Global Deletion Research Articles

Absence of the mitochondrial translocator protein 18kDa in mice does not affect body weight or food intake responses to altered energy availability.

Changes in mitochondrial function in a variety of cells/tissues are critical for orchestrating systemic energy homeostasis and are linked to the development of obesity and many of its comorbidities. The mitochondrial translocator protein of 18kDa (TSPO) is expressed in organs throughout the body, including the brain, liver, adipose tissue, gonads and adrenal glands, where it is implicated in regulating steroidogenesis and cellular metabolism. Prior work from our group and others has shown that, in rodents, TSPO levels are altered in adipose tissue by obesity and that modulation of TSPO activity may impact systemic glucose homeostasis. Furthermore, in vitro studies in a variety of cell types have implicated TSPO in mediating cellular energetics and substrate utilisation. Although mice with germline global TSPO deficiency (TSPO-/- ) have no reported changes in body weight under standard husbandry conditions, we hypothesised that, given the roles of TSPO in regulating mitochondrial function and cellular metabolic flexibility, these animals may have alterations in their systemic response to altered energy availability, either nutritional excess or insufficiency. In agreement with published work, compared to wild-type (TSPO+/+ ) littermates, TSPO-/- mice of both sexes did not exhibit differences in body weight on standard chow. Furthermore, following a 12-hour overnight fast, there was no difference in weight loss or compensatory food intake during re-feeding. Five weeks of feeding a high-fat diet (HFD) did not reveal any impact of the absence of TSPO on body weight gain in either male or female mice. Basal blood glucose levels and glucose clearance in a glucose tolerance test were influenced by feeding a HFD diet but not by genotype. In conclusion, in the paradigms examined, germline global deletion of TSPO did not change the physiological response to deviations in systemic energy availability at the whole organism level.

The Expression Pattern of Cypher in the Multiple Organs of Mice

Cypher, which is named ZASP/LDB3, one of the members of PDZ-LIM family, was well known to express in cardiac and skeletal muscles which played a functional role in muscles. It has shown that global deletion of Cypher in mice leads to lethality due to dysfunction of the skeletal muscles. Recently, the MGD database has been shown that conditional deletion of Cypher or gene mutations leads to human disease, the etiology and mechanism of which remained unknown. Therefore, it might be very important that we systemically investigated the expression of Cypher in organs or tissues from developmental stages through adulthood at first. We explored the expression pattern of Cypher by [Formula: see text]-gal staining of Cypher LacZ knockin mice combing with chemical staining, immunostaining and in situ hybridization, suggesting that Cypher might play a functional role in those organs. Further investigation will be done by cooperation with other groups by using the conditional deletion of Cypher or the point mutation of genes in the organs, which studied Cypher expression according to the MGD database.

TNFR2/14-3-3ε signaling complex instructs macrophage plasticity in inflammation and autoimmunity.

TNFR1 and TNFR2 have received prominent attention because of their dominance in the pathogenesis of inflammation and autoimmunity. TNFR1 has been extensively studied and primarily mediates inflammation. TNFR2 remains far less studied, although emerging evidence demonstrates that TNFR2 plays an antiinflammatory and immunoregulatory role in various conditions and diseases. Herein, we report that TNFR2 regulates macrophage polarization, a highly dynamic process controlled by largely unidentified intracellular regulators. Using biochemical copurification and mass spectrometry approaches, we isolated the signaling molecule 14-3-3ε as a component of TNFR2 complexes in response to progranulin stimulation in macrophages. In addition, 14-3-3ε was essential for TNFR2 signaling-mediated regulation of macrophage polarization and switch. Both global and myeloid-specific deletion of 14-3-3ε resulted in exacerbated inflammatory arthritis and counteracted the protective effects of progranulin-mediated TNFR2 activation against inflammation and autoimmunity. TNFR2/14-3-3ε signaled through PI3K/Akt/mTOR to restrict NF-κB activation while simultaneously stimulating C/EBPβ activation, thereby instructing macrophage plasticity. Collectively, this study identifies 14-3-3ε as a previously unrecognized vital component of the TNFR2 receptor complex and provides new insights into the TNFR2 signaling, particularly its role in macrophage polarization with therapeutic implications for various inflammatory and autoimmune diseases with activation of the TNFR2/14-3-3ε antiinflammatory pathway.

