Lidia Hernandez

A progeroid syndrome in mice is caused by defects in A-type lamins.

Numerous studies of the underlying causes of ageing have been attempted by examining diseases associated with premature ageing, such as Werners syndrome and Hutchinson–Gilford progeria syndrome (HGPS). HGPS is a rare genetic disorder resulting in phenotypes suggestive of accelerated ageing, including shortened stature, craniofacial disproportion, very thin skin, alopecia and osteoporosis, with death in the early teens predominantly due to atherosclerosis. However, recent reports suggest that developmental abnormalities may also be important in HGPS. Here we describe the derivation of mice carrying an autosomal recessive mutation in the lamin A gene (Lmna) encoding A-type lamins, major components of the nuclear lamina. Homozygous mice display defects consistent with HGPS, including a marked reduction in growth rate and death by 4 weeks of age. Pathologies in bone, muscle and skin are also consistent with progeria. The Lmna mutation resulted in nuclear morphology defects and decreased lifespan of homozygous fibroblasts, suggesting premature cell death. Here we present a mouse model for progeria that may elucidate mechanisms of ageing and development in certain tissue types, especially those developing from the mesenchymal cell lineage.

Disruption of the mouse necdin gene results in early post-natal lethality.

Prader-Willi syndrome (PWS) is a neurobehavioural disorder characterized by neonatal respiratory depression, hypotonia and failure to thrive in infancy, followed by hyperphagia and obesity among other symptoms. PWS is caused by the loss of one or more paternally expressed genes on chromosome 15q11–q13, which can be due to gene deletions, maternal uniparental disomy or mutations disrupting the imprinting mechanism. Imprinted genes mapped to this region include SNRPN (refs 3 ,4), ZNF127 ( ref. 5), IPW (ref. 6) and NDN (which encodes the DNA-binding protein necdin; refs 7,8,9,10). The mouse homologues of these genes map to mouse chromosome 7 in a region syntenic with human chromosome 15q11–q13 (refs 7,11). Imprinting of the human genes is under the control of an imprinting center (IC), a long-range, cis-acting element located in the 5′ region of SNRPN (ref. 12). A related control element was isolated in the mouse Snrpn genomic region which, when deleted on the paternally inherited chromosome, resulted in the loss of expression of all four genes and early post-natal lethality. To determine the possible contribution of Ndn to the PWS phenotype, we generated Ndn mutant mice. Heterozygous mice inheriting the mutated maternal allele were indistinguishable from their wild-type littermates. Mice carrying a paternally inherited Ndn deletion allele demonstrated early post-natal lethality. This is the first example of a single gene being responsible for phenotypes associated with PWS.

Zac1 (Lot1), a Potential Tumor Suppressor Gene, and the Gene for ɛ-Sarcoglycan Are Maternally Imprinted Genes: Identification by a Subtractive Screen of Novel Uniparental Fibroblast Lines

ABSTRACT Imprinted genes are expressed from one allele according to their parent of origin, and many are essential to mammalian embryogenesis. Here we show that the ɛ-sarcoglycan gene (Sgce) andZac1 (Lot1) are both paternally expressed imprinted genes. They were identified in a subtractive screen for imprinted genes using a cDNA library made from novel parthenogenetic and wild-type fibroblast lines. Sgce is a component of the dystrophin-sarcoglycan complex, Zac1 is a nuclear protein inducing growth arrest and/or apoptosis, and Zac1 is a potential tumor suppressor gene. Sgce and Zac1 are expressed predominantly from their paternal alleles in all adult mouse tissues, except that Zac1 is biallelic in the liver andSgce is weakly expressed from the maternal allele in the brain. Sgce and Zac1 are broadly expressed in embryos, with Zac1 being highly expressed in the liver primordium, the umbilical region, and the neural tube.Sgce, however, is strongly expressed in the allantoic region on day 9.5 but becomes more widely expressed throughout the embryo by day 11.5. Sgce is located at the proximal end of mouse chromosome 6 and is a candidate gene for embryonic lethality associated with uniparental maternal inheritance of this region.Zac1 maps to the proximal region of chromosome 10, identifying a new imprinted locus in the mouse, homologous with human chromosome 6q24-q25. In humans, unipaternal disomy for this region is associated with fetal growth retardation and transient neonatal diabetes mellitus. In addition, loss of expression of ZAChas been described for a number of breast and ovarian carcinomas, suggesting that ZAC is a potential tumor suppressor gene.

