Anti-apoptotic Molecule Bcl-2 Regulates the Differentiation, Activation, and Survival of Both Osteoblasts and Osteoclasts

The anti-apoptotic molecule Bcl-2 inhibits apoptosis by preventing cytochrome c release from mitochondria. Although several studies have indicated the importance of Bcl-2 in maintaining skeletal integrity, the detailed cellular and molecular mechanisms remain elusive. Bcl-2^−/− mice are growth-retarded and exhibit increased bone volume of the primary spongiosa, mainly due to the decreased number and dysfunction of osteoclasts. Osteoblast function is also impaired in Bcl-2^−/− mice. Ex vivo studies on osteoblasts and osteoclasts showed that Bcl-2 promoted the differentiation, activation, and survival of both cell types. Because Bcl-2^−/− mice die before 6 weeks of age due to renal failure and cannot be compared with adult wild type mice, we generated Bcl-2^−/−Bim^+/− mice, in which a single Bim allele was inactivated, and compared them with their Bcl-2^+/−Bim^+/− littermates. Loss of a single Bim allele restored normal osteoclast function in Bcl-2^−/− mice but did not restore the impaired function of osteoblasts, and the mice exhibited osteopenia. These data demonstrate that Bcl-2 promotes the differentiation, activity, and survival of both osteoblasts and osteoclasts. The balance between Bcl-2 and Bim regulates osteoclast apoptosis and function, whereas other pro-apoptotic members are important for osteoblasts.     

Absence of Bim sensitizes mice to experimental Trypanosoma cruzi infection

Chagas disease is a life-threatening disorder caused by the protozoan parasite Trypanosoma cruzi. Parasite-specific antibodies, CD8⁺ T cells, as well as IFN-γ and nitric oxide (NO) are key elements of the adaptive and innate immunity against the extracellular and intracellular forms of the parasite. Bim is a potent pro-apoptotic member of the Bcl-2 family implicated in different aspects of the immune regulation, such as negative selection of self-reactive thymocytes and elimination of antigen-specific T cells at the end of an immune response. Interestingly, the role of Bim during infections remains largely unidentified. To explore the role of Bim in Chagas disease, we infected WT, Bim^+/−, Bim^−/− mice with trypomastigotes forms of the Y strain of T. cruzi. Strikingly, our data revealed that Bim^−/− mice exhibit a delay in the development of parasitemia followed by a deficiency in the control of parasite load in the bloodstream and a decreased survival compared to WT and Bim^+/− mice. At the peak of parasitemia, peritoneal macrophages of Bim^−/− mice exhibit decreased NO production, which correlated with a decrease in the pro-inflammatory Small Peritoneal Macrophage (SPM) subset. A similar reduction in NO secretion, as well as in the pro-inflammatory cytokines IFN-γ and IL-6, was also observed in Bim^−/− splenocytes. Moreover, an impaired anti-T. cruzi CD8⁺ T-cell response was found in Bim^−/− mice at this time point. Taken together, our results suggest that these alterations may contribute to the establishment of a delayed yet enlarged parasitic load observed at day 9 after infection of Bim^−/− mice and place Bim as an important protein in the control of T. cruzi infections.Subject terms: Cell death and immune response, Infectious diseases     

Preferential control of induced regulatory T cell homeostasis via a Bim/Bcl-2 axis

Apoptosis has an essential role in controlling T cell homeostasis, especially during the contraction phase of an immune response. However, its contribution to the balance between effector and regulatory populations remains unclear. We found that Rag1^−/− hosts repopulated with Bim^−/− conventional CD4⁺ T cells (Tconv) resulted in a larger induced regulatory T cell (iTreg) population than mice given wild-type (WT) Tconv. This appears to be due to an increased survival advantage of iTregs compared with activated Tconv in the absence of Bim. Downregulation of Bcl-2 expression and upregulation of Bim expression were more dramatic in WT iTregs than activated Tconv in the absence of IL-2 in vitro. The iTregs generated following Tconv reconstitution of Rag1^−/− hosts exhibited lower Bcl-2 expression and higher Bim/Bcl-2 ratio than Tconv, which indicates that iTregs were in an apoptosis-prone state in vivo. A significant proportion of the peripheral iTreg pool exhibits low Bcl-2 expression indicating increased sensitivity to apoptosis, which may be a general characteristic of certain Treg subpopulations. In summary, our data suggest that iTregs and Tconv differ in their sensitivity to apoptotic stimuli due to their altered ratio of Bim/Bcl-2 expression. Modulating the apoptosis pathway may provide novel therapeutic approaches to alter the balance between effector T cells and Tregs.     

