Proto-oncogene Sno expression, alternative isoforms and immediate early serum response.

The mouse Sno gene, a Ski proto-oncogene homolog, expresses two isoforms, SnoN and SnoN2 (also called sno -dE3), which differ from each other in a location downstream from the site of alternative splicing previously described in the human SNO gene. SnoN2 is missing a 138 nt coding segment present in mouse SnoN and human SNON . We have cloned and sequenced the human ortholog of mouse SnoN2 , the existence of which was predicted from conservation of the alternative splice donor site that produces the SnoN2 isoform. Mouse SnoN2 and SnoN are expressed throughout embryonic development, in neonatal muscle and in many adult tissues. SnoN2 is the major species in most tissues, but SnoN and SnoN2 are expressed at approximately equal levels in brain. In human tissues, SNON2 is the less abundantly expressed isoform. Expression of mouse SnoN and SnoN2 mRNAs is induced with immediate early kinetics upon serum stimulation of quiescent fibroblasts, even in the presence of the protein synthesis inhibitor cycloheximide, while Ski is not. Interestingly, although both isoforms of Sno are induced, SnoN2 induction is much higher than SnoN . These data are consistent with a role for Sno in the response to proliferation stimuli.     

Defective T-cell activation is associated with augmented transforming growth factor Beta sensitivity in mice with mutations in the Sno gene

The proto-oncogene Sno has been shown to be a negative regulator of transforming growth factor beta (TGF-beta) signaling in vitro, using overexpression and artificial reporter systems. To examine Sno function in vivo, we made two targeted deletions at the Sno locus: a 5' deletion, with reduced Sno protein (hypomorph), and an exon 1 deletion removing half the protein coding sequence, in which Sno protein is undetectable in homozygotes (null). Homozygous Sno hypomorph and null mutant mice are viable without gross developmental defects. We found that Sno mRNA is constitutively expressed in normal thymocytes and splenic T cells, with increased expression 1 h following T-cell receptor ligation. Although thymocyte and splenic T-cell populations appeared normal in mutant mice, T-cell proliferation in response to activating stimuli was defective in both mutant strains. This defect could be reversed by incubation with either anti-TGF-beta antibodies or exogenous interleukin-2 (IL-2). Together, these findings suggest that Sno-dependent suppression of TGF-beta signaling is required for upregulation of growth factor production and normal T-cell proliferation following receptor ligation. Indeed, both IL-2 and IL-4 levels are reduced in response to anti-CD3 epsilon stimulation of mutant T cells, and transfected Sno activated an IL-2 reporter system in non-T cells. Mutant mouse embryo fibroblasts also exhibited a reduced cell proliferation rate that could be reversed by administration of anti-TGF-beta. Our data provide strong evidence that Sno is a significant negative regulator of antiproliferative TGF-beta signaling in both T cells and other cell types in vivo.     

Beclin 1 deficiency is associated with increased hypoxia-induced angiogenesis

Beclin 1, a tumor suppressor protein, acts as an initiator of autophagy in mammals. Heterozygous disruption of Beclin 1 accelerates tumor growth, but the underlying mechanisms remain unclear. We examined the role of Beclin 1 in tumor proliferation and angiogenesis, using a primary mouse melanoma tumor model. Beclin 1 (Becn1 (+/-) ) hemizygous mice displayed an aggressive tumor growth phenotype with increased angiogenesis under hypoxia, associated with enhanced levels of circulating erythropoietin but not vascular endothelial growth factor, relative to wild-type mice. Using in vivo and ex vivo assays, we demonstrated increased angiogenic activity in Becn1 (+/-) mice relative to wild-type mice. Endothelial cells from Becn1 (+/-) mice displayed increased proliferation, migration and tube formation in response to hypoxia relative to wild-type cells. Moreover, Becn1 (+/-) cells subjected to hypoxia displayed increased hypoxia-inducible factor-2α (HIF-2α) expression relative to HIF-1α. Genetic interference of HIF-2α but not HIF-1α, dramatically reduced hypoxia-inducible proliferation, migration and tube formation in Becn1 (+/-) endothelial cells. We demonstrated that mice deficient in the autophagic protein Beclin 1 display a pro-angiogenic phenotype associated with the upregulation of HIF-2α and increased erythropoietin production. These results suggest a relationship between Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development.     

