Double Mutant Mice Research Articles

MiR-1/133a clusters cooperatively specify the cardiomyogenic lineage by adjustment of myocardin levels during embryonic heart development.

miRNAs are small RNAs directing many developmental processes by posttranscriptional regulation of protein-coding genes. We uncovered a new role for miR-1-1/133a-2 and miR-1-2/133a-1 clusters in the specification of embryonic cardiomyocytes allowing transition from an immature state characterized by expression of smooth muscle (SM) genes to a more mature fetal phenotype. Concomitant knockout of miR-1-1/133a-2 and miR-1-2/133a-1 released suppression of the transcriptional co-activator myocardin, a major regulator of SM gene expression, but not of its binding partner SRF. Overexpression of myocardin in the embryonic heart essentially recapitulated the miR-1/133a mutant phenotype at the molecular level, arresting embryonic cardiomyocytes in an immature state. Interestingly, the majority of postulated miR-1/133a targets was not altered in double mutant mice, indicating that the ability of miR-1/133a to suppress target molecules strongly depends on the cellular context. Finally, we show that myocardin positively regulates expression of miR-1/133a, thus constituting a negative feedback loop that is essential for early cardiac development.

Cdc42 regulates schwann cell radial sorting and myelin sheath folding through NF2/merlin‐dependent and independent signaling

The Rho family GTPase Cdc42 has been implicated in developmental Schwann cell (SC) proliferation, providing sufficient SCs for radial sorting of axons preceding SC differentiation in the peripheral nervous system. We generated Cdc42 conditional knockout (Cdc42-CKO) mice and confirmed aberrant axon sorting in Cdc42-CKO nerves. In adult Cdc42-CKO nerves, blood vessels were enlarged, and mature Remak bundles containing small axons were absent. Abnormal infoldings and outfoldings of myelin sheaths developed in Cdc42-CKO nerves, mimicking pathological features of Charcot-Marie-Tooth (CMT) disease. The NF2/merlin tumor suppressor has been implicated up- and down-stream of Cdc42. In Cdc42-CKO;NF2-del double mutant mice, radial sorting defects seen in Cdc42-CKO nerves were rescued, while changes in myelin sheaths in Cdc42-CKO nerves were not. Phosphorylation of Focal adhesion kinase (FAK) and P-GSK3β, as well as expression of β-catenin were decreased in Cdc42-CKO nerves, and these changes were rescued by NF2/merlin mutation in Cdc42-CKO;NF2-del double mutant mice. Thus, Cdc42 regulates SC radial sorting in vivo through NF2/merlin dependent signaling pathways, while Cdc42 modulation of myelin sheath folding is NF2/merlin independent.

A novel epidermal growth factor receptor-signaling platform and its targeted translation in pancreatic cancer

Epidermal growth factor (EGF)-induced EGFR tyrosine kinase receptor activation in cancer cell survival responses has become a strategic molecular-targeting clinical therapeutic intent, but the failures of these targeted approaches in the clinical setting demand alternate strategies. Here, we uncover a novel neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP-9) cross-talk in alliance with GPCR neuromedin B, which is essential for EGF-induced receptor activation and cellular signaling. Neu1 and MMP-9 form a complex with EGFR on the cell surface. Tamiflu (oseltamivir phosphate), anti-Neu1 antibodies, broad range MMP inhibitor galardin (GM6001), neuromedin B GPCR specific antagonist BIM-23127, the selective inhibitor of whole heterotrimeric G-protein complex BIM-46174 and MMP-9 specific inhibitor dose-dependently inhibited Neu1 activity associated with EGF stimulated 3T3–hEGFR cells. Tamiflu, anti-Neu1 antibodies and MMP9i attenuated EGFR phosphorylation associated with EGF-stimulated cells. Preclinical data provide the proof-of-evidence for a therapeutic targeting of Neu1 with Tamiflu in impeding human pancreatic cancer growth and metastatic spread in heterotopic xenografts of eGFP-MiaPaCa-2 tumors growing in RAGxCγ double mutant mice. Tamiflu-treated cohort exhibited a reduction of phosphorylation of EGFR-Tyr1173, Stat1-Tyr701, Akt-Thr308, PDGFRα-Tyr754 and NFκBp65-Ser311 but an increase in phospho-Smad2-Ser465/467 and -VEGFR2-Tyr1175 in the tumor lysates from the xenografts of human eGFP-MiaPaCa-2 tumor-bearing mice. The findings identify a novel promising alternate therapeutic treatment of human pancreatic cancer.

