, 1.38±0.24; TRPV1-/-, 0.83±0.09) in the pancreas when fed a CON diet (p<0.05). Sucrose intake further increased levels of pancreatic phosphorylated JNK (WT, 0.89±0.19; TRPV1-/-, 1.05±0.19) and decreased glut2 contents (WT, 0.39±0.04; TRPV1-/-, 0.26±0.16) in both strains (p<0.05). Sucrose intake increased pancreatic TGF-β levels in both strains with exacerbation in TRPV1-/- mice (p<0.05), and increased pancreatic phosphorylated Smad2/3 and collagen levels in TRPV1 -/- mice only (p<0.05). Quantitative immunostaining revealed that sucrose intake enhanced pancreatic infiltration of CD3 positive cells in both strains and macrophage in TRPV1 -/- mice only (p<0.05). Thus, TRPV1 ablation impairs insulin release and glucose tolerance at baseline possibly via increased pancreatic phosphorylated JNK signaling and decreased glut2 expression. Sucrose intake aggravates impairment in insulin release and glucose tolerance when TRPV1 is ablation, possibly via enhancing ER stress, macrophage infiltration, and activation of the TGF-β/Smad pathway leading to pancreatic fibrosis, suggesting preserving TRPV1 expression and function may attenuate pancreatic injury via alleviating inflammatory responses.

Genetic control of murine invariant natural killer T-cell development dynamically differs dependent on the examined tissue type

Previous studies using gene-targeted mutant mice revealed several molecules important for the development or function of invariant natural killer T (iNKT) cells. However, these gene knockout mice represent cases that are rare in humans. Thus, it remains unclear how naturally occurring allelic variants of these genes or others regulate the numerical and functional diversity of iNKT cells in both mice and humans. Studies in humans are mostly limited to iNKT cells in peripheral blood (PB). It is not known if the relative distribution of iNKT cells between PB and other lymphoid organs is correlated or under common genetic control. To initially address these questions, we analyzed iNKT cells in the spleen, thymus and PB of 38 inbred mouse strains. Percentages of iNKT cells in these three anatomical sites varied significantly in a strain-dependent manner. The correlation between PB and spleen was moderate, and none was observed between PB and thymus. Similarly, proportions of the CD4-expressing subset of iNKT cells differed significantly among inbred strains. The percentages of CD4-positive iNKT cells displayed a strong correlation between PB and spleen, although it remained poor between PB and thymus. Genome-wide association studies across strains identified only partially overlapping loci associated with variability of iNKT cell frequencies within and between differing anatomical sites.

Mitotic chromosome condensation mediated by the retinoblastoma protein is tumor-suppressive

Condensation and segregation of mitotic chromosomes is a critical process for cellular propagation, and, in mammals, mitotic errors can contribute to the pathogenesis of cancer. In this report, we demonstrate that the retinoblastoma protein (pRB), a well-known regulator of progression through the G1 phase of the cell cycle, plays a critical role in mitotic chromosome condensation that is independent of G1-to-S-phase regulation. Using gene targeted mutant mice, we studied this aspect of pRB function in isolation, and demonstrate that it is an essential part of pRB-mediated tumor suppression. Cancer-prone Trp53(-/-) mice succumb to more aggressive forms of cancer when pRB's ability to condense chromosomes is compromised. Furthermore, we demonstrate that defective mitotic chromosome structure caused by mutant pRB accelerates loss of heterozygosity, leading to earlier tumor formation in Trp53(+/-) mice. These data reveal a new mechanism of tumor suppression, facilitated by pRB, in which genome stability is maintained by proper condensation of mitotic chromosomes.