Adipocyte NR1D1 dictates adipose tissue expansion during obesity.

The circadian clock component NR1D1 (REVERBα) is considered a dominant regulator of lipid metabolism, with global Nr1d1 deletion driving dysregulation of white adipose tissue (WAT) lipogenesis and obesity. However, a similar phenotype is not observed under adipocyte-selective deletion (Nr1d1Flox2-6:AdipoqCre), and transcriptional profiling demonstrates that, under basal conditions, direct targets of NR1D1 regulation are limited, and include the circadian clock and collagen dynamics. Under high-fat diet (HFD) feeding, Nr1d1Flox2-6:AdipoqCre mice do manifest profound obesity, yet without the accompanying WAT inflammation and fibrosis exhibited by controls. Integration of the WAT NR1D1 cistrome with differential gene expression reveals broad control of metabolic processes by NR1D1 which is unmasked in the obese state. Adipocyte NR1D1 does not drive an anticipatory daily rhythm in WAT lipogenesis, but rather modulates WAT activity in response to alterations in metabolic state. Importantly, NR1D1 action in adipocytes is critical to the development of obesity-related WAT pathology and insulin resistance.

IL4Rα signaling promotes neonatal cardiac regeneration and cardiomyocyte cell cycle activity

Neonatal heart regeneration depends on proliferation of pre-existing cardiomyocytes, yet the mechanisms driving regeneration and cardiomyocyte proliferation are not comprehensively understood. We recently reported that the anti-inflammatory cytokine, interleukin 13 (IL13), promotes neonatal cardiac regeneration; however, the signaling pathway and cell types mediating this regenerative response remain unknown. Here, we hypothesized that expression of the type II heterodimer receptor for IL13, comprised of IL4Rα and IL13Rα1, expressed directly on cardiomyocytes mediates cardiomyocyte cell cycle and heart regeneration in neonatal mice. Our data demonstrate that indeed global deletion of one critical subunit of the type II receptor, IL4Rα (IL4Rα−/−), decreases cardiomyocyte proliferation during early postnatal development and significantly impairs cardiac regeneration following injury in neonatal mice. While multiple myocardial cell types express IL4Rα, we demonstrate that IL4Rα deletion specifically in cardiomyocytes mediates cell cycle activity and neonatal cardiac regeneration. This demonstrates for the first time a functional role for IL4Rα signaling directly on cardiomyocytes in vivo. Reciprocally, we examined the therapeutic benefit of activating the IL4Rα receptor in non-regenerative hearts via IL13 administration. Following myocardial infarction, administration of IL13 reduced scar size and promoted cardiomyocyte DNA synthesis and karyokinesis, but not complete cytokinesis, in 6-day old non-regenerative mice. Our data demonstrate a novel role for IL4Rα signaling directly on cardiomyocytes during heart regeneration and suggest the potential for type II receptor activation as one potential therapeutic target for promoting myocardial repair.

Global Deletion of 11β-HSD1 Prevents Muscle Wasting Associated with Glucocorticoid Therapy in Polyarthritis.

