Mutant Mouse Regional Resource Center Research Articles

Estrogen-related receptor γ (errγ) is a catabolic regulator of osteoarthritis pathogenesis

Purpose: The estrogen-related receptor (ERR) family of orphan nuclear receptor is composed of ERRα, ERRβ and ERRγ, which are known to regulate various isoform-specific functions under normal and pathophysiological conditions. However, the functions of ERRs in regulating joint cartilage homeostasis and/or OA pathogenesis have not yet been studied. Here, we investigate the involvement of ERRs in the pathogenesis of osteoarthritis (OA) in mice. Methods: Human OA cartilage was sourced from individuals undergoing arthroplasty. C57BL/6J mice were used for the experimental OA studies. Esrrg+/- mice (B6.129P2-Esrrgtm1Dgen/Mmnc) were purchased from the Mutant Mouse Regional Resource Center (MMRRC). Col2a1-Esrrg Tg mice were generated using the Col2a1 promoter and enhancer. Human OA cartilage was frozen, sectioned at a thickness of 5 μm, and fixed in paraformaldehyde. Sulfate proteoglycans were detected by alcian blue staining, and cartilage destruction in mice was examined using safranin-O staining. Femoral condyles and tibial plateaus were obtained from mice, and chondrocytes were isolated from cartilage tissue by digestion with 0.2% collagenase. Results: Among the ERR family members, ERRγ was specifically upregulated in cartilage from human OA patients and various mouse models of OA. Gain-of-function (adenovirus-mediated overexpression in joint tissues or Col2a1-Esrrg Tg mice) and loss-of-function (Esrrg+/- mice or shRNA-mediated knockdown) approaches clearly indicated that ERRγ acts as a novel catabolic regulator of OA pathogenesis, at least in part, by the upregulating matrix-degrading enzymes, MMP3 and MMP13, in articular chondrocytes. Conclusions: Our results are the first to demonstrate that ERRγ acts as a catabolic regulator of cartilage degeneration and OA pathogenesis, and collectively support the idea that ERRγ could be a therapeutic target for OA

Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction

Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD(-/-) mice. LCAD(-/-) mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD(-/-) mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD(-/-) surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD(-/-) lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.

Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth

Olfm1, a secreted highly conserved glycoprotein, is detected in peripheral and central nervous tissues and participates in neural progenitor maintenance, cell death in brain, and optic nerve arborization. In this study, we identified Olfm1 as a molecule promoting axon growth through interaction with the Nogo A receptor (NgR1) complex. Olfm1 is coexpressed with NgR1 in dorsal root ganglia and retinal ganglion cells in embryonic and postnatal mice. Olfm1 specifically binds to NgR1, as judged by alkaline phosphatase assay and coimmunoprecipitation. The addition of Olfm1 inhibited the growth cone collapse of dorsal root ganglia neurons induced by myelin-associated inhibitors, indicating that Olfm1 attenuates the NgR1 receptor functions. Olfm1 caused the inhibition of NgR1 signaling by interfering with interaction between NgR1 and its coreceptors p75NTR or LINGO-1. In zebrafish, inhibition of optic nerve extension by olfm1 morpholino oligonucleotides was partially rescued by dominant negative ngr1 or lingo-1. These data introduce Olfm1 as a novel NgR1 ligand that may modulate the functions of the NgR1 complex in axonal growth.

Effect of vitamin C deficiency during postnatal development on adult behavior: functional phenotype of Gulo(–/–) knockout mice

On page 270, we erroneously listed The Jackson Laboratory as the source of the Gulo(–/–) knockout mouse line, on a C57BL/6J background, created by Dr. Maeda. We regret this oversight. The correct source was the Mutant Mouse Regional Resource Center at the University of California Davis (Davis, CA, USA) which is the holder and distributor of this mouse line. The official name of the line is B6.129P2-Gulotm1Mae/Mmucd.