Abstract 5416: Ablation of TRPV1 Exacerbates Pressure Overload-Induced Cardiac Hypertrophy

Background We have recently shown that the transient receptor potential vanilloid type 1 (TRPV1), a ligand-gated cation channel expressed in sensory nerves innervating the heart, plays an important role in protecting the heart from ischemia/reperfusion injury. However, the role of TRPV1 in pressure overload-induced hypertrophy of the heart is unknown. Objective - This study tests the hypothesis that TRPV1 protects the heart from pressure overload-induced hypertrophy and functional deterioration using gene-targeted TRPV1-null mutant (TRPV1 −/− ) or wild-type (WT) mice. Methods and Results Four weeks after transverse aortic constriction ( TAC ) in TRPV1 −/− and WT mice, echo cardiography demonstrated that cardiac fractional shortening (FS) and ejection fraction (EF) were suppressed in a greater extent in TRPV1 −/− than WT mice (FS: 14 ± 3% in TRPV1 −/− vs. 18 ± 2% in WT mice, P <0.05; EF: 34 ± 6% in TRPV1 −/− vs. 43 ± 3% mice, P <0.05), while there was no difference in FS and EF in TRPV1 −/− and WT sham-operated mice. The ratio of heart weight to body weight (mg/g) was greater in TRPV1 −/− than in WT mice (7.39±1.34 in TRPV1 −/− vs. 6.26±1.07 in WT mice, P <0.05), and the increase in heart weigh was accompanied by an increase in cardiomyocyte cross-sectional areas in TRPV1 −/− compared to WT mice (248±8.2 in TRPV1 −/− vs 224±8.7 in WT μm 2 , P <0.05). Immunohistochemistry study showed enhanced cellular infiltration and fibrosis in TRPV1 −/− compared to WT mice. Consistently, hydroxyproline assay showed that the collagen level was higher in TRPV1 −/− than WT mice (1.7±0.3 in TRPV1 −/− vs 1.3±0.4 μg/mg dry tissue in WT, P <0.05), and ELISA assay showed that interleukin-6 levels were higher in TRPV1 −/− than in WT mice (14.2±4.6 in TRPV1 −/− vs. 10.6±2.6 pg/mg protein in WT, P <0.05). Conclusions These data show that ablation of TRPV1 results in exaggerated cardiac hypertrophy, fibrosis, and dysfunction induced by pressure overload, which may result from enhanced inflammatory responses in the absence of TRPV1.

T 세포 발생 연구를 위한 유전자 적중 마우스

T 세포의 발생은 흉선에서 이루어지는데, 골수나 태아의 간으로부터 유래한 전구세포가 여러 단계의 복잡한 발달단계를 거쳐서 단일 CD4나 CD8 분자를 세포 표면에 발현하는 성숙한 T세포로 분화하여 2차 림프기관으로 이동한다. T세포 수용체로부터 유래하는 신호와 하위 신호전달 경로는 성숙한 T 세포 및 흉선 T 세포의 발생과 분화에 중요하다. 유전자 적중 마우스를 이용한 연구들을 통해서 복잡한 T 세포 발생을 조절하는 중요한 신호전달 경로를 파악하여 메카니즘에 대한 이해가 깊게 되었다. 유전자의 발현 시기와 조직이나 세포를 고려한 신호전달을 이해하기 위해 보다 정교한 유전자 조작로 기법의 개발이 중요하다고 사료된다. T cells develop in thymus, where immature progenitors undergo multi-stage developmental processes until mature CD4+ and CD8+ single positive T cells are generated and egress out to the periphery. Signals from T cell receptor are pivotal for T cell development in thymus as well as in periphery. Gene targeted mutant mice have contributed the understanding underlying mechanisms by dissecting the signaling pathways which governing these complex processes. Development of refined mutational system will be imperative to analyze the signaling pathways by taking consideration of context of 'timing and/or space' of gene function.

Global growth deficiencies in mice lacking the ribosomal protein HIP/RPL29

Because of their deleterious effects on developing organisms, ribosomal protein (RP) mutations have been poorly described in mammals, and only a few heterozygous mutations have been shown to be viable. This observation is believed to be due to the fact that each RP is an essential component in the assembly of a functional stable ribosome. Here, we created gene targeted mutant mice lacking HIP/RPL29, an RP associated with translationally active ribosomes in eukaryotes. In contrast to other RP mutants, HIP/RPL29 null mice are viable but are up to 50% smaller than their control littermates at weaning age. In null embryos, delayed global growth is first observed around mid-gestation, and postnatal lethality due to low birth weight results in distortion of the Mendelian ratio. Prenatal growth defects are not fully compensated for during adulthood, and null animals display proportionately smaller organs and stature, and reach sexual maturity considerably later when compared with their control siblings. Additionally, HIP/RPL29 null embryonic fibroblasts have decreased rates of proliferation and protein synthesis and exhibit reduced steady state levels of core RPs. Altogether, our findings provide conclusive genetic evidence that HIP/RPL29 functions as an important regulator of global growth by modulating the rate of protein synthesis.