Mutation of Prkar1a causes osteoblast neoplasia driven by dysregulation of protein kinase A

Carney complex (CNC) is an autosomal dominant neoplasia syndrome caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A (PKA). This genetic defect induces skin pigmentation, endocrine tumors, myxomas, and schwannomas. Some patients with the complex also develop myxoid bone tumors termed osteochondromyxomas. To study the link between the PRKAR1A mutations and tumor formation, we generated a mouse model of this condition. Prkar1a(+/-) mice develop bone tumors with high frequency, although these lesions have not yet been characterized, either from human patients or from mice. Bone tumors from Prkar1a(+/-) mice were heterogeneous, including elements of myxomatous, cartilaginous, and bony differentiation that effaced the normal bone architecture. Immunohistochemical analysis identified an osteoblastic origin for the abnormal cells associated with islands of bone. To better understand these cells at the biochemical level, we isolated primary cultures of tumoral bone and compared them with cultures of bone from wild-type animals. The tumor cells exhibited the expected decrease in Prkar1a protein and exhibited increased PKA activity. At the phenotypic level, we observed that tumor cells behaved as incompletely differentiated osteoblasts and were able to form tumors in immunocompromised mice. Examination of gene expression revealed down-regulation of markers of bone differentiation and increased expression of locally acting growth factors, including members of the Wnt signaling pathway. Tumor cells exhibited enhanced growth in response to PKA-stimulating agents, suggesting that tumorigenesis in osteoblast precursor cells is driven by effects directly mediated by the dysregulation of PKA.     

p18Ink4c, but not p27Kip1, collaborates with Men1 to suppress neuroendocrine organ tumors

Mutant mice lacking both cyclin-dependent kinase (CDK) inhibitors p18(Ink4c) and p27(Kip1) develop a tumor spectrum reminiscent of human multiple endocrine neoplasia (MEN) syndromes. To determine how p18 and p27 genetically interact with Men1, the tumor suppressor gene mutated in familial MEN1, we characterized p18-Men1 and p27-Men1 double mutant mice. Compared with their corresponding single mutant littermates, the p18(-/-); Men1(+/-) mice develop tumors at an accelerated rate and with an increased incidence in the pituitary, thyroid, parathyroid, and pancreas. In the pituitary and pancreatic islets, phosphorylation of the retinoblastoma (Rb) protein at both CDK2 and CDK4/6 sites was increased in p18(-/-) and Men1(+/-) cells and was further increased in p18(-/-); Men1(+/-) cells. The remaining wild-type Men1 allele was lost in most tumors from Men1(+/-) mice but was retained in most tumors from p18(-/-); Men1(+/-) mice. Combined mutations of p27(-/-) and Men1(+/-), in contrast, did not exhibit noticeable synergistic stimulation of Rb kinase activity, cell proliferation, and tumor growth. These results demonstrate that functional collaboration exists between p18 and Men1 and suggest that Men1 may regulate additional factor(s) that interact with p18 and p27 differently.     

Involvement of p38alpha mitogen-activated protein kinase in lung metastasis of tumor cells