Uncoupling of neuroinflammation from axonal degeneration in mice lacking the myelin protein tetraspanin‐2

Deficiency of the major constituent of central nervous system (CNS) myelin, proteolipid protein (PLP), causes axonal pathology in spastic paraplegia type-2 patients and in Plp1(null) -mice but is compatible with almost normal myelination. These observations led us to speculate that PLP's role in myelination may be partly compensated for by other tetraspan proteins. Here, we demonstrate that the abundance of the structurally related tetraspanin-2 (TSPAN2) is highly increased in CNS myelin of Plp1(null) -mice. Unexpectedly, Tspan2(null) -mutant mice generated by homologous recombination in embryonic stem cells displayed low-grade activation of astrocytes and microglia in white matter tracts while they were fully myelinated and showed no signs of axonal degeneration. To determine overlapping functions of TSPAN2 and PLP, Tspan2(null) *Plp1(null) double-mutant mice were generated. Strikingly, the activation of astrocytes and microglia was strongly enhanced in Tspan2(null) *Plp1(null) double-mutants compared with either single-mutant, but the levels of dysmyelination and axonal degeneration were not increased. In this model, glial activation is thus unlikely to be caused by axonal pathology, and vice versa does not potentiate axonal degeneration. Our results support the concept that multiple myelin proteins have distinct roles in the long-term preservation of a healthy CNS, rather than in myelination per se.

Deletion of Sema3a or plexinA1/plexinA3 Causes Defects in Sensory Afferent Projections of Statoacoustic Ganglion Neurons

Statoacoustic ganglion (SAG) neurons project sensory afferents to appropriate targets in the inner ear to form functional vestibular and auditory circuits. Neuropilin1 (Npn1), a receptor for class 3 semaphorins, is required to generate appropriate afferent projections in SAG neurons; however, the ligands and coreceptors involved in Npn1 functioning remain unknown. Here we show that both plexinA1 and plexinA3 are expressed by SAG neurons, and plexinA1/plexinA3 double mutant mice show defects in afferent projections of SAG neurons in the inner ear. In control mice, sensory afferents of SAG neurons terminate at the vestibular sensory patches, whereas in plexinA1/plexinA3 double mutants, they extend more dorsally in the inner ear beyond normal vestibular target areas. Moreover, we find that semaphorin3a (Sema3a) is expressed in the dorsal otocyst, and Sema3a mutant mice show defects in afferent projections of SAG neurons similar to those observed in plexinA1/plexinA3 double mutants and in mice lacking a functional Npn1 receptor. Taken together, these genetic findings demonstrate that Sema3a repellent signaling plays a role in the establishment of proper afferent projections in SAG neurons, and this signaling likely occurs through a receptor complex involving Npn1 and either plexinA1 or plexinA3.

Mouse genetics and proteomic analyses demonstrate a critical role for complement in a model of DHRD/ML, an inherited macular degeneration