Synergistic Roles for G-protein γ3 and γ7 Subtypes in Seizure Susceptibility as Revealed in Double Knock-out Mice

The functions of different G-protein αβγ subunit combinations are traditionally ascribed to their various α components. However, the discovery of similarly diverse γ subtypes raises the possibility that they may also contribute to specificity. To test this possibility, we used a gene targeting approach to determine whether the closely related γ(3) and γ(7) subunits can perform functionally interchangeable roles in mice. In contrast to single knock-out mice that show normal survival, Gng3(-/-)Gng7(-/-) double knock-out mice display a progressive seizure disorder that dramatically reduces their median life span to only 75 days. Biochemical analyses reveal that the severe phenotype is not due to redundant roles for the two γ subunits in the same signaling pathway but rather is attributed to their unique actions in different signaling pathways. The results suggest that the γ(3) subunit is a component of a G(i/o) protein that is required for γ-aminobutyric acid, type B, receptor-regulated neuronal excitability, whereas the γ(7) subunit is a component of a G(olf) protein that is responsible for A(2A) adenosine or D(1) dopamine receptor-induced neuro-protective response. The development of this mouse model offers a novel experimental framework for exploring how signaling pathways integrate to produce normal brain function and how their combined dysfunction leads to spontaneous seizures and premature death. The results underscore the critical role of the γ subunit in this process.

Mastl Kinase Gene Disruption Causes Embryonic Lethality.

AbstractAbstract 1567Microtubule-associated serine/threonine like kinase (MASTL kinase) was originally identified by genetic linkage as the probable mutant gene responsible for the platelet phenotype in a family with autosomal-dominant thrombocytopenia. MASTL kinase is the mammalian ortholog of Drosophila Greatwall kinase, which is required for cell cycle regulation. Affected members of the thrombocytopenic family carried a mutant MASTL kinase allele with a single nucleotide change that converted a glutamic acid codon to one specifying aspartic acid at amino acid 167 of the protein; no unaffected members of the family carried the mutant allele. Affected family members had a 60% reduction in the numbers of circulating platelets and megakaryocytes that were less mature and less polyploid than normal megakaryocytes. We have sought to determine how MASTL kinase affects platelet production and circulation by generating a strain of mice deficient in MASTL kinase. For this, we obtained embryonic stem (ES) cells containing a gene trap mutation within the murine Mastl gene from the Mutant Mouse Regional Resource Center of the National Center for Research Resources (MMRRC, University of California, San Diego). The mutation was generated by an insertional disruption, and the resulting mutant allele carries a beta–galactosidase/neomycin fusion expression cassette within the fourth intron of the Mastl kinase gene. Chimeric animals were bred and their offspring were genotyped by Southern blotting, confirming germ-line transmission. F1 heterozygote offspring were produced and appear developmentally normal and healthy, with platelet counts indistinguishable from those of their wild-type littermates. We have yet to observe a single weaned animal that is homozygous for the mutant allele in the F1 mice, indicating that MASTL kinase deficiency results in embryonic lethality. We observed decidual swellings at 13.5 days post coitus (dpc), which contained no traces of either embryos or yolk sac membranes. While this has made it impossible to genotype these malformed embryos, it does indicate that the embryos are viable to 3.5–5.5 dpc and are able to implant within the uterine horn. This unexpected phenotype demonstrates a significant and previously unknown role for Mastl kinase in embryonic development. We hypothesize that Mastl kinase may be involved in regulating cell differentiation during embryogenesis. In terms of role of MASTL kinase in familial thrombocytopenia, the mutant animals have allowed us to conclude that the thrombocytopenic syndrome is very unlikely to be the result of haploinsufficiency of MASTL kinase because heterozygous animals have normal platelet counts. Because of the dominant nature of the syndrome, this suggests that the mechanism involves either gain-of-function of the mutant allele or a dominant-negative inhibition of MASTL function specific to the megakaryocyte, two possibilities we are examining. Disclosures:No relevant conflicts of interest to declare.

Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis

We report here a high-throughput method for the modification of bacterial artificial chromosomes (BACs) that uses a novel two-plasmid approach. In this protocol, a vector modified in our laboratory to hold an R6Kγ origin of replication and a marker recombination cassette is inserted into a BAC in a single recombination step. Temporal control of recombination is achieved through the use of a second plasmid, pSV1.RecA, which possesses a recombinase gene and a temperature-sensitive origin of replication. This highly efficient protocol has allowed us to successfully modify more than 2,000 BACs, from which over 1,000 BAC transgenic mice have been generated. A complete cycle from BAC choice to embryo implantation takes about 5 weeks. Marker genes introduced into the mice include EGFP and EGFP-L10a. All vectors used in this project can be obtained from us by request, and the EGFP reporter mice are available through the Mutant Mouse Regional Resource Center (NINDS/GENSAT collection). CNS anatomical expression maps of the mice are available to the public at http://www.gensat.org/.

Abstract 3283: Developmental and metabolic deregulation in 14-3-3zeta knockout mouse

Abstract 14-3-3s are a family of proteins conserved throughout eukaryotics. By binding to specific phosphorylated serine/threonine containing motifs in the target proteins, 14-3-3s modulates their molecular structure, stability, subcellular localization and/or enzymatic activity. Our lab recently found that 14-3-3ζ (zeta) is overexpressed in ∼45% breast cancers and 14-3-3ζ overexpression predicts poor patient survival. Additionally, 14-3-3ζ prevents anoikis by suppressing p53 and promotes epithelial mesenchymal transition by activating TGB-β signaling pathway, which plays important roles during tumor formation and progression. These led us to we hypothesizing that 14-3-3ζ plays important role in breast cancer. In our initial attempt to systematically study the role of 14-3-3ζ in breast cancer in vivo, we have recently generated 14-3-3ζ conventional knockout mouse by Gene Trap. We obtained the ES cell line RRR334 (which has no functional 14-3-3ζ based on the 5’ RACE data of the Baygenomics) from the Mutant Mouse Regional Resource Center. We confirmed silencing of 14-3-3ζ in the clone by RT-PCR. The integration site of the Gene Trap vector was located about 5kb downstream of exon 3 inside the intron 3 and PCR genotyping primers were designed accordingly. The ES cells were injected into C57Bl/6 albino mouse blastocysts. Chimera mice with high ES cell contributions based on their fur color were mated with C57Bl/6 albino mice to determine germ line transmission. When we mated the founder 14-3-3ζ heterozygous (+/−) male with 14-3-3ζ +/− female, only ∼ 5% of the offspring's were 14-3-3ζ homozygous (−/−) mice, indicating 14-3-3ζ −/− mice are embryonic lethal. This lethality hinders our study of the role of 14-3-3ζ in tumorigenesis and progression. To overcome this, we outbred to CD-1 mouse strain and the resulting mice are viable and fertile. Interestingly, the 14-3-3ζ knockout mice are smaller than their littermates. This growth retardation did not seem to be mediated by deregulated growth hormone level. The 14-3-3ζ null mice also had lower blood glucose level and their livers and brain had a reduced glucose uptake. Our preliminary studies indicated that HIF1-α, a well known oncogene and a master regulator of metabolism (e.g., glycolysis), is downregulated in 14-3-3ζ null mice. We are currently further characterizing the phenotypes of 14-3-3ζ knockout mouse and also studying the molecular mechanism of how loss of 14-3-3ζ leads to growth retardation and how 14-3-3ζ regulates glucose metabolism. We are also studying whether loss of 14-3-3ζ has any impact on tumor formation and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3283.