To study the role of p38 mitogen-activated protein kinase (p38) activity during the process of metastasis, p38alpha(+/-) mice were subjected to an in vivo metastasis assay. The number of lung colonies of tumor cells intravenously injected in p38alpha(+/-) mice was markedly decreased compared with that in wild-type (WT) mice. On the other hand, the time-dependent increase in tumor volume after subcutaneous tumor cells transplantation was comparable between WT and p38alpha(+/-) mice. Platelets of p38alpha(+/-) mice were poorly bound to tumor cells in vitro and in vivo compared with those of WT mice. E- and P-selectin mRNAs were markedly induced in the lung after intravenous injection of tumor cells. However, the induction of these selectin mRNAs in p38alpha(+/-) mice was weaker than that in WT mice. Furthermore, the resting expression levels of E-selectin in lung endothelial cells and P-selectin in platelets of p38alpha(+/-) mice were suppressed compared with those of WT mice. The number of tumor cells attached on lung endothelial cells of p38alpha(+/-) mice was significantly reduced compared with that of WT mice. The transmigrating activity of tumor cells through lung endothelial cells of p38alpha(+/-) mice was similar to that of WT mice. These results suggest that p38alpha plays an important role in extravasation of tumor cells, possibly through regulating the formation of tumor-platelet aggregates and their interaction with the endothelium involved in a step of hematogenous metastasis.     

The role of Dpp and its inhibitors during eggshell patterning in Drosophila

The Drosophila eggshell is patterned by the combined action of the epidermal growth factor [EGF; Gurken (Grk)] and transforming growth factor beta [TGF-beta; Decapentaplegic (Dpp)] signaling cascades. Although Grk signaling alone can induce asymmetric gene expression within the follicular epithelium, here we show that the ability of Grk to induce dorsoventral polarity within the eggshell strictly depends on Dpp. Dpp, however, specifies at least one anterior region of the eggshell in the absence of Grk. Dpp forms an anteriorposterior morphogen gradient within the follicular epithelium and synergizes with the dorsoventral gradient of Grk signaling. High levels of Grk and Dpp signaling induce the operculum, whereas lower levels of both pathways induce the dorsal appendages. We provide evidence that the crosstalk between both pathways occurs at least at two levels. First, Dpp appears to directly enhance the levels of EGF pathway activity within the follicular epithelium. Second, Dpp and EGF signaling collaborate in controlling the expression of Dpp inhibitors. One of these inhibitors is Drosophila sno (dSno), a homolog of the Ski/Sno family of vertebrate proto-oncogenes, which synergizes with daughters against dpp and brinker to set the posterior and lateral limits of the region, giving rise to dorsal follicle cells.     

Embryonic fibroblasts from Gpx4+/- mice: a novel model for studying the role of membrane peroxidation in biological processes

A previous study using mice null for Gpx4 indicates that PHGPx plays a critical role in antioxidant defense and is essential for the survival of the mouse. In the present study, we further analyzed the stress response of MEFs (murine embryonic fibroblasts) derived from mice heterozygous for the Gpx4 gene (Gpx4(+/-) mice). MEFs from Gpx4(+/-) mice have a 50% reduction in PHGPx expression without any changes in the activities of other major antioxidant defense enzymes. Compared to MEFs from Gpx4(+/+) mice, MEFs from Gpx4(+/-) mice were more sensitive to exposure to the oxidizing agent t-butyl hydroperoxide (t-BuOOH), and t-BuOOH exposure induced increased apoptosis in MEFs from Gpx4(+/-) mice. When cultured at low cell density, MEFs from Gpx4(+/-) mice also showed retarded growth under normal culture conditions (20% oxygen) that was reversed by culturing under low oxygen (2% oxygen). In addition, oxidative damage was increased in the MEFs from the Gpx4(+/-) mice, as indicated by increased levels of F(2)-isoprostanes and 8-oxo-2-deoxyguanosine in these cells. Our data demonstrate that MEFs from Gpx4(+/-) mice are more sensitive to oxidative stress because of reduced expression of PHGPx.     