Efficient method to generate single‐copy transgenic mice by site‐specific integration in embryonic stem cells

Transgenic and gene-targeted mutant mice provide powerful tools for analysis of the cellular processes involved in early development and in the pathogenesis of many diseases. Here we describe a transgene integration strategy mediated by site-specific recombination that allows establishment of multiple embryonic stem (ES) cell lines carrying tetracycline-inducible genes targeted to a specific locus to assure predictable temporal and spatial expression in ES cells and mice. Using homologous recombination we inserted an frt homing site into which tetracycline-inducible transgenes can be integrated efficiently in the presence of FLPe recombinase. This strategy and the vectors described here are generally applicable to any locus in ES cells and should allow for the rapid production of mice with transgenes efficiently targeted to a defined site.

Phosphorylation of the Stat1 Transactivation Domain Is Required for Full-Fledged IFN-γ-Dependent Innate Immunity

Stat1 is phosphorylated on serine 727 within its transactivating domain (TAD) in response to interferons or other immunological signals. We generated gene-targeted mutant mice expressing a serine727-alanine mutant of Stat1. These animals showed increased mortality upon infection with Listeria monocytogenes and impaired clearance of the bacteria from spleen and liver. The Stat1S727A mice were more resistant to the LPS-induced septic shock syndrome, suggesting that Stat1 serine phosphorylation promotes inflammatory responses. Expression of IFN-γ-induced genes was strongly reduced in macrophages expressing Stat1 S727A. While mutation of Stat1 at S727 did not reduce its binding to chromatin, association with the coactivator CBP and histone acetylation at the interferon-responsive GBP promoter was strongly reduced, suggesting defective recruitment of histone acetylases as the mechanism underlying IFN-γ hyporesponsiveness. Our data demonstrate that the increase in transcription factor activity caused by Stat serine phosphorylation contributes to macrophage activation and to IFN-γ-dependent immune responses in vivo.

Intracellular Domains of NMDA Receptor Subtypes Are Determinants for Long-Term Potentiation Induction

NMDA receptors (NMDARs) are essential for modulating synaptic strength at central synapses. At hippocampal CA3-to-CA1 synapses of adult mice, different NMDAR subtypes with distinct functionality assemble from NR1 with NR2A and/or NR2B subunits. Here we investigated the role of these NMDA receptor subtypes in long-term potentiation (LTP) induction. Because of the higher NR2B contribution in the young hippocampus, LTP of extracellular field potentials could be enhanced by repeated tetanic stimulation in young but not in adult mice. Similarly, NR2B-specific antagonists reduced LTP in young but only marginally in adult wild-type mice, further demonstrating that in mature CA3-to-CA1 connections LTP induction results primarily from NR2A-type signaling. This finding is also supported by gene-targeted mutant mice expressing C-terminally truncated NR2A subunits, which participate in synaptic NMDAR channel formation and Ca2+ signaling, as indicated by immunopurified synaptic receptors, postembedding immunogold labeling, and spinous Ca2+ transients in the presence of NR2B blockers. These blockers abolished LTP in the mutant at all ages, revealing that, without the intracellular C-terminal domain, NR2A-type receptors are deficient in LTP signaling. Without NR2B blockade, CA3-to-CA1 LTP was more strongly reduced in adult than young mutant mice but could be restored to wild-type levels by repeated tetanic stimulation. Thus, besides NMDA receptor-mediated Ca2+ influx, subtype-specific signaling is critical for LTP induction, with the intracellular C-terminal domain of the NR2 subunits directing signaling pathways with an age-dependent preference.