Macular degenerations, inherited and age related, are important causes of vision loss. Human genetic studies have suggested perturbation of the complement system is important in the pathogenesis of age-related macular degeneration. The mechanisms underlying the involvement of the complement system are not understood, although complement and inflammation have been implicated in drusen formation. Drusen are an early clinical hallmark of inherited and age-related forms of macular degeneration. We studied one of the earliest stages of macular degeneration which precedes and leads to the formation of drusen, i.e. the formation of basal deposits. The studies were done using a mouse model of the inherited macular dystrophy Doyne Honeycomb Retinal Dystrophy/Malattia Leventinese (DHRD/ML) which is caused by a p.Arg345Trp mutation in EFEMP1. The hallmark of DHRD/ML is the formation of drusen at an early age, and gene targeted Efemp1(R345W/R345W) mice develop extensive basal deposits. Proteomic analyses of Bruch's membrane/choroid and Bruch's membrane in the Efemp1(R345W/R345W) mice indicate that the basal deposits comprise normal extracellular matrix (ECM) components present in abnormal amounts. The proteomic analyses also identified significant changes in proteins with immune-related function, including complement components, in the diseased tissue samples. Genetic ablation of the complement response via generation of Efemp1(R345W/R345W):C3(-/-) double-mutant mice inhibited the formation of basal deposits. The results demonstrate a critical role for the complement system in basal deposit formation, and suggest that complement-mediated recognition of abnormal ECM may participate in basal deposit formation in DHRD/ML and perhaps other macular degenerations.

The permissive role of prolactin as a regulator of luteinizing hormone action in the female mouse ovary and extragonadal tumorigenesis

Transgenic female mice overexpressing the hCGβ subunit (hCGβ(+)) and producing elevated levels of luteinizing hormone (LH)/hCG bioactivity present as young adults with enhanced ovarian steroidogenesis, precocious puberty, and infertility. They subsequently develop pituitary prolactinomas, high circulating prolactin (PRL) levels, and marked mammary gland lobuloalveolar development followed by adenocarcinomas. None of these phenotypes appear in gonadectomized mice, indicating that the hCG-induced aberrations of ovarian function are responsible for the extragonadal phenotypes. PRL receptor-deficient (PRLR(-/-)) female mice are sterile, despite ovulating, due to a failure of embryo implantation, as a consequence of decreased ovarian LH receptor (Lhcgr) expression and inadequate corpus luteum formation and progesterone production. To study further the presumed permissive role of PRL in the maintenance of gonadal responsiveness to LH/hCG stimulation, we crossed the hCGβ(+) and PRLR(-/-) mice. The double-mutant hCGβ(+)/PRLR(-/-) females remained sterile with an ovarian phenotype similar to PRLR(-/-) mice, indicating that LH action, Lhcgr expression, and consequent luteinization are not possible without simultaneous PRL signaling. The high frequency of pituitary prolactinomas in PRLR(-/-) mice was not affected by transgenic hCGβ expression. In contrast, none of the hCGβ(+)/PRLR(-/-) females showed either mammary gland lobuloalveolar development or tumors, and the increased mammary gland Wnt-5b expression, possibly responsible for the tumorigenesis in hCGβ(+) mice, was absent in double-mutant mice. Hence, high LH/hCG stimulation is unable to compensate for missing PRL signaling in the maintenance of luteal function. PRL thus appears to be a major permissive regulator of LH action in the ovary and of its secondary extragonadal effects.

A TRPC1 Protein-dependent Pathway Regulates Osteoclast Formation and Function

Ca(2+) signaling is essential for bone homeostasis and skeletal development. Here, we show that the transient receptor potential canonical 1 (TRPC1) channel and the inhibitor of MyoD family, I-mfa, function antagonistically in the regulation of osteoclastogenesis. I-mfa null mice have an osteopenic phenotype characterized by increased osteoclast numbers and surface, which are normalized in mice lacking both Trpc1 and I-mfa. In vitro differentiation of pre-osteoclasts derived from I-mfa-deficient mice leads to an increased number of mature osteoclasts and higher bone resorption per osteoclast. These parameters return to normal levels in osteoclasts derived from double mutant mice. Consistently, whole cell currents activated in response to the depletion of intracellular Ca(2+) stores are larger in pre-osteoclasts derived from I-mfa knock-out mice compared with currents in wild type mice and normalized in cells derived from double mutant mice, suggesting a cell-autonomous effect of I-mfa on TRPC1 in these cells. A new splice variant of TRPC1 (TRPC1ε) was identified in early pre-osteoclasts. Heterologous expression of TRPC1ε in HEK293 cells revealed that it is unique among all known TRPC1 isoforms in its ability to amplify the activity of the Ca(2+) release-activated Ca(2+) (CRAC) channel, mediating store-operated currents. TRPC1ε physically interacts with Orai1, the pore-forming subunit of the CRAC channel, and I-mfa is recruited to the TRPC1ε-Orai1 complex through TRPC1ε suppressing CRAC channel activity. We propose that the positive and negative modulation of the CRAC channel by TRPC1ε and I-mfa, respectively, fine-tunes the dynamic range of the CRAC channel regulating osteoclastogenesis.

