Disrupting the Interaction between Retinoblastoma Protein and Raf-1 Leads to Defects in Progenitor Cell Proliferation and Survival during Early Inner Ear Development

The retinoblastoma protein (pRb) is required for cell-cycle exit of embryonic mammalian hair cells but is not required for hair cell fate determination and early differentiation, and this provides a strategy for hair cell regeneration by manipulating the pRb pathway. To reveal the mechanism of pRb functional modification in the inner ear, we compared the effects of attenuated pRb phosphorylation by an inhibitor of the Mitogen-Activated Protein (MAP) kinase pathway and an inhibitor of the Rb–Raf-1 interaction on cultured chicken otocysts. We demonstrated that the activity of pRb is correlated with its phosphorylation state, which is regulated by a newly established cell cycle-independent pathway mediated by the physical interaction between Raf-1 and pRb. The phosphorylation of pRb plays an important role during the early stage of inner ear development, and attenuated phosphorylation in progenitor cells leads to cell cycle arrest and increased apoptosis along with a global down-regulation of the genes involved in cell cycle progression. Our study provides novel routes to modulate pRb function for hair cell regeneration.     

Brca2 Deficiency Leads to T Cell Loss and Immune Dysfunction

Germline mutations in the breast cancer type 2 susceptibility gene (BRCA2) are linked to familial breast cancer and the progressive bone marrow failure syndrome Fanconi anaemia. Established Brca2 mouse knockout models show embryonic lethality, but those with a truncating mutation at the C-terminus survive to birth and develop thymic lymphoma at an early age. To overcome early lethality and investigate the function of BRCA2, we used T cell-specific conditional Brca2 knockout mice, which were previously shown to develop thymic lymphoma at a low penetrance. In the current study we showed that the number of peripheral T cells, particularly naïve pools, drastically declined with age. This decline was primarily ascribed to improper peripheral maintenance. Furthermore, heterozygous mice with one wild-type Brca2 allele manifested reduced T cell numbers, suggesting that Brca2 haploinsufficiency might also result in T cell loss. Our study reveals molecular events occurring in Brca2-deficient T cells and suggests that both heterozygous and homozygous Brca2 mutation may lead to dysfunction in T cell populations.     

Cell Cycle Arrest during Measles Virus Infection: a G0-Like Block Leads to Suppression of Retinoblastoma Protein Expression

One of the major mechanisms by which measles virus (MV) infection causes disease and death is suppression of the immune response. The nonresponsiveness of MV-infected human lymphocytes to mitogens and a partial block in the G₀/G₁ phase of the cell cycle observed in vitro is thought to reflect in vivo immunosuppression. In order to molecularly dissect MV-induced immunosuppression, we analyzed expression of surface activation markers and cell cycle-regulatory proteins in MV-infected human T lymphocytes. MV Edmonston (MV-Ed) could induce and maintain a high level of the early activation marker CD69 in the absence of proliferation. Expression of cyclins D3 and E, which positively control entry into S phase, was also significantly decreased. Analysis of inhibitors of progression into S phase showed that a high level of p27 was maintained in the G₀/G₁-blocked subpopulation of MV-Ed-infected cells compared to the proliferating MV-infected cells. Furthermore, cell cycle-related upregulation of retinoblastoma (Rb) protein synthesis did not occur in the MV-Ed-infected lymphocytes. Acridine orange staining, which distinguishes cells in G₀ from cells in G₁, showed that RNA levels were not upregulated following activation, which is consistent with cells remaining in a G₀ state. Although expression of surface activation markers indicated entry into the cycle, intracellular Rb and RNA levels suggested a quiescent state. These results indicate that MV can uncouple activation of T lymphocytes from transition of G₀ to G₁.     