Glucocorticoids provide indispensable anti-inflammatory therapies. However, metabolic adverse effects including muscle wasting restrict their use. The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) modulates peripheral glucocorticoid responses through pre-receptor metabolism. This study investigates how 11β-HSD1 influences skeletal muscle responses to glucocorticoid therapy for chronic inflammation. We assessed human skeletal muscle biopsies from patients with rheumatoid arthritis and osteoarthritis for 11β-HSD1 activity ex vivo. Using the TNF-α-transgenic mouse model (TNF-tg) of chronic inflammation, we examined the effects of corticosterone treatment and 11β-HSD1 global knock-out (11βKO) on skeletal muscle, measuring anti-inflammatory gene expression, muscle weights, fiber size distribution, and catabolic pathways. Muscle 11β-HSD1 activity was elevated in patients with rheumatoid arthritis and correlated with inflammation markers. In murine skeletal muscle, glucocorticoid administration suppressed IL6 expression in TNF-tg mice but not in TNF-tg11βKO mice. TNF-tg mice exhibited reductions in muscle weight and fiber size with glucocorticoid therapy. In contrast, TNF-tg11βKO mice were protected against glucocorticoid-induced muscle atrophy. Glucocorticoid-mediated activation of catabolic mediators (FoxO1, Trim63) was also diminished in TNF-tg11βKO compared to TNF-tg mice. In summary, 11β-HSD1 knock-out prevents muscle atrophy associated with glucocorticoid therapy in a model of chronic inflammation. Targeting 11β-HSD1 may offer a strategy to refine the safety of glucocorticoids.

14-3-3 epsilon is an intracellular component of TNFR2 receptor complex and its activation protects against osteoarthritis

ObjectivesOsteoarthritis (OA) is the most common joint disease; however, the indeterminate nature of mechanisms by which OA develops has restrained advancement of therapeutic targets. TNF signalling has been implicated in...

Fli1 Is Essential for the Maintenance of Hematopoietic Stem Cell Homeostasis and Function

The ETS family of transcription factors is known to play an essential role in hematopoietic and vascular development. One such factor that is widely expressed in all vascular beds and almost all hematopoietic lineages, extending from long term hematopoietic stem cells to terminally differentiated peripheral blood cells, is Fli1. Global deletion of Fli1 leads to embryonic lethality at E12.5 due to dramatic hemorrhaging caused by poor vascular integrity and platelet dysfunction. The role of Fli1 in adults has been explored in several different cells types ranging from megakaryocytes to B and T cells. It has also been implicated in transcriptional regulation of hematopoietic stem and progenitor cells through combinatorial analysis, however its exact contribution to stem cell maintenance and function remains unclear. We hypothesized that Fli1 plays a dual role in regulating both the seed and the soil - specifically hematopoietic stem cell (HSC) function in a cell autonomous manner as well as the niche required for nurturing these cells. In this study, we focused our attention on the cell autonomous function of Fli1. We found that global deletion of Fli1 leads to lethality as a result of complete peripheral blood failure in addition to aberrant vasculature. Using the cre-lox system and various transplantation strategies, we identify Fli1 as one of the critical regulators of adult hematopoietic stem cell function. Specific deletion of Fli1 in the hematopoietic compartment alone is sufficient to induce significant reduction in peripheral blood counts, accompanied by hemorrhage, resulting in lethality. On further inspection, Fli1-/- HSCs are unable to expand ex vivo or engraft in a competitive setting. Additionally, Fli1 is essential for hematopoietic reconstitution post radiation and its deletion abolishes the ability of stem cells to reconstitute the bone marrow and contribute to peripheral blood lineages. ChIP-seq analysis demonstrates Fli1 occupancy in almost all essential hematopoietic gene enhancers and promoters. By correlating Fli1-/- HSC RNA seq analysis with the ChIP occupancy data we can conclude that Fli1 is, in fact, one of the master regulators of the HSC transcriptional program essential to maintain the balance between self-renewal and differentiation. Taken together, our results indicate that Fli1 is required for maintenance of HSC homeostasis and function, making it one of the unique transcription factors to play a critical role in HSC function in adults as well as during development. DisclosuresNo relevant conflicts of interest to declare.

Limbic progesterone receptor activity enhances neuronal excitability and seizures.