A Deficiency in the Autophagy Gene Atg16L1 Enhances Resistance to Enteric Bacterial Infection

Polymorphisms in the essential autophagy gene Atg16L1 have been linked with susceptibility to Crohn's disease, a major type of inflammatory bowel disease (IBD). Although the inability to control intestinal bacteria is thought to underlie IBD, the role of Atg16L1 during extracellular intestinal bacterial infections has not been sufficiently examined and compared to the function of other IBD susceptibility genes, such as Nod2, which encodes a cytosolic bacterial sensor. We find that Atg16L1 mutant mice are resistant to intestinal disease induced by the model bacterial pathogen Citrobacter rodentium. An Atg16L1 deficiency alters the intestinal environment to mediate an enhanced immune response that is dependent on monocytic cells, but this hyperimmune phenotype and its protective effects are lost in Atg16L1/Nod2 double-mutant mice. These results reveal an immunosuppressive function of Atg16L1 and suggest that gene variants affecting the autophagy pathway may have been evolutionarily maintained to protect against certain life-threatening infections.

Hepatic Remnant Lipoprotein Clearance by Heparan Sulfate Proteoglycans and Low-Density Lipoprotein Receptors Depend on Dietary Conditions in Mice

Chylomicron and very low-density lipoprotein remnants are cleared from the circulation in the liver by heparan sulfate proteoglycan (HSPG) receptors (syndecan-1), the low-density lipoprotein receptor (LDLR), and LDLR-related protein-1 (LRP1), but the relative contribution of each class of receptors under different dietary conditions remains unclear. Triglyceride-rich lipoprotein clearance was measured in AlbCre(+)Ndst1(f/f), Ldlr(-/-), and AlbCre(+)Lrp1(f/f) mice and mice containing combinations of these mutations. Triglyceride measurements in single and double mutant mice showed that HSPGs and LDLR dominate clearance under fasting conditions and postprandial conditions, but LRP1 contributes significantly when LDLR is absent. Mice lacking hepatic expression of all 3 receptors (AlbCre(+)Ndst1(f/f) Lrp1(f/f) Ldlr(-/-)) displayed dramatic hyperlipidemia (870 ± 270 mg triglyceride/dL; 1300 ± 350 mg of total cholesterol/dL) and exhibited persistent elevated postprandial triglyceride levels because of reduced hepatic clearance. Analysis of the particles accumulating in mutants showed that HSPGs preferentially clear a subset of small triglyceride-rich lipoproteins (≈ 20-40 nm diameter), whereas LDLR and LRP1 clear larger particles (≈ 40-60 nm diameter). Finally, we show that HSPGs play a major role in clearance of triglyceride-rich lipoproteins in mice fed normal chow or under postprandial conditions but seem to play a less significant role on a high-fat diet. These data show that HSPGs, LDLR, and LRP1 clear distinct subsets of particles, that HSPGs work independently of LDLR and LRP1, and that HSPGs, LDLR, and LRP1 are the 3 major hepatic triglyceride-rich lipoprotein clearance receptors in mice.