Cardiac-specific Deletion of LKB1 Leads to Hypertrophy and Dysfunction

LKB1 encodes a serine/threonine kinase, which functions upstream of the AMP-activated protein kinase (AMPK) superfamily. To clarify the role of LKB1 in heart, we generated and characterized cardiac myocyte-specific LKB1 knock-out (KO) mice using α-myosin heavy chain-Cre deletor strain. LKB1-KO mice displayed biatrial enlargement with atrial fibrillation and cardiac dysfunction at 4 weeks of age. Left ventricular hypertrophy was observed in LKB1-KO mice at 12 weeks but not 4 weeks of age. Collagen I and III mRNA expression was elevated in atria at 4 weeks, and atrial fibrosis was seen at 12 weeks. LKB1-KO mice displayed cardiac dysfunction and atrial fibrillation and died within 6 months of age. Indicative of a prohypertrophic environment, the phosphorylation of AMPK and eEF2 was reduced, whereas mammalian target of rapamycin (mTOR) phosphorylation and p70S6 kinase phosphorylation were increased in both the atria and ventricles of LKB1-deficient mice. Consistent with vascular endothelial growth factor mRNA and protein levels being significantly reduced in LKB1-KO mice, these mice also exhibited a reduction in capillary density of both atria and ventricles. In cultured cardiac myocytes, LKB1 silencing induced hypertrophy, which was ameliorated by the expression of a constitutively active form AMPK or by treatment with the inhibitor of mTOR, rapamycin. These findings indicate that LKB1 signaling in cardiac myocytes is essential for normal development of the atria and ventricles. Cardiac hypertrophy and dysfunction in LKB1-deficient hearts are associated with alterations in AMPK and mTOR/p70S6 kinase/eEF2 signaling and with a reduction in vascular endothelial growth factor expression and vessel rarefaction.     

Newt Myotubes Reenter the Cell Cycle by Phosphorylation of the Retinoblastoma Protein

Withdrawal from the cell cycle is an essential aspect of vertebrate muscle differentiation and requires the retinoblastoma (Rb) protein that inhibits expression of genes needed for cell cycle entry. It was shown recently that cultured myotubes derived from the Rb^−/−mouse reenter the cell cycle after serum stimulation (Schneider, J.W., W. Gu, L. Zhu, V. Mahdavi, and B. Nadal-Ginard. 1994. Science (Wash. DC). 264:1467– 1471). In contrast with other vertebrates, adult urodele amphibians such as the newt can regenerate their limbs, a process involving cell cycle reentry and local reversal of differentiation. Here we show that myotubes formed in culture from newt limb cells are refractory to several growth factors, but they undergo S phase after serum stimulation and accumulate 4N nuclei. This response to serum is inhibited by contact with mononucleate cells. Despite the phenotypic parallel with Rb^−/− mouse myotubes, Rb is expressed in the newt myotubes, and its phosphorylation via cyclin-dependent kinase 4/6 is required for cell cycle reentry. Thus, the postmitotic arrest of urodele myotubes, although intact in certain respects, can be undermined by a pathway that is inactive in other vertebrates. This may be important for the regenerative ability of these animals.     

Early Low Protein Diet Aggravates Unbalance between Antioxidant Enzymes Leading to Islet Dysfunction

Background

Islets from adult rat possess weak antioxidant defense leading to unbalance between superoxide dismutase (SOD) and hydrogen peroxide-inactivating enzymatic activities, catalase (CAT) and glutathione peroxidase (GPX) rending them susceptible to oxidative stress. We have shown that this vulnerability is influenced by maternal diet during gestation and lactation.

Methodology/Principal Findings

The present study investigated if low antioxidant activity in islets is already observed at birth and if maternal protein restriction influences the development of islet antioxidant defenses. Rats were fed a control diet (C group) or a low protein diet during gestation (LP) or until weaning (LPT), after which offspring received the control diet. We found that antioxidant enzymatic activities varied with age. At birth and after weaning, normal islets possessed an efficient GPX activity. However, the antioxidant capacity decreased thereafter increasing the potential vulnerability to oxidative stress. Maternal protein malnutrition changed the antioxidant enzymatic activities in islets of the progeny. At 3 months, SOD activity was increased in LP and LPT islets with no concomitant activation of CAT and GPX. This unbalance could lead to higher hydrogen peroxide production, which may concur to oxidative stress causing defective insulin gene expression due to modification of critical factors that modulate the insulin promoter. We found indeed that insulin mRNA level was reduced in both groups of malnourished offspring compared to controls. Analyzing the expression of such critical factors, we found that c-Myc expression was strongly increased in islets from both protein-restricted groups compared to controls.