Hepatic granulomas induced by Schistosoma mansoni in mice deficient for connexin 43 present lower cell proliferation and higher collagen content

Granuloma formation involves a coordinated interaction between monocytes and macrophages, epithelioid cells, lymphocytes, eosinophils, neutrophils and fibroblasts. It has been established that extracellular communication via cytokines is important for the assembly of granulomas. However, the importance of gap junctions and intercellular communication to granuloma formation and development had never been assessed. Connexins are proteins that form gap junctions, and connexin 43 (Cx43) is present in macrophages, lymphoid cells, myelogenous cells, fibroblasts and others. We analyzed the effect of heterologous deletion of Gja1 (Cx43 gene) on the formation and development of hepatic granulomas induced by Schistosoma mansoni eggs. Heterozygous (Cx43(+/-)) and wild-type (Cx43(+/+)) mice were infected subcutaneously with S. mansoni cercarie and evaluated after 6, 8 and 12 weeks. Granuloma cells express Cx43, as revealed by real-time PCR in isolated granulomas, and by immunohistochemistry. Cx43 expression was reduced in Cx43(+/-) mice, as expected. No differences in the average area of granulomas or number of cells per granuloma were observed between mice of different genotypes. However, granuloma cells from Cx43(+/-) mice displayed a reduced index of the proliferating cell nuclear antigen (PCNA) labeling at 8 and 12 weeks post-infection. Moreover, Cx43(+/-) granulomas unexpectedly presented a higher degree of fibrosis, quantified by morphometric analysis in Sirius Red-stained slides. Our results indicate that the deletion of one allele of the Cx43 gene, and possibly the reduced gap junction intercellular communication capacity (GJIC), may impair the interactions between granuloma cells, reducing their proliferation and increasing their collagen content, thereby modifying the characteristics of S. mansoni granuloma in mice.     

The yeast antiviral proteins Ski2p, Ski3p, and Ski8p exist as a complex in vivo

The yeast superkiller (SKI) genes were originally identified from mutations allowing increased production of killer toxin encoded by M "killer" virus, a satellite of the dsRNA virus L-A. XRN1 (SKI1) encodes a cytoplasmic 5'-exoribonuclease responsible for the majority of cytoplasmic RNA turnover, whereas SKI2, SKI3, and SKI8 are required for normal 3'-degradation of mRNA and for repression of translation of poly(A) minus RNA. Ski2p is a putative RNA helicase, Ski3p is a tetratricopeptide repeat (TPR) protein, and Ski8p contains five WD-40 (beta-transducin) repeats. An xrn1 mutation in combination with a ski2, ski3, or ski8 mutation is lethal, suggesting redundancy of function. Using functional epitope-tagged Ski2, Ski3, and Ski8 proteins, we show that Ski2p, Ski3p, and Ski8p can be coimmunoprecipitated as an apparent heterotrimeric complex. With epitope-tagged Ski2p, there was a 1:1:1 stoichiometry of the proteins in the complex. Ski2p did not associate with Ski3p in the absence of Ski8p, nor did Ski2p associate with Ski8p in the absence of Ski3p. However, the Ski3p/Ski8p interaction did not require Ski2p. In addition, ski6-2 or ski4-1 mutations or deletion of SKI7 did not affect complex formation. The identification of a complex composed of Ski2p, Ski3p, and Ski8p explains previous results showing phenotypic similarity between mutations in SKI2, SKI3, and SKI8. Indirect immunofluorescence of Ski3p and subcellular fractionation of Ski2p and Ski3p suggest that Ski2p and Ski3p are cytoplasmic. These data support the idea that Ski2p, Ski3p, and Ski8p function in the cytoplasm in a 3'-mRNA degradation pathway.     