The Impact of Bdnf Gene Deficiency to the Memory Impairment and Brain Pathology of APPswe/PS1dE9 Mouse Model of Alzheimer’s Disease

Brain-derived neurotrophic factor (BDNF) importantly regulates learning and memory and supports the survival of injured neurons. Reduced BDNF levels have been detected in the brains of Alzheimer’s disease (AD) patients but the exact role of BDNF in the pathophysiology of the disorder remains obscure. We have recently shown that reduced signaling of BDNF receptor TrkB aggravates memory impairment in APPswe/PS1dE9 (APdE9) mice, a model of AD. The present study examined the influence of Bdnf gene deficiency (heterozygous knockout) on spatial learning, spontaneous exploratory activity and motor coordination/balance in middle-aged male and female APdE9 mice. We also studied brain BDNF protein levels in APdE9 mice in different ages showing progressive amyloid pathology. Both APdE9 and Bdnf mutations impaired spatial learning in males and showed a similar trend in females. Importantly, the effect was additive, so that double mutant mice performed the worst. However, APdE9 and Bdnf mutations influenced spontaneous locomotion in contrasting ways, such that locomotor hyperactivity observed in APdE9 mice was normalized by Bdnf deficiency. Obesity associated with Bdnf deficiency did not account for the reduced hyperactivity in double mutant mice. Bdnf deficiency did not alter amyloid plaque formation in APdE9 mice. Before plaque formation (3 months), BDNF protein levels where either reduced (female) or unaltered (male) in the APdE9 mouse cortex. Unexpectedly, this was followed by an age-dependent increase in mature BDNF protein. Bdnf mRNA and phospho-TrkB levels remained unaltered in the cortical tissue samples of middle-aged APdE9 mice. Immunohistological studies revealed increased BDNF immunoreactivity around amyloid plaques indicating that the plaques may sequester BDNF protein and prevent it from activating TrkB. If similar BDNF accumulation happens in human AD brains, it would suggest that functional BDNF levels in the AD brains are even lower than reported, which could partially contribute to learning and memory problems of AD patients.

Characterization of a novel NKG2D and NKp46 double-mutant mouse reveals subtle variations in the NK cell repertoire

AbstractThe immunoreceptors NKG2D and NKp46 are known for their capacity to activate natural killer (NK) cell cytotoxicity and secretory responses in the contexts of tumors and infections, yet their roles in NK cell education remain unclear. Here, we provide the first characterization of mice deficient for both NKG2D and NKp46 receptors to address the relevance of their concomitant absence during NK cell development and function. Our findings reveal that NK cells develop normally in double-mutant (DKO) mice. Mice lacking NKG2D but not NKp46 showed subtle differences in the percentages of NK cells expressing inhibitory Ly49 receptors and the adhesion molecule DNAM-1. A slightly increased percentage of terminally differentiated NK cells and functional response to in vitro stimuli was observed in some experiments. These alterations were modest and did not affect NK cell function in vivo in response to mouse cytomegalovirus infection. NKp46 deficiency alone, or in combination with NKG2D deficiency, had no effect on frequency or function of NK cells.

Developmental role for endocannabinoid signaling in regulating glucose metabolism and growth.

Treatment of ob/ob (obese) mice with a cannabinoid receptor 1 (Cnr1) antagonist reduces food intake, suggesting a role for endocannabinoid signaling in leptin action. We further evaluated the role of endocannabinoid signaling by analyzing the phenotype of Cnr1 knockout ob/ob mice. Double mutant animals show a more severe growth retardation than ob/ob mice with similar levels of adiposity and reduced IGF-I levels without alterations of growth hormone (GH) levels. The double mutant mice are also significantly more glucose intolerant than ob/ob mice. This is in contrast to treatment of ob/ob mice with a Cnr1 antagonist that had no effect on glucose metabolism, suggesting a possible requirement for endocannabinoid signaling during development for normal glucose homeostasis. Double mutant animals also showed similar leptin sensitivity as ob/ob mice, suggesting that there are developmental changes that compensate for the loss of Cnr1 signaling. These data establish a role for Cnr1 during development and suggest that compensatory changes during development may mitigate the requirement for Cnr1 in mediating the effects of leptin. The data also suggest a developmental role for Cnr1 to promote growth, regulate the GH/IGF-I axis, and improve β-cell function and glucose homeostasis in the setting of leptin deficiency.

Contributions of VLDLR and LRP8 in the establishment of retinogeniculate projections.