Conclusion and Significance

Modification in antioxidant activity by maternal low protein diet could predispose to pancreatic islet dysfunction later in life and provide new insights to define a molecular mechanism responsible for intrauterine programming of endocrine pancreas.     

Mitochondrial Dysfunction Leads to Deconjugation of Quercetin Glucuronides in Inflammatory Macrophages

Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-O-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. In vitro experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that β-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of β-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the β-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular β-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides in vivo in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites.     

Lipopolysaccharide Exposure during Pregnancy Leads to Aortic Dysfunction in Offspring Rats

Background

Prenatal exposure to Lipopolysaccharide (LPS) produces hypertension in adult offspring rats. The present study was to explore the effects of prenatal inflammation on morphological and functional changes in the aorta from offspring rats and to further assess its susceptibility to cardiovascular diseases.

Methods and Results

Pregnant rats were treated intraperitoneally on gestation Days 8, 10 and 12 with saline, LPS (0.79 mg/kg), or pyrrolidine dithiocarbamate (PDTC, 100 mg/kg)+LPS, respectively. Aortic ring reactivity and histopathological alteration were analyzed in offspring at the age of 12 weeks. The detections of connexin (Cx) 37, Cx40, Cx43, and Cx45, including immunofluorescent patterns, protein levels and mRNA expression in the aorta, were performed as well. Furthermore, the expressions of Nuclear factor (NF)-κB (p65), IκBα, phospho-IκBα and IκBβ were determined. The results showed that prenatal LPS exposure leads to morphological abnormalities and impaired aortic reactivity in offspring. Prenatal LPS exposure also decreased the protein and mRNA expression of Cx37 in the aorta from offspring rats. NF-κB and phospho-IκBα levels were both increased, IκBα level, however, was decreased in the aorta of offspring from the maternal LPS exposure compared to the controls. Simultaneously, PDTC treatment markedly reversed the action of LPS.

Conclusions

Decreased expression of Cx37 contributed to the aortic dysfunction of prenatal LPS exposure offspring, which should be associated with NF-κB activation.     

The Retinoblastoma Protein Is Linked to the Activation of Ras

The inner membrane-bound protein Ras integrates various extracellular signals that are subsequently communicated from the cytoplasm to the nucleus via the Raf/MEK/MAPK cascade. Here we show that the retinoblastoma protein pRb, previously reported to be a nuclear target of this pathway, can in turn influence the activation state of Ras. Rb-deficient fibroblasts display elevated levels (up to 30-fold) of activated Ras during G(1). Expression of wild-type pRb or a number of pRb mutants defective in E2F regulation reverses this effect. We provide evidence that the mid-G(1) activation of Ras in Rb-deficient cells, which occurs at the level of guanine nucleotide binding, differs from that of epidermal growth factor-induced stimulation of Ras, being dependent on protein synthesis. The aberrant levels of Ras activity associated with loss of pRb may be responsible for the differentiation defects in Rb-deficient cells, because suppression of Ras activity in Rb(-/-) fibroblasts restores the transactivation function of MyoD and the expression of a late marker of skeletal muscle differentiation. These data suggest that nuclear-cytoplasmic communication between pRb and Ras is bidirectional.     

Disruption of Sarcoendoplasmic Reticulum Calcium ATPase Function in Drosophila Leads to Cardiac Dysfunction

Abnormal sarcoendoplasmic reticulum Calcium ATPase (SERCA) function has been associated with poor cardiac function in humans. While modifiers of SERCA function have been identified and studied using animal models, further investigation has been limited by the absence of a model system that is amenable to large-scale genetic screens. Drosophila melanogaster is an ideal model system for the investigation of SERCA function due to the significant homology to human SERCA and the availability of versatile genetic screening tools. To further the use of Drosophila as a model for examining the role of SERCA in cardiac function, we examined cardiac function in adult flies. Using optical coherence tomography (OCT) imaging in awake, adult Drosophila, we have been able to characterize cardiac chamber dimensions in flies with disrupted in Drosophila SERCA (CaP60A). We found that the best studied CaP60A mutant, the conditional paralytic mutant CaP60A^kum170, develops marked bradycardia and chamber enlargement that is closely linked to the onset of paralysis and dependent on extra cardiac CaP60A. In contrast to prior work, we show that disruption of CaP60A in a cardiac specific manner results in cardiac dilation and dysfunction rather than alteration in heart rate. In addition, the co-expression of a calcium release channel mutation with CaP60A ^kum170 is sufficient to rescue the cardiac phenotype but not paralysis. Finally, we show that CaP60A overexpression is able to rescue cardiac function in a model of Drosophila cardiac dysfunction similar to what is observed in mammals. Thus, we present a cardiac phenotype associated with Drosophila SERCA dysfunction that would serve as additional phenotyping for further large-scale genetic screens for novel modifiers of SERCA function.     