Emerging evidence raises the possibility that progesterone receptor (PR) signaling may contribute to the reproductive hormone fluctuation-linked seizure precipitation, called catamenial epilepsy. Therefore, we studied PR isoform expression in limbic regions involved in temporal lobe epilepsy and the effect of PR activation on neuronal activity and seizures. We evaluated PR expression in the limbic regions, entorhinal cortex (EC), hippocampus, and amygdala in female rats using quantitative real-time polymerase chain reaction (qRT-PCR). A selective agonist, Nestorone (16-methylene-17 alpha-acetoxy-19-nor-pregn-4-ene-3,20-dione) activated PRs, and the effect on excitability and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission of EC neurons was studied using electrophysiology. Finally, we assessed PR regulation of epileptic seizures and status epilepticus (SE) induced by lithium-pilocarpine in female rats with the global deletion of PRs (PR knockout; PRKO) using video electroencephalography (-EEG). Limbic regions EC, hippocampus, and amygdala robustly expressed PR messenger RNA (mRNA). Nestorone (16-methylene-17 alpha-acetoxy-19-nor-pregn-4-ene-3,20-dione) treatment reduced the action potential threshold of layer II/III EC neurons and increased the frequency of AMPA receptor-mediated synaptic currents of ovariectomized and estrogen-primed female rats. Female rats lacking PRs (PRKO) experienced a shorter duration, less intense, and less fatal SE than wild-type (WT) animals. Furthermore, Nestorone treatment caused seizure exacerbation in the WT epileptic animals, but not in the PRKO epileptic animals. Activation of PRs expressed in the EC and hippocampus increased neuronal excitability and worsened seizures. These receptors may play a role in catamenial epilepsy.

Dual targeting of salt inducible kinases and CSF1R uncouples bone formation and bone resorption.

Bone formation and resorption are typically coupled, such that the efficacy of anabolic osteoporosis treatments may be limited by bone destruction. The multi-kinase inhibitor YKL-05-099 potently inhibits salt inducible kinases (SIKs) and may represent a promising new class of bone anabolic agents. Here, we report that YKL-05-099 increases bone formation in hypogonadal female mice without increasing bone resorption. Postnatal mice with inducible, global deletion of SIK2 and SIK3 show increased bone mass, increased bone formation, and, distinct from the effects of YKL-05-099, increased bone resorption. No cell-intrinsic role of SIKs in osteoclasts was noted. In addition to blocking SIKs, YKL-05-099 also binds and inhibits CSF1R, the receptor for the osteoclastogenic cytokine M-CSF. Modeling reveals that YKL-05-099 binds to SIK2 and CSF1R in a similar manner. Dual targeting of SIK2/3 and CSF1R induces bone formation without concomitantly increasing bone resorption and thereby may overcome limitations of most current anabolic osteoporosis therapies.

Deletion of Fibroblast growth factor 9 globally and in skeletal muscle results in enlarged tuberosities at sites of deltoid tendon attachments.

The growth of most bony tuberosities, like the deltoid tuberosity (DT), rely on the transmission of muscle forces at the tendon-bone attachment during skeletal growth. Tuberosities distribute muscle forces and provide mechanical leverage at attachment sites for joint stability and mobility. The genetic factors that regulate tuberosity growth remain largely unknown. In mouse embryos with global deletion of fibroblast growth factor 9 (Fgf9), the DT size is notably enlarged. In this study, we explored the tissue-specific regulation of DT size using both global and targeted deletion of Fgf9. We showed that cell hypertrophy and mineralization dynamics of the DT, as well as transcriptional signatures from skeletal muscle but not bone, were influenced by the global loss of Fgf9. Loss of Fgf9 during embryonic growth led to increased chondrocyte hypertrophy and reduced cell proliferation at the DT attachment site. This endured hypertrophy and limited proliferation may explain the abnormal mineralization patterns and locally dysregulated expression of markers of endochondral development in Fgf9null attachments. We then showed that targeted deletion of Fgf9 in skeletal muscle leads to postnatal enlargement of the DT. Taken together, we discovered that Fgf9 may play an influential role in muscle-bone cross-talk during embryonic and postnatal development.