Loss of BH3-only Protein Bim Inhibits Apoptosis of Hemopoietic Cells in the Fetal Liver and Male Germ Cells but Not Neuronal Cells in Bcl-x�Cdeficient Mice

Members of the Bcl-2 family include pro- and antiapoptotic proteins that regulate programmed cell death of developing tissues and death in response to cellular damage. In developing mice, the antiapoptotic Bcl-x_L is necessary for survival of neural and hematopoietic cells, and consequently, bcl-x–deficient mice die around Day 13.5 of embryogenesis. Furthermore, adult bcl-x^+/− heterozygous male mice have reduced fertility because of testicular degeneration. Bax, a multi-BH (Bcl-2 homology) domain proapoptotic member of the Bcl-2 family, is regulated by Bcl-x_L and is required for the neuropathological abnormalities seen in bcl-x–deficient embryos. The BH3 domain only subgroup of the Bcl-2 family includes proapoptotic members that are essential for the initiation of apoptotic signaling. In this study, we investigated the role for Bim, a BH3 domain only protein, in the embryonic lethality and increased developmental cell death in bcl-x–deficient animals and the perturbed testicular function in bcl-x^+/− adults. Our studies show that bim deficiency attenuates hematopoietic cell death in the fetal liver of bcl-x–deficient animals, indicating that Bim contributes to programmed cell death in this cell population. In addition, we found that testicular degeneration of adult bcl-x^+/− males was rescued by concomitant Bim deficiency. However, concomitant Bim deficiency had no effect on the embryonic lethality and widespread nervous system abnormalities caused by bcl-x deficiency. Our work identifies Bim as an important regulator of bcl-x deficiency–induced cell death during hematopoiesis and testicular development. (J Histochem Cytochem 56:921–927, 2008)     

Insulin receptor substrate 2 (IRS2)-deficient mice show sensorineural hearing loss that is delayed by concomitant protein tyrosine phosphatase 1B (PTP1B) loss of function

The insulin receptor substrate (IRS) proteins are key mediators of insulin and insulinlike growth factor 1 (IGF-1) signaling. Protein tyrosine phosphatase (PTP)-1B dephosphorylates and inactivates both insulin and IGF-1 receptors. IRS2-deficient mice present altered hepatic insulin signaling and β-cell failure and develop type 2–like diabetes. In addition, IRS2 deficiency leads to developmental defects in the nervous system. IGF1 gene mutations cause syndromic sensorineural hearing loss in humans and mice. However, the involvement of IRS2 and PTP1B, two IGF-1 downstream signaling mediators, in hearing onset and loss has not been studied. Our objective was to study the hearing function and cochlear morphology of Irs2-null mice and the impact of PTP1B deficiency. We have studied the auditory brainstem responses and the cochlear morphology of systemic Irs2^−/−Ptpn1^+/+, Irs2^+/+Ptpn1^−/−and Irs2^−/−Ptpn1^−/− mice at different postnatal ages. The results indicated that Irs2^−/−Ptpn1^+/+ mice present a profound congenital sensorineural deafness before the onset of diabetes and altered cochlear morphology with hypoinnervation of the cochlear ganglion and aberrant stria vascularis, compared with wild-type mice. Simultaneous PTP1B deficiency in Irs2^−/−Ptpn1^−/− mice delays the onset of deafness. We show for the first time that IRS2 is essential for hearing and that PTP1B inhibition may be useful for treating deafness associated with hyperglycemia and type 2 diabetes.     

Incudomalleal joint formation: the roles of apoptosis,migration and downregulation

Background  

The middle ear of mammals is composed of three endochondrial ossicles, the stapes, incus and malleus. Joints link the malleus to the incus and the incus to the stapes. In the mouse the first arch derived malleus and incus are formed from a single Sox9 and Type II collagen expressing condensation that later subdivides to give rise to two separate ossicles. In contrast the stapes forms from a separate condensation derived from the second branchial arch. Fusion of the malleus and incus is observed in a number of human syndromes and results in conductive hearing loss. Understanding how this joint forms during normal development is thus an important step in furthering our understanding of such defects.     

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH ^−/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH ^−/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH ^−/− iPS cell lines, we aggregated FAH ^−/−-iPS cells with tetraploid embryos and obtained entirely FAH ^−/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH ^−/− mice. Then, we transduced FAH cDNA into the FAH ^−/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models.     