Loss of Prion Protein Leads to Age-Dependent Behavioral Abnormalities and Changes in Cytoskeletal Protein Expression

The cellular prion protein (PrPC) is a highly conserved protein whose exact physiological role remains elusive. In the present study, we investigated age-dependent behavioral abnormalities in PrPC-knockout (Prnp0/0) mice and wild-type (WT) controls. Prnp0/0 mice showed age-dependent behavioral deficits in memory performance, associative learning, basal anxiety, and nest building behavior. Using a hypothesis-free quantitative proteomic investigation, we found that loss of PrPC affected the levels of neurofilament proteins in an age-dependent manner. In order to understand the biochemical basis of these observations, we analyzed the phosphorylation status of neurofilament heavy chain (NF-H). We found a reduction in NF-H phosphorylation in both Prnp0/0 mice and in PrPC-deficient cells. The expression of Fyn and phospho-Fyn, a potential regulator for NF phosphorylation, was associated with PrPC ablation. The number of β-tubulin III-positive neurons in the hippocampus was diminished in Prnp0/0 mice relative to WT mice. These data indicate that PrPC plays an important role in cytoskeletal organization, brain function, and age-related neuroprotection. Our work represents the first direct biochemical link between these proteins and the observed behavioral phenotypes.     

Activation of Endothelial Cell Kinin Receptors Leads to Intracellular Calcium Increases and Filamin Translocation: Regulation by Protein Kinase C

Membrane-associated cytoskeletal proteins provide support for endothelial cell (EC) junctional cell adhesion molecules. Nonmuscle filamin is a dimeric actin cross-linking protein that interacts with F-actin and membrane glycoproteins. Both bradykinin and des-Arg⁹-bradykinin cause filamin redistribution from the plasma membrane to the cytosol of confluent EC. Kinin-induced filamin translocation parallels the dynamics of intracellular Ca²⁺ increases. Pretreatment with kinin receptor antagonists blocks the Ca²⁺ response as well as filamin translocation induced by kinins. Protein kinase C activation prior to kinin stimulation attenuates intracellular Ca²⁺ increases and filamin translocation. BAPTA, a cell-permeable Ca²⁺ chelator, attenuates bradykinin-induced intracellular Ca²⁺ increases and filamin translocation. This study demonstrates that bovine pulmonary artery ECs express both kinin B1 and B2 receptors, and that activation of either receptor leads to intracellular Ca²⁺ increases. This Ca²⁺ signalling, which is downregulated by protein kinase C activation, is essential for kinin-induced filamin translocation.     

Depletion of the SR-Related Protein TbRRM1 Leads to Cell Cycle Arrest and Apoptosis-Like Death in Trypanosoma brucei

Arginine-Serine (RS) domain-containing proteins are RNA binding proteins with multiple functions in RNA metabolism. In mammalian cells this group of proteins is also implicated in regulation and coordination of cell cycle and apoptosis. In trypanosomes, an early branching group within the eukaryotic lineage, this group of proteins is represented by 3 members, two of them are SR proteins and have been recently shown to be involved in rRNA processing as well as in pre-mRNA splicing and stability. Here we report our findings on the 3rd member, the SR-related protein TbRRM1. In the present study, we showed that TbRRM1 ablation by RNA-interference in T. brucei procyclic cells leads to cell-cycle block, abnormal cell elongation compatible with the nozzle phenotype and cell death by an apoptosis-like mechanism. Our results expand the role of the trypanosomal RS-domain containing proteins in key cellular processes such as cell cycle and apoptosis-like death, roles also carried out by the mammalian SR proteins, and thus suggesting a conserved function in this phylogenetically conserved protein family.     