Endothelial arginase 2 mediates retinal ischemia/reperfusion injury by inducing mitochondrial dysfunction

ObjectiveRetinal ischemic disease is a major cause of vision loss. Current treatment options are limited to late-stage diseases, and the molecular mechanisms of the initial insult are not fully understood. We have previously shown that the deletion of the mitochondrial arginase isoform, arginase 2 (A2), limits neurovascular injury in models of ischemic retinopathy. Here, we investigated the involvement of A2-mediated alterations in mitochondrial dynamics and function in the pathology. MethodsWe used wild-type (WT), global A2 knockout (A2KO-) mice, cell-specific A2 knockout mice subjected to retinal ischemia/reperfusion (I/R), and bovine retinal endothelial cells (BRECs) subjected to an oxygen-glucose deprivation/reperfusion (OGD/R) insult. We used western blotting to measure levels of cell stress and death markers and the mitochondrial fragmentation protein, dynamin related protein 1 (Drp1). We also used live cell mitochondrial labeling and Seahorse XF analysis to evaluate mitochondrial fragmentation and function, respectively. ResultsWe found that the global deletion of A2 limited the I/R-induced disruption of retinal layers, fundus abnormalities, and albumin extravasation. The specific deletion of A2 in endothelial cells was protective against I/R-induced neurodegeneration. The OGD/R insult in BRECs increased A2 expression and induced cell stress and cell death, along with decreased mitochondrial respiration, increased Drp1 expression, and mitochondrial fragmentation. The overexpression of A2 in BREC also decreased mitochondrial respiration, promoted increases in the expression of Drp1, mitochondrial fragmentation, and cell stress and resulted in decreased cell survival. In contrast, the overexpression of the cytosolic isoform, arginase 1 (A1), did not affect these parameters. ConclusionsThis study is the first to show that A2 in endothelial cells mediates retinal ischemic injury through a mechanism involving alterations in mitochondrial dynamics and function.

Sam68 promotes hepatic gluconeogenesis via CRTC2

Hepatic gluconeogenesis is essential for glucose homeostasis and also a therapeutic target for type 2 diabetes, but its mechanism is incompletely understood. Here, we report that Sam68, an RNA-binding adaptor protein and Src kinase substrate, is a novel regulator of hepatic gluconeogenesis. Both global and hepatic deletions of Sam68 significantly reduce blood glucose levels and the glucagon-induced expression of gluconeogenic genes. Protein, but not mRNA, levels of CRTC2, a crucial transcriptional regulator of gluconeogenesis, are >50% lower in Sam68-deficient hepatocytes than in wild-type hepatocytes. Sam68 interacts with CRTC2 and reduces CRTC2 ubiquitination. However, truncated mutants of Sam68 that lack the C- (Sam68ΔC) or N-terminal (Sam68ΔN) domains fails to bind CRTC2 or to stabilize CRTC2 protein, respectively, and transgenic Sam68ΔN mice recapitulate the blood-glucose and gluconeogenesis profile of Sam68-deficient mice. Hepatic Sam68 expression is also upregulated in patients with diabetes and in two diabetic mouse models, while hepatocyte-specific Sam68 deficiencies alleviate diabetic hyperglycemia and improves insulin sensitivity in mice. Thus, our results identify a role for Sam68 in hepatic gluconeogenesis, and Sam68 may represent a therapeutic target for diabetes.

MyD88 Is Not Required for Muscle Injury-Induced Endochondral Heterotopic Ossification in a Mouse Model of Fibrodysplasia Ossificans Progressiva.

Excess inflammation and canonical BMP receptor (BMPR) signaling are coinciding hallmarks of the early stages of injury-induced endochondral heterotopic ossification (EHO), especially in the rare genetic disease fibrodysplasia ossificans progressiva (FOP). Multiple inflammatory signaling pathways can synergistically enhance BMP-induced Smad1/5/8 activity in multiple cell types, suggesting the importance of pathway crosstalk in EHO and FOP. Toll-like receptors (TLRs) and IL-1 receptors mediate many of the earliest injury-induced inflammatory signals largely via MyD88-dependent pathways. Thus, the hypothesis that MyD88-dependent signaling is required for EHO was tested in vitro and in vivo using global or Pdgfrα-conditional deletion of MyD88 in FOP mice. As expected, IL-1β or LPS synergistically increased Activin A (ActA)-induced phosphorylation of Smad 1/5 in fibroadipoprogenitors (FAPs) expressing Alk2R206H. However, conditional deletion of MyD88 in Pdgfrα-positive cells of FOP mice did not significantly alter the amount of muscle injury-induced EHO. Even more surprisingly, injury-induced EHO was not significantly affected by global deletion of MyD88. These studies demonstrate that MyD88-dependent signaling is dispensable for injury-induced EHO in FOP mice.