BackgroundRetinal ganglion cells (RGCs), the output neurons of the retina, project to over 20 distinct brain nuclei, including the lateral geniculate nucleus (LGN), a thalamic region comprised of three functionally distinct subnuclei: the ventral LGN (vLGN), the dorsal LGN (dLGN) and the intergeniculate leaflet (IGL). We previously identified reelin, an extracellular glycoprotein, as a critical factor that directs class-specific targeting of these subnuclei. Reelin is known to bind to two receptors: very-low-density lipoprotein receptor (VLDLR) and low-density lipoprotein receptor-related protein 8 (LRP8), also known as apolipoprotein E receptor 2 (ApoER2). Here we examined the roles of these canonical reelin receptors in retinogeniculate targeting.ResultsTo assess the roles of VLDLR and LRP8 in retinogeniculate targeting, we used intraocular injections of fluorescently conjugated cholera toxin B subunit (CTB) to label all RGC axons in vivo. Retinogeniculate projections in mutant mice lacking either VLDLR or LRP8 appeared similar to controls; however, deletion of both receptors resulted in dramatic defects in the pattern of retinal innervation in LGN. Surprisingly, defects in vldlr−/−;lrp8−/− double mutant mice were remarkably different than those observed in mice lacking reelin. First, we failed to observe retinal axons exiting the medial border of the vLGN and IGL to invade distant regions of non-retino-recipient thalamus. Second, an ectopic region of binocular innervation emerged in the dorsomedial pole of vldlr−/−;lrp8−/− mutant dLGN. Analysis of retinal projection development, retinal terminal sizes and LGN cytoarchitecture in vldlr−/−;lrp8−/− mutants, all suggest that a subset of retinal axons destined for the IGL are misrouted to the dorsomedial pole of dLGN in the absence of VLDLR and LRP8. Such mistargeting is likely the result of abnormal migration of IGL neurons into the dorsomedial pole of dLGN in vldlr−/−;lrp8−/− mutants.ConclusionsIn contrast to our expectations, the development of both the LGN and retinogeniculate projections appeared dramatically different in mutants lacking either reelin or both canonical reelin receptors. These results suggest that there are reelin-independent functions of VLDLR and LRP8 in LGN development, and VLDLR- and LRP8-independent functions of reelin in class-specific axonal targeting.

GATA transcription factors: New key regulators in pancreas organogenesis.

GATA transcription factors: New key regulators in pancreas organogenesis.

Genetic disruption of Abl nuclear import reduces renal apoptosis in a mouse model of cisplatin-induced nephrotoxicity

DNA damage activates nuclear Abl tyrosine kinase to stimulate intrinsic apoptosis in cancer cell lines and mouse embryonic stem cells. To examine the in vivo function of nuclear Abl in apoptosis, we generated Abl-μNLS (μ, mutated in nuclear localization signals) mice. We show here that cisplatin-induced apoptosis is defective in the renal proximal tubule cells (RPTC) from the Ablμ/μ mice. When injected with cisplatin, we found similar levels of platinum in the Abl+/+ and the Ablμ/μ kidneys, as well as similar initial inductions of p53 and PUMAα expression. However, the accumulation of p53 and PUMAα could not be sustained in the Ablμ/μ kidneys, leading to reductions in renal apoptosis and tubule damage. Co-treatment of cisplatin with the Abl kinase inhibitor, imatinib, reduced the accumulation of p53 and PUMAα in the Abl+/+ but not in the Ablμ/μ kidneys. The residual apoptosis in the Ablμ/μ mice was not further reduced in the Ablμ/μ; p53−/− double-mutant mice, suggesting that nuclear Abl and p53 are epistatic to each other in this apoptosis response. Although apoptosis and tubule damage were reduced, cisplatin-induced increases in phospho-Stat-1 and blood urea nitrogen were similar between the Abl+/+ and the Ablμ/μ kidneys, indicating that RPTC apoptosis is not the only factor in cisplatin-induced nephrotoxicity. These results provide in vivo evidence for the pro-apoptotic function of Abl, and show that its nuclear localization and tyrosine kinase activity are both required for the sustained expression of p53 and PUMAα in cisplatin-induced renal apoptosis.