Hearts of Hypoxia-inducible Factor Prolyl 4-Hydroxylase-2 Hypomorphic Mice Show Protection against Acute Ischemia-Reperfusion Injury

Hypoxia-inducible factor (HIF) has a pivotal role in oxygen homeostasis and cardioprotection mediated by ischemic preconditioning. Its stability is regulated by HIF prolyl 4-hydroxylases (HIF-P4Hs), the inhibition of which is regarded as a promising strategy for treating diseases such as anemia and ischemia. We generated a viable Hif-p4h-2 hypomorph mouse line (Hif-p4h-2(gt/gt)) that expresses decreased amounts of wild-type Hif-p4h-2 mRNA: 8% in the heart; 15% in the skeletal muscle; 34-47% in the kidney, spleen, lung, and bladder; 60% in the brain; and 85% in the liver. These mice have no polycythemia and show no signs of the dilated cardiomyopathy or hyperactive angiogenesis observed in mice with broad spectrum conditional Hif-p4h-2 inactivation. We focused here on the effects of chronic Hif-p4h-2 deficiency in the heart. Hif-1 and Hif-2 were stabilized, and the mRNA levels of glucose transporter-1, several enzymes of glycolysis, pyruvate dehydrogenase kinase 1, angiopoietin-2, and adrenomedullin were increased in the Hif-p4h-2(gt/gt) hearts. When isolated Hif-p4h-2(gt/gt) hearts were subjected to ischemia-reperfusion, the recovery of mechanical function and coronary flow rate was significantly better than in wild type, while cumulative release of lactate dehydrogenase reflecting the infarct size was reduced. The preischemic amount of lactate was increased, and the ischemic versus preischemic [CrP]/[Cr] and [ATP] remained at higher levels in Hif-p4h-2(gt/gt) hearts, indicating enhanced glycolysis and an improved cellular energy state. Our data suggest that chronic stabilization of Hif-1alpha and Hif-2alpha by genetic knockdown of Hif-p4h-2 promotes cardioprotection by induction of many genes involved in glucose metabolism, cardiac function, and blood pressure.

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Polycystin-1 Regulates Skeletogenesis through Stimulation of the Osteoblast-specific Transcription Factor RUNX2-II

Polycystin-1 (PC1) may play an important role in skeletogenesis through regulation of the bone-specific transcription factor Runx2-II. In the current study we found that PC1 co-localizes with the calcium channel polycystin-2 (PC2) in primary cilia of MC3T3-E1 osteoblasts. To establish the role of Runx2-II in mediating PC1 effects on bone, we crossed heterozygous Pkd1(m1Bei) and Runx2-II mice to create double heterozygous mice (Pkd1(+/m1Bei)/Runx2-II(+/-)) deficient in both PC1 and Runx2-II. Pkd1(+/m1Bei)/Runx2-II(+/-) mice exhibited additive reductions in Runx2-II expression that was associated with impaired endochondral bone development, defective osteoblast-mediated bone formation, and osteopenia. In addition, we found that basal intracellular calcium levels were reduced in homozygous Pkd1(m1Bei) osteoblasts. In contrast, overexpression of a PC1 C-tail construct increased intracellular calcium and selectively stimulated Runx2-II P1 promoter activity in osteoblasts through a calcium-dependent mechanism. Site-directed mutagenesis of critical amino acids in the coiled-coil domain of PC1 required for coupling to PC2 abolished PC1-mediated Runx2-II P1 promoter activity. Additional promoter analysis mapped the PC1-responsive region to the "osteoblast-specific" enhancer element between -420 and -350 bp that contains NFI and AP-1 binding sites. Chromatin immunoprecipitation assays confirmed the calcium-dependent binding of NFI to this region. These findings indicate that PC1 regulates osteoblast function through intracellular calcium-dependent control of Runx2-II expression. The overall function of the primary cilium-polycystin complex may be to sense and transduce environmental clues into signals regulating osteoblast differentiation and bone development.

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health

Endocrine-Related Resources from the National Institutes of Health