34-OR: Role of Arid5b in Thermogenesis and Adipose Tissue Browning

Background: Adipose tissue considerably influence metabolic homeostasis and play a central role in regulating whole-body energy and glucose metabolism. Previously, we have reported that mice with the global deletion of Arid5b are lean and have less fat in white and brown adipose tissues. However, this role is unknown in adipocyte specific Arid5b knockout (5bFKO) mice. Aim: We investigated the effects of adipose-specific deficiency of Arid5b on systemic glucose homeostasis and adipose tissue metabolism. Result: Deletion of Arid5b in adipose tissue using adiponectin promoter-driven Cre recombinase significantly improved glucose tolerance with significant reduction of insulin level. Body weight of 5bFKO mice was significantly reduced at the later age. Core body temperature of 5bFKO mice was significantly higher compared to the control mice. Robust increase in the protein expression of uncoupling protein 1 (Ucp1) was observed in inguinal white adipose tissue (iWAT) of 5bFKO mice housed at ambient temperature. This phenotype was further confirmed by prolonged cold exposure where Ucp1 and mitochondrial proteins such as pyruvate dehydrogenase (PDH), mitochondrially encoded cytochrome c oxidase (MTCO1) and ATP5A were substantially increased in iWAT of adipocyte specific Arid5b deficient mice. Gene expression of beige adipogenesis markers such as cell death inducing DFFA like effector A (Cidea) and iodothyronine deiodinase 2 (Dio2) were also significantly increased indicating browning of iWAT. Furthermore, loss of Arid5B in fat led to significant reduction in macrophages and total immune cells in iWAT. All together these results show that the deletion of Arid5b could promote browning of iWAT. Conclusion: These findings demonstrate that Arid5b regulates thermogenic genes expression to promote beige adipocytes formation and controls adipose inflammation. Inhibition of Arid5b may provide a novel therapeutic approach for obesity and diabetes. Disclosure M. Shukare: None. R. Huang: None. A. Ehsani: None. G. Zhang: None. J. Chalise: None. R. H. Whitson: None. K. Itakura: None.

Cardiac metallothionein overexpression rescues diabetic cardiomyopathy in Akt2-knockout mice.

To efficiently prevent diabetic cardiomyopathy (DCM), we have explored and confirmed that metallothionein (MT) prevents DCM by attenuating oxidative stress, and increasing expression of proteins associated with glucose metabolism. To determine whether Akt2 expression is critical to MT prevention of DCM, mice with either global Akt2 gene deletion (Akt2‐KO), or cardiomyocyte‐specific overexpressing MT gene (MT‐TG) or both combined (MT‐TG/Akt2‐KO) were used. Akt2‐KO mice exhibited symptoms of DCM (cardiac remodelling and dysfunction), and reduced expression of glycogen and glucose metabolism‐related proteins, despite an increase in total Akt (t‐Akt) phosphorylation. Cardiac MT overexpression in MT‐TG/Akt2‐KO mice prevented DCM and restored glucose metabolism‐related proteins expression and baseline t‐Akt phosphorylation. Furthermore, phosphorylation of ERK1/2 increased in the heart of MT‐TG/Akt2‐KO mice, compared with Akt2‐KO mice. As ERK1/2 has been implicated in the regulation of glucose transport and metabolism this increase could potentially underlie MT protective effect in MT‐TG/Akt2‐KO mice. Therefore, these results show that although our previous work has shown that MT preserving Akt2 activity is sufficient to prevent DCM, in the absence of Akt2 MT may stimulate alternative or downstream pathways protecting from DCM in a type 2 model of diabetes, and that this protection may be associated with the ERK activation pathway.

Global Deletion of Pannexin 3 Resulting in Accelerated Development of Aging-Induced Osteoarthritis in Mice.