Altered functional differentiation of mesoangioblasts in a genetic myopathy

Mutations underlying genetic cardiomyopathies might affect differentiation commitment of resident progenitor cells. Cardiac mesoangioblasts (cMabs) are multipotent progenitor cells resident in the myocardium. A switch from cardiac to skeletal muscle differentiation has been recently described in cMabs from β-sarcoglycan-null mice (βSG^−/−), a murine model of genetic myopathy with early myocardial involvement. Although complementation with βSG gene was inconsequential, knock-in of miRNA669a (missing in βSG^−/− cMabs) partially rescued the mutation-induced molecular phenotype. Here, we undertook a detailed evaluation of functional differentiation of βSG^−/− cMabs and tested the effects of miRNA669a-induced rescue in vitro. To this end, cMabs were compared with neonatal cardiomyocytes (CMs) and skeletal muscle C2C12 cells, representative of cardiac and skeletal muscle respectively. Consistent with previous data on molecular patterns, electrophysiological and Ca²⁺-handling properties of βSG^−/− cMabs were closer to C2C12 cells than to CM ones. Nevertheless, subtler aspects, including action potential contour, Ca²⁺-spark properties and RyR isoform expression, distinguished βSG^−/− cMabs from C2C12 cells. Contrary to previous reports, wild-type cMabs failed to show functional differentiation towards either cell type. Knock-in of miRNA669a in βSG^−/− cMabs rescued the wild-type functional phenotype, i.e. it completely prevented development of skeletal muscle functional responses. We conclude that miRNA669a expression, ablated by βSG deletion, may prevent functional differentiation of cMabs towards the skeletal muscle phenotype.     

Pro-apoptotic Bax is the major and Bak an auxiliary effector in cytokine deprivation-induced mast cell apoptosis

The process of apoptosis in immune cells like mast cells is essential to regain homeostasis after an inflammatory response. The intrinsic pathway of apoptosis is ultimately controlled by the pro-apoptotic Bcl-2 family members Bax and Bak, which upon activation oligomerize to cause increased permeabilization of the mitochondria outer membrane leading to cell death. We examined the role of Bax and Bak in cytokine deprivation-induced apoptosis in mast cells using connective tissue-like mast cells and mucosal-like mast cells derived from bax^−/−, bak^−/− and bax^−/−bak^−/− mice. Although both Bax and Bak were expressed at readily detectable protein levels, we found a major role for Bax in mediating mast cell apoptosis induced by cytokine deprivation. We analyzed cell viability by propidium iodide exclusion and flow cytometry after deprivation of vital cytokines for each mast cell population. Upon cytokine withdrawal, bak^−/− mast cells died at a similar rate as wild type, whereas bax^−/− and bax^−/−bak^−/− mast cells were partially or completely resistant to apoptosis, respectively. The total resistance seen in bax^−/−bak^−/− mast cells is comparable with mast cells deficient of both pro-apoptotic Bim and Puma or mast cells overexpressing anti-apoptotic Bcl-2. These results show that Bax has a predominant and Bak a minor role in cytokine deprivation-induced apoptosis in both connective tissue-like and mucosal-like mast cells.     

Tcrδ translocations that delete the Bcl11b haploinsufficient tumor suppressor gene promote atm-deficient T cell acute lymphoblastic leukemia

ATM is the master regulator of the cellular response to DNA double strand breaks (DSBs). Deficiency of ATM predisposes humans and mice to αβ T lymphoid cancers with clonal translocations between the T cell receptor (TCR) α/δ locus and a 450 kb region of synteny on human chromosome 14 and mouse chromosome 12. While these translocations target and activate the TCL1 oncogene at 14q32 to cause T cell pro-lymphocytic leukemia (T-PLL), the TCRα/δ;14q32 translocations in ATM-deficient T cell acute lymphoblastic leukemia (T-ALL) have not been characterized and their role in cancer pathogenesis remains unknown. The corresponding lesion in Atm-deficient mouse T-ALLs is a chromosome t(12;14) translocation with Tcrδ genes fused to sequences on chromosome 12; although these translocations do not activate Tcl1, they delete the Bcl11b haploinsufficient tumor suppressor gene. To assess whether Tcrδ translocations that inactivate one copy of Bcl11b promote transformation of Atm-deficient cells, we analyzed Atm^−/− mice with mono-allelic Bcl11b deletion initiating in thymocytes concomitant with Tcrδ recombination. Inactivation of one Bcl11b copy had no effect on the predisposition of Atm^−/− mice to clonal T-ALLs. Yet, none of these T-ALLs had a clonal chromosome t(12;14) translocation that deleted Bcl11b indicating that Tcrδ translocations that inactivate a copy of Bcl11b promote transformation of Atm-deficient thymocytes. Our data demonstrate that antigen receptor locus translocations can cause cancer by deleting a tumor suppressor gene. We discuss the implications of these findings for the etiology and therapy of T-ALLs associated with ATM deficiency and TCRα/δ translocations targeting the 14q32 cytogenetic region.     