Inhibin and inhibin alpha-chain precursors are produced by immature rat Sertoli cells in culture.

Stimulation of Sertoli cells from immature rats with dibutyryl cyclic (dbc) AMP resulted in a decrease in the ratio of inhibin biological (B):immunological (I) activities in vitro. To establish the basis for this decrease, culture medium from Sertoli cells stimulated with dbcAMP was fractionated by dye-affinity chromatography, reverse-phase HPLC, and preparative PAGE. Two peaks of inhibin activity were identified: a predominantly bioactive 29-kDa peak I material (B:I ratio = 5.0) and a bio-inactive, immunoactive 27-kDa peak II material (B:I ratio = 0.1). Evidence of a subunit structure was established by iodination and immunopurification using an inhibin alpha-subunit antiserum. On reduction, peak I (29-kDa) material showed bands of 19 kDa and 14 kDa, whereas peak II (27-kDa) material showed a single 20-kDa band. On the basis of HPLC retention position, molecular mass, evidence of subunit structures and their molecular masses, and inhibin in vitro bio- and immunoactivities, peak I and II materials were most likely inhibin and the alpha-subunit precursor protein pro-alpha C. Western blotting of Sertoli cell culture medium using antiserum directed against the NH2 terminal region (alpha N) of the alpha-subunit precursor also indicated the presence of 24-kDa alpha N. It is concluded that after dbcAMP stimulation, Sertoli cells produce 29-kDa inhibin and the alpha-subunit precursor proteins pro-alpha C and alpha N. The production of the alpha-subunit precursor in addition to inhibin provides an explanation for the decrease in the inhibin B:I ratio following dbcAMP stimulation of Sertoli cells in culture.     

Transfection of N-Methyl-d-Aspartate Receptors in a Nonneuronal Cell Line Leads to Cell Death

Abstract: Neurons grown in culture die when they are exposed to high concentrations (0.1–1 m M ) of the neurotransmitter l -glutamate. A similar phenomenon may occur in the mammalian brain during ischemia and other injuries that cause excessive glutamate release. Activation of N -methyl- d -aspartate (NMDA) receptors and the consequent Ca²⁺ influx are thought to play a critical role in the process of neuronal toxicity. Events subsequent to the Ca²⁺ influx are not well understood. We have discovered that nonneuronal kidney cells expressing NMDA receptors after DNA transfection undergo cell death unless they are protected by drugs that block the NMDA receptor ion channel. Furthermore, transfected cells expressing a mutated NMDA receptor that conducts less Ca²⁺ are less vulnerable to cell death. In addition, we find that even though several active forms of NMDA receptors can be synthesized in these cells after transfection with different cloned subunits, not all receptor types are equally toxic. These experiments suggest that Ca²⁺ influx through NMDA channels may be toxic to nonneuronal cells and that the NMDA receptor expression may be the major neuron-specific component of excitotoxicity.     

Absence of PTHrP Nuclear Localization and Carboxyl Terminus Sequences Leads to Abnormal Brain Development and Function

We assessed whether the nuclear localization sequences (NLS) and C terminus of parathyroid hormone-related protein (PTHrP) play critical roles in brain development and function. We used histology, immunohistochemistry, histomorphometry, Western blots and electrophysiological recordings to compare the proliferation and differentiation of neural stem cells, neuronal hippocampal synaptic transmission, and brain phenotypes including shape and structures, in Pthrp knock-in mice, which express PTHrP (1-84), a truncated form of the protein that is missing the NLS and the C-terminal region of the protein, and their wild-type littermates. Results showed that Pthrp knock-in mice display abnormal brain shape and structures; decreased neural cell proliferative capacity and increased apoptosis associated with up-regulation of cyclin dependent kinase inhibitors p16, p21, p27 and p53 and down-regulation of the Bmi-1 oncogene; delayed neural cell differentiation; and impaired hippocampal synaptic transmission and plasticity. These findings provide in vivo experimental evidence that the NLS and C-terminus of PTHrP are essential not only for the regulation of neural cell proliferation and differentiation, but also for the maintenance of normal neuronal synaptic transmission and plasticity.