Osteoarthritis (OA) results in pathologic changes in the joint tissue. The mechanisms driving disease progression remain largely unclear, and thus disease-modifying treatments are lacking. Pannexin 3 (Panx3) was identified as a potential mediator of cartilage degeneration in OA, and our previous study in mice indicated that deletion of the Panx3 gene delayed surgically induced cartilage degeneration. This study was undertaken to examine the role of Panx3 in other OA subtypes, particularly primary OA during aging, in a mouse model of aging-induced OA. Wild-type (WT) and Panx3-/- C57BL/6J (Black-6) mice, ages 18-24 months, were analyzed by micro-computed tomography to investigate bone mineral density and body composition. Joints were harvested from the mice, and histopathologic analysis of the joint tissue for OA development was conducted with a specific focus on changes in articular cartilage, subchondral bone, and synovial tissue. Global loss of Panx3 in aging mice was not associated with increased mortality or changes in body composition. Mice lacking Panx3 had shorter appendicular skeletons than WT mice, but overall the body compositions appeared quite similar. Panx3 deletion dramatically accelerated cartilage degeneration and subchondral bone thickening with aging in both 18-month-old and 24-month-old mice, while promoting synovitis in 18-month-old mice. These observations in a mouse model of OA suggest that Panx3 has a protective role against the development of primary aging-associated OA. It appears that Panx3 has opposing context-specific roles in joint health following traumatic injury versus that associated with aging. These data strongly suggest that there are differences in the molecular pathways driving different subtypes of OA, and therefore a detailed understanding of these pathways could directly improve strategies for OA diagnosis, therapy, and research.

Intestinal Growth in Glucagon Receptor Knockout Mice Is Not Associated With the Formation of AOM/DSS-Induced Tumors.

Treatment with exogenous GLP-2 has been shown to accelerate the growth of intestinal adenomas and adenocarcinomas in experimental models of colonic neoplasia, however, the role of endogenous GLP-2 in tumor promotion is less well known. Mice with a global deletion of the glucagon receptor (Gcgr-/-) display an increase in circulating GLP-1 and GLP-2. Due to the intestinotrophic nature of GLP-2, we hypothesized that Gcgr-/- mice would be more susceptible to colonic dysplasia in a model of inflammation-induced colonic carcinogenesis. Female Gcgr-/- mice were first characterized for GLP-2 secretion and in a subsequent study they were given a single injection with the carcinogen azoxymethane (7.5 mg/kg) and treated with dextran sodium sulfate (DSS) (3%) for six days (n=19 and 9). A cohort of animals (n=4) received a colonoscopy 12 days following DSS treatment and all animals were sacrificed after six weeks. Disruption of glucagon receptor signaling led to increased GLP-2 secretion (p<0.0001) and an increased concentration of GLP-2 in the pancreas of Gcgr-/- mice, coinciding with an increase in small intestinal (p<0.0001) and colonic (p<0.05) weight. Increased villus height was recorded in the duodenum (p<0.001) and crypt depth was increased in the duodenum and jejunum (p<0.05 and p<0.05). Disruption of glucagon receptor signaling did not affect body weight during AOM/DSS treatment, neither did it affect the inflammatory score assessed during colonoscopy or the number of large and small adenomas present at the end of the study period. In conclusion, despite the increased endogenous GLP-2 secretion Gcgr-/- mice were not more susceptible to AOM/DSS-induced tumors.

The Role of Nuclear Receptor Corepressors NCoR1 and SMRT on Physiologic Function in the Adult Mouse