Correction: Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity

The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a^−/−Myo3b^−/− mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b^−/− mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a^−/−Myo3b^−/− cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a^−/−Myo3b^−/− stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.     

2��-O Methylation of the Viral mRNA Cap by West Nile Virus Evades Ifit1-Dependent and -Independent Mechanisms of Host Restriction In Vivo

Prior studies have shown that 2′-O methyltransferase activity of flaviviruses, coronaviruses, and poxviruses promotes viral evasion of Ifit1, an interferon-stimulated innate immune effector protein. Viruses lacking 2′-O methyltransferase activity exhibited attenuation in primary macrophages that was rescued in cells lacking Ifit1 gene expression. Here, we examined the role of Ifit1 in restricting pathogenesis in vivo of wild type WNV (WNV-WT) and a mutant in the NS5 gene (WNV-E218A) lacking 2′-O methylation of the 5′ viral RNA cap. While deletion of Ifit1 had marginal effects on WNV-WT pathogenesis, WNV-E218A showed increased replication in peripheral tissues of Ifit1 ^−/− mice after subcutaneous infection, yet this failed to correlate with enhanced infection in the brain or lethality. In comparison, WNV-E218A was virulent after intracranial infection as judged by increased infection in different regions of the central nervous system (CNS) and a greater than 16,000-fold decrease in LD₅₀ values in Ifit1 ^−/− compared to wild type mice. Ex vivo infection experiments revealed cell-type specific differences in the ability of an Ifit1 deficiency to complement the replication defect of WNV-E218A. In particular, WNV-E218A infection was impaired in both wild type and Ifit1 ^−/− brain microvascular endothelial cells, which are believed to participate in blood-brain barrier (BBB) regulation of virus entry into the CNS. A deficiency of Ifit1 also was associated with increased neuronal death in vivo, which was both cell-intrinsic and mediated by immunopathogenic CD8⁺ T cells. Our results suggest that virulent strains of WNV have largely evaded the antiviral effects of Ifit1, and viral mutants lacking 2′-O methylation are controlled in vivo by Ifit1-dependent and -independent mechanisms in different cell types.     

Survival and differentiation defects contribute to neutropenia in glucose-6-phosphatase-β (G6PC3) deficiency in a model of mouse neutrophil granulocyte differentiation

Differentiation of neutrophil granulocytes (neutrophils) occurs through several steps in the bone marrow and requires a coordinate regulation of factors determining survival and lineage-specific development. A number of genes are known whose deficiency disrupts neutrophil generation in humans and in mice. One of the proteins encoded by these genes, glucose-6-phosphatase-β (G6PC3), is involved in glucose metabolism. G6PC3 deficiency causes neutropenia in humans and in mice, linked to enhanced apoptosis and ER stress. We used a model of conditional Hoxb8 expression to test molecular and functional differentiation as well as survival defects in neutrophils from G6PC3^−/− mice. Progenitor lines were established and differentiated into neutrophils when Hoxb8 was turned off. G6PC3^−/− progenitor cells underwent substantial apoptosis when differentiation was started. Transgenic expression of Bcl-X_L rescued survival; however, Bcl-X_L-protected differentiated cells showed reduced proliferation, immaturity and functional deficiency such as altered MAP kinase signaling and reduced cytokine secretion. Impaired glucose utilization was found and was associated with ER stress and apoptosis, associated with the upregulation of Bim and Bax; downregulation of Bim protected against apoptosis during differentiation. ER-stress further caused a profound loss of expression and secretion of the main neutrophil product neutrophil elastase during differentiation. Transplantation of wild-type Hoxb8-progenitor cells into irradiated mice allowed differentiation into neutrophils in the bone marrow in vivo. Transplantation of G6PC3^−/− cells yielded few mature neutrophils in bone marrow and peripheral blood. Transgenic Bcl-X_L permitted differentiation of G6PC3^−/− cells in vivo. However, functional deficiencies and differentiation abnormalities remained. Differentiation of macrophages from Hoxb8-dependent progenitors was only slightly disturbed. A combination of defects in differentiation and survival thus underlies neutropenia in G6PC3^−/− deficiency, both originating from a reduced ability to utilize glucose. Hoxb8-dependent cells are a model to study differentiation and survival of the neutrophil lineage.