β-Actin and Fascin-2 Cooperate to Maintain Stereocilia Length

Stereocilia are actin-based protrusions on auditory sensory hair cells that are deflected by sound waves to initiate the conversion of mechanical energy to neuronal signals. Stereocilia maintenance is essential because auditory hair cells are not renewed in mammals. This process requires both β-actin and γ-actin as knock-out mice lacking either isoform develop distinct stereocilia pathology during aging. In addition, stereocilia integrity may hinge on immobilizing actin, which outside of a small region at stereocilia tips turns over with a very slow, months-long half-life. Here, we establish that β-actin and the actin crosslinking protein fascin-2 cooperate to maintain stereocilia length and auditory function. We observed that mice expressing mutant fascin-2 (p.R109H) or mice lacking β-actin share a common phenotype including progressive, high-frequency hearing loss together with shortening of a defined subset of stereocilia in the hair cell bundle. Fascin-2 binds β-actin and γ-actin filaments with similar affinity in vitro and fascin-2 does not depend on β-actin for localization in vivo. Nevertheless, double-mutant mice lacking β-actin and expressing fascin-2 p.R109H have a more severe phenotype suggesting that each protein has a different function in a common stereocilia maintenance pathway. Because the fascin-2 p.R109H mutant binds but fails to efficiently crosslink actin filaments, we propose that fascin-2 crosslinks function to slow actin depolymerization at stereocilia tips to maintain stereocilia length.

Fancd2 and p21 function independently in maintaining the size of hematopoietic stem and progenitor cell pool in mice

Fanconi anemia patients suffer from progressive bone marrow failure. An overactive p53 response to DNA damage contributes to the progressive elimination of Fanconi anemia hematopoietic stem and progenitor cells (HSPC), and hence presents a potential target for therapeutic intervention. To investigate whether the cell cycle regulatory protein p21 is the primary mediator of the p53-dependent stem cell loss, p21/Fancd2 double-knockout mice were generated. Surprisingly double mutant mice displayed even more severe loss of HSPCs than Fancd2−/− single mutants. p21 deletion did not rescue the abnormal cell cycle profile and had no impact on the long-term repopulating potential of Fancd2−/− bone marrow cells. Collectively, our data indicate that p21 has an indispensable role in maintaining a normal HSPC pool and suggest that other p53-targeted factors, not p21, mediate the progressive elimination of HSPC in Fanconi anemia.

Abstract 5184: Mechanistic analysis of liver inflammation and cancer formation in mice with heterozygous lose of β-spectrin (β2SP).