Mgat5 and Pten interact to regulate cell growth and polarity

Phosphatase and tensin homolog (Pten) phosphatase opposes intracellular phosphoinositide 3-kinase (PI3K)/Akt signaling and is a potent tumor suppressor, while Golgi beta1,6 N-acetylglucosaminyltransferase V (Mgat5) is positively associated with cancer progression and metastasis. beta1,6GlcNAc-branched N-glycans on receptor glycoproteins promote their surface residency and sensitizes cells to growth factor signaling. Here we demonstrate that the Pten heterozygosity in mouse embryonic fibroblasts enhances cell adhesion-dependent PI3K/Akt signaling, cell spreading, and proliferation, while Pten/Mgat5 double mutant cells are normalized. However, planar asymmetry typical of fibroblasts and invasive carcinomas is not fully rescued, suggesting that Mgat5 and Pten function together to regulate the membrane dynamics of PI3K/Akt signaling typical of motile cells. Pten heterozygosity was associated with increased surface beta1,6GlcNAc-branched N-glycans, suggesting positive feedback from PI3K signaling to N-glycan branching. In vivo, Mgat5(-/-) Pten(+/-) and Mgat5(+/-)Pten(+/-)mutant mice showed a small but significant increase in longevity compared with Pten(+/-) mice. Taken together, our results reveal that Mgat5 and Pten interact in an opposing manner to regulate cellular sensitivities to extracelluar growth cues.     

Function of GRIM-19, a Mitochondrial Respiratory Chain Complex I Protein, in Innate Immunity

Mitochondria respiratory chain (RC), consisting of five multisubunit complexes, is crucial for cellular energy production, reactive oxygen species generation, and regulation of apoptosis. Recently, a few mitochondrial proteins have been reported to be essential for innate immunity, but the function of mitochondrial RC in innate immunity is largely unknown. By knock-out of GRIM-19, a newly identified subunit protein of mitochondrial complex I, in mice, we found that heterogeneous mice (GRIM-19(+/-)) are prone to spontaneous urinary tract infection, mostly by Staphylococcus saprophyticus. Macrophages derived from these mice have compromised mitochondrial complex I activity and increased reactive oxygen species level. Bacterial infection induces a rapid up-regulation of GRIM-19 and complex I activity in the wild-type macrophages, but both are reduced in the macrophages from GRIM-19(+/-) mice. These cells also have decreased intracellular killing ability against S. saprophyticus. The defects for this probably occur in the fusion of bacteria to lysosome, but not in the bacterial engulfment and macrophage migration. In addition, production of proinflammatory cytokines, such as interleukin (IL)-1, IL-12, IL-6, and interferon (IFN)-γ, induced by both bacterial infection and lipopolysaccharide (LPS) and monodansylcadaverine treatment, is also decreased in the GRIM19(+/-) macrophages. Inhibition of mitochondrial RC activity by inhibitors shows a similar reduction on the cytokine production. Due to low cytokine production, the inflammatory response caused by in vivo bacterial challenge in the bladders of GRIM-19(+/-) mice is compromised. This study provides genetic evidence for a critical role of mitochondrial RC in innate immunity.     

The mitochondrial SDHD gene is required for early embryogenesis, and its partial deficiency results in persistent carotid body glomus cell activation with full responsiveness to hypoxia

The SDHD gene encodes one of the two membrane-anchoring proteins of the succinate dehydrogenase (complex II) of the mitochondrial electron transport chain. This gene has recently been proposed to be involved in oxygen sensing because mutations that cause loss of its function produce hereditary familiar paraganglioma, a tumor of the carotid body (CB), the main arterial chemoreceptor that senses oxygen levels in the blood. Here, we report the generation of a SDHD knockout mouse, which to our knowledge is the first mammalian model lacking a protein of the electron transport chain. Homozygous SDHD(-/-) animals die at early embryonic stages. Heterozygous SDHD(+/-) mice show a general, noncompensated deficiency of succinate dehydrogenase activity without alterations in body weight or major physiological dysfunction. The responsiveness to hypoxia of CBs from SDHD(+/-) mice remains intact, although the loss of an SDHD allele results in abnormal enhancement of resting CB activity due to a decrease of K(+) conductance and persistent Ca(2+) influx into glomus cells. This CB overactivity is linked to a subtle glomus cell hypertrophy and hyperplasia. These observations indicate that constitutive activation of SDHD(+/-) glomus cells precedes CB tumor transformation. They also suggest that, contrary to previous beliefs, mitochondrial complex II is not directly involved in CB oxygen sensing.