Endothelial cell activation in inflammation: lessons from mutant mouse models

Dysregulation of the inflammatory response is a causative or contributing factor in many cardiovascular pathologies. Molecular mechanisms involved in these processes indicate that the vascular endothelium is an important facet in the regulation of inflammation. Gene targeted mutational studies in the mouse have shown that adhesion and signaling molecule expression within the endothelium participate in the pathogenic process of cardiovascular disease. These studies provide insight into genetic pathways that may be therapeutically relevant in both treatment and diagnostic regimens. However, we have also learned that the role of these pathways in endothelium during inflammatory diseases is complex, requiring further study to better understand specific mechanisms involved in endothelial cell dysfunction during cardiovascular disease. Alternative gene targeting techniques, such as the Cre–loxP system, are beginning to allow tissue specific investigation of genetic pathways within the endothelium; however, extensive use of this technology is limited. This review discusses the role of the endothelium during inflammation and the insights that have been gained from the use of gene targeted mutant mice.

Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D.

The vitamin D hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the biologically active form of vitamin D, is essential for an intact mineral metabolism. Using gene targeting, we sought to generate vitamin D receptor (VDR) null mutant mice carrying the reporter gene lacZ driven by the endogenous VDR promoter. Here we show that our gene-targeted mutant mice express a VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. Expression of the lacZ reporter gene was widely distributed during embryogenesis and postnatally. Strong lacZ expression was found in bones, cartilage, intestine, kidney, skin, brain, heart, and parathyroid glands. Homozygous mice are a phenocopy of mice totally lacking the VDR protein and showed growth retardation, rickets, secondary hyperparathyroidism, and alopecia. Feeding of a diet high in calcium, phosphorus, and lactose normalized blood calcium and serum PTH levels, but revealed a profound renal calcium leak in normocalcemic homozygous mutants. When mice were treated with pharmacological doses of vitamin D metabolites, responses in skin, bone, intestine, parathyroid glands, and kidney were absent in homozygous mice, indicating that the mutant receptor is nonfunctioning and that vitamin D signaling pathways other than those mediated through the classical nuclear receptor are of minor physiological importance. Furthermore, rapid, nongenomic responses to 1,25-(OH)(2)D(3) in osteoblasts were abrogated in homozygous mice, supporting the conclusion that the classical VDR mediates the nongenomic actions of 1,25-(OH)(2)D(3).

Rethinking the role of tumour necrosis factor-alpha in ultraviolet (UV) B-induced immunosuppression: altered immune response in UV-irradiated TNFR1R2 gene-targeted mutant mice.

Ultraviolet (UV) B-induced immunosuppression, implicated in the pathogenesis of skin cancers, is postulated to be mediated in part by cis-urocanic acid (cis-UCA) via tumour necrosis factor (TNF)-alpha. TNF-alpha produces morphological changes in Langerhans cells indistinguishable from those induced by UVB exposure and antibodies against TNF-alpha have been demonstrated to inhibit UVB-induced immunosuppression in vivo. To clarify further the role of TNF-alpha in UVB-induced immunosuppression and in cis-UCA immunosuppression. We performed a contact hypersensitivity (CHS) assay on gene-targeted mutant mice (TNFR1R2-/-) lacking genes for both receptors (p55 and p75) for TNF-alpha. Mice were either irradiated with UVB or injected intradermally with cis-UCA, sensitized with 2,4-dinitrofluorobenzene, challenged on the ears and the response was measured. The TNFR1R2-/- mice showed hyporesponsiveness in the CHS response compared with wild-type (P < 0.001), confirming the proinflammatory role of TNF-alpha. However, significant suppression of CHS was seen after irradiation and after cis-UCA injection in both locally (sensitization on irradiated site; P < 0.05) and systemically (sensitization on non-irradiated site; P < 0.05) sensitized wild-type and gene-targeted mice. These results demonstrate that TNF-alpha signalling is only partially involved in UVB-induced immunosuppression and does not play a major part in the cis-UCA immunosuppression mechanism.