Thyroid hormone (TH) plays an essential role in maintaining homeostasis and regulating metabolism in all organ systems beginning with embryogenesis and continuing throughout life. TH action is mediated by the thyroid hormone receptor (TR), which is a nuclear receptor, and it’s coregulators. The nuclear receptor corepressor 1 (NCoR1) and the silencing mediator of retinoid and thyroid hormone receptors (SMRT) are two critical corepressors of the TR that inhibit gene transcription in the absence of TH. Repression is mediated by complexing with histone deacetylase 3 (HDAC3), which is stabilized by NCoR1 and SMRT. NCoR1 and SMRT are critical for maintaining metabolic homeostasis and act to mediate energy expenditure, insulin sensitivity, and body weight. We sought to elucidate the roles of NCoR1 and SMRT in maintaining global physiologic function in the adult mouse. In order to study the post-natal role of these corepressors, we used a tamoxifen-inducible Cre recombinase (UBC-Cre-ERT2) to knock-out (KO) NCoR1, SMRT, or NCoR1 and SMRT together in adult mice because global deletion of either corepressor during embryogenesis is lethal. Mice were injected with tamoxifen at 8 weeks of age to KO either NCoR1 (NCoR1-KO; NKO), SMRT (SMRT-KO; SKO), or both NCoR1 and SMRT (double KO; DKO) and metabolic parameters were analyzed. While postnatal deletion of either NCoR1 or SMRT did not impact mortality, KO of both NCoR1 and SMRT resulted in a rapidly lethal phenotype heralded by weight loss, hypoglycemia and hypothermia. Metabolic phenotyping confirmed a loss of body mass and in particular fat mass in addition to a reduction in energy expenditure and increase in fecal caloric density. Further analysis showed the rapid development of hepatosteatosis and disturbances in lipid metabolism with a profound increase in beta-oxidation. We also found a reduction in HDAC3 protein levels in the DKO mice but no rapidly lethal phenotype in HDAC3 KO mice. Overall, we show that NCoR1 and SMRT together are critical for life as their deletion results in a rapidly lethal phenotype. While NCoR1 and SMRT are required to stabilize the corepressor complex, including HDAC3, HDAC3 KO resulted in a distinct and separate phenotype.

Nuclear Receptor Corepressors NCoR1 and SMRT Plays Unique Roles in Central Nervous System

The nuclear corepressor 1 (NCoR1) and the silencing mediator of retinoid and thyroid hormone receptors (SMRT) are critical coregulators of the thyroid hormone receptor (TR), mediating transcriptional repression via histone deacetylation. Thyroid hormone (TH) plays an essential role in many physiological processes via the TR. How the corepressors regulate TR signaling is not fully understood, especially in central nervous system (CNS). To determine the role of NCoR1 and SMRT in the CNS, we used mice with conditional NCoR1 (NCoR1lox/lox) and SMRT (SMRTlox/lox) alleles in combination with mice that express Cre recombinase in a neuronal specific fashion (Snap25-Cre). Global deletion of NCoR1 or SMRT during embryogenesis results in lethality. We also showed that NCoR1/SMRT double knock-out mice die within two weeks after induction of Cre activity in adult mice. Now, we found that neuronal specific NCoR1 or SMRT KO mice survive without obvious impairment of neuronal development. However, NCoR1/SMRT double knock-out mice die within postnatal 1-2 weeks and have impaired body growth. Thus, both NCoR1 and SMRT have important roles in maintaining normal neuronal function. Recently, cased of mutations in NCoR1 and SMRT in humans have been reported. These cases report phenotypes including Autism Spectrum Disorder (ASD) and intellectual disability. The cerebellum has been thought to contribute to motor control and learning. Surprisingly, it has also been shown to be a key brain structure involved in social cognition and its dysfunction may play a role in ASD. The Purkinje cell is the main neuron in the cerebellum. Thus, we generated cerebellar Purkinje cell specific NCoR1/SMRT knock-out mice using L7/Pcp2-Cre mice. In contrast to neuronal specific KO mice, both NCoR1 or SMRT single or double knock-out mice survive until adulthood. SMRT Purkinje cell knock-out mice showed abnormalities in 3ch social interaction test indicating impaired social functioning, similar to some ASD symptoms. Electrophysiological testing showed current injection evoked more action potentials in SMRT KO mice. These results suggest Purkinje cell dysfunction caused by SMRT deletion may result in social disability. Our data demonstrate for the first time that NCoR1 and SMRT have separate functions in different areas of the brain but also have some redundant function when knocked out together in all neurons.