Abstract Acquisition of specific genetic alterations has been shown to play a pivotal role in the progression of various stages of liver cancer, and studies have proved that alteration of transforming growth factor-β (TGF-β) signaling pathway can lead to the development of gastrointestinal cancers including liver cancer. β-Spectrin (β2SP) is one of adapter proteins for Smad3/Smad4 complex formation during TGF-β signal transduction. Forty % of β2SP+/- mice develop liver cancer, and the underlying molecular mechanisms remain largely unclear. Hypothesis: Compared to wild-type (WT) mice, β2SP+/- mice had significantly higher number of inflammatory nodules in the liver and this pathological feature might be one of mechanisms contributing to liver cancer formation seen in β2SP+/- mice. Inflammation plays a crucial role at different stages of tumor development. We hypothesize that an increase in liver inflammation leads to liver cancer formation in β2SP+/- mice. We proposed to study the mechanism by which β2SP regulates liver inflammation and suppresses liver cancer development. Experimental Procedures and Results: (1) We dissected livers from 18-month-old WT and β2SP+/- mice and processed for paraffin embedding and the tissue slides were used for hematoxylin and eosin (H&E) staining. We observed inflammatory nodules in the liver of near 60% β2SP+/- mice compared to less than 5% in WT mice. (2) To identify the type of leucocytes infiltrated in the liver tissues, we carried out immunohistochemistry staining by using antibodies directly against macrophage, T-cell receptor and B-cell antigen, and found that the number of T- and B-lymphocytes in the liver was significantly higher in β2SP+/- mice than that in WT mice. (3) Microarray and quantitative real-time RT-PCR analyses indicated that hepatic expression of interleukin-1 α (IL-1α) and IL-1β mRNA was highly upregulated only in β2SP+/- mice. (4) Western blot analyses showed that STAT3 activation in the liver was higher in β2SP+/- mice than that in WT mice. Double mutant mice with β2SP+/- and liver specific deletion of STAT3 (LSTAT3-/-) had reduced inflammatory nodules and IL-1 expression compared to β2SP+/- mice, suggesting that STAT3 is one of the regulators in mediating the cross-talk between β2SP and IL-1 in the regulation of inflammatory response in liver. Conclusion: Our results suggest that hepatic expression of IL-1α and IL-1β is induced in β2SP+/- mice, which is likely due to the elevated activation of STAT3 and contributes to the increased hepatic inflammation in β2SP+/- mice. Citation Format: Ling Lin, Hua Wang, Bin Gao, Xiuling Zhi, Aiwu Ruth He. Mechanistic analysis of liver inflammation and cancer formation in mice with heterozygous lose of β-spectrin (β2SP). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5184. doi:10.1158/1538-7445.AM2013-5184

Abstract LB-258: The human Aurora-A kinase Phe31Ile polymorphism affects cancer susceptibility in a knock-in mouse model.

Abstract Aurora-A kinase is a serine/threonine kinase that is essential for faithful chromosome segregation during mitosis. A single nucleotide polymorphism T91A (amino acid Phe31→Ile) present in the coding region of human Aurora-A is associated with susceptibility to multiple cancer types, yet the precise biochemical mechanisms by which these different polymorphisms function are still not well characterized. To investigate the functional differences between the two alleles of Aurora-A, we have generated a knock-in mouse that carries the human-specific Aurora-A Phe31 allele. Normally, rodents only carry the Ile allele. Homozygous (F/F) and heterozygous (F/I) mice for this knock-in allele were born healthy and fertile with no obvious phenotype. Growth rates of MEFs isolated from F/F, F/I, and I/I embryos showed no significant differences. To investigate the oncogenic properties of each variant in tumor development in the context of different p53 status, we generated a series of Aurora-A knock-in/p53-deficient double mutant mice. All mice were exposed to a single dose of 4Gy whole-body γ-radiation at 5 weeks of age and observed daily until they were moribund, then sacrificed and autopsied. While the I/I genotype conferred increased risk (reduced latency) compared to the F/F genotype in the p53+/- mice (p=0.037), the reverse was observed in the p53-/- mice (p=0.009); 43% of the I/I mice versus 25% of the F/F mice developed lymphomas in the p53+/- background, while 55% of the I/I mice versus 76% of the F/F mice developed lymphomas in the p53-/- background. These results support our hypothesis that the two polymorphisms confer differential susceptibility to lymphoma development in a p53-dependent manner. Interestingly, Aurora-A+/- heterozygous null mice (F/- and I/- on the p53+/- background) showed increased incidence and decreased latency of tumor formation compared to the Aurora-A+/+ mice. These data suggest dual roles for Aurora-A as both an oncogene and a haploinsufficient tumor suppressor in lymphomagenesis. There exist complex regulatory mechanisms between Aurora-A and p53, emphasizing that a balanced level of Aurora-A is critical for cell survival and maintenance of genome integrity. These data highlight the critical role of interactions between common inherited genetic variants in determining tumor susceptibility, a phenomenon that may underlie the “Missing heritability” noted in GWAS in human populations. Citation Format: Ihn Young Song, Atul Kumar, Reyno Delrosario, Jian-Hua Mao, Allan Balmain. The human Aurora-A kinase Phe31Ile polymorphism affects cancer susceptibility in a knock-in mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-258. doi:10.1158/1538-7445.AM2013-LB-258