Dual control of LIF expression and LIF receptor function regulate Stat3 activation at the onset of uterine receptivity and embryo implantation

Leukemia inhibitory factor (LIF) expression in the uterus is essential for embryo implantation in mice. Here we describe the spatial and temporal regulation of LIF signaling in vivo by using tissues isolated from uteri on different days over the implantation period. During this time, LIF receptors are expressed predominantly in the luminal epithelium (LE) of the uterus. Isolated epithelium responds to LIF by phosphorylation and nuclear translocation of signal transducer and activator of transcription (Stat) 3, but not by an increase in mitogen-activated protein kinase levels. The related cytokines Il-6, ciliary neurotrophic factor, as well as epidermal growth factor, do not activate Stat3, although epidermal growth factor stimulates mitogen-activated protein kinase. In vivo Stat3 activation is induced by LIF alone, resulting in the localization of Stat3 specifically to the nuclei of the LE coinciding with the onset of uterine receptivity. The responsiveness of the LE to LIF is regulated temporally, with Stat activation being restricted to day 4 of pregnancy despite the presence of constant levels of LIF receptor throughout the preimplantation period. Uterine receptivity is therefore under dual control and is regulated by both the onset of LIF expression in the endometrial glands and the release from inhibition of receptor function in the LE.

The lipid phosphatase LPP3 regulates extra-embryonic vasculogenesis and axis patterning

Bioactive phospholipids, which include sphingosine-1-phosphate, lysophosphatidic acid, ceramide and their derivatives regulate a wide variety of cellular functions in culture such as proliferation, apoptosis and differentiation. The availability of these lipids and their products is regulated by the lipid phosphate phosphatases (LPPs). Here we show that mouse embryos deficient for LPP3 fail to form a chorio-allantoic placenta and yolk sac vasculature. A subset of embryos also show a shortening of the anterior-posterior axis and frequent duplication of axial structures that are strikingly similar to the phenotypes associated with axin deficiency, a critical regulator of Wnt signaling. Loss of LPP3 results in a marked increase in β-catenin-mediated TCF transcription, whereas elevated levels of LPP3 inhibit β-catenin-mediated TCF transcription. LPP3 also inhibits axis duplication and leads to mild ventralization in Xenopus embryo development. Although LPP3 null fibroblasts show altered levels of bioactive phospholipids, consistent with loss of LPP3 phosphatase activity, mutant forms of LPP3, specifically lacking phosphatase activity, were able to inhibit β-catenin-mediated TCF transcription and also suppress axis duplication, although not as effectively as intact LPP3. These results reveal that LPP3 is essential to formation of the chorio-allantoic placenta and extra-embryonic vasculature. LPP3 also mediates gastrulation and axis formation, probably by influencing the canonical Wnt signaling pathway. The exact biochemical roles of LPP3 phosphatase activity and its undefined effect on β-catenin-mediated TCF transcription remain to be determined.

Functional coupling between the extracellular matrix and nuclear lamina by Wnt signaling in Progeria

The segmental premature aging disease Hutchinson-Gilford Progeria (HGPS) is caused by a truncated and farnesylated form of Lamin A. In a mouse model for HGPS, a similar Lamin A variant causes the proliferative arrest and death of postnatal, but not embryonic, fibroblasts. Arrest is due to an inability to produce a functional extracellular matrix (ECM), because growth on normal ECM rescues proliferation. The defects are associated with inhibition of canonical Wnt signaling, due to reduced nuclear localization and transcriptional activity of Lef1, but not Tcf4, in both mouse and human progeric cells. Defective Wnt signaling, affecting ECM synthesis, may be critical to the etiology of HGPS because mice exhibit skeletal defects and apoptosis in major blood vessels proximal to the heart. These results establish a functional link between the nuclear envelope/lamina and the cell surface/ECM and may provide insights into the role of Wnt signaling and the ECM in aging.

Nuclear factor κB transcription factors are coexpressed and convey a poor outcome in ovarian cancer

Recent work has suggested a role for nuclear factor κB (NF‐κB) in the propagation of ovarian cancer cell lines, but the significance and mechanism of NF‐κB in ovarian cancer is unknown. The authors hypothesized that the NF‐κB pathway is over activated in aggressive ovarian cancers.

Paternal and maternal genomes confer opposite effects on proliferation, cell-cycle length, senescence, and tumor formation

Loss of imprinting is the silencing of active imprinted genes or the activation of silent imprinted genes, and it is one of the most common epigenetic changes associated with the development of a wide variety of tumors. Here, we have analyzed the effects that global imprinted gene expression has on cell proliferation and transformation. Primary mouse embryonic fibroblasts (MEFs), whose entire genome is either exclusively paternal (androgenetic) or maternal (parthenogenetic), exhibit dramatically contrasting patterns of growth. In comparison with biparental MEFs, andro-genetic proliferation is characterized by a shorter cell cycle, increased saturation density, spontaneous transformation, and formation of tumors at low passage number. Parthenogenetic MEFs reach a lower saturation density, senesce, and die. The maternally expressed imprinted genes p57kip2 and M6P/Igf2r retard proliferation and reduce the long-term growth of MEFs. In contrast, the paternally expressed growth factor Igf2 is essential for the long-term proliferation of all genotypes. Increased Igf2 expression in primary MEFs not only stimulates proliferation, but also results in their rapid conversion to malignancy with tumor formation of short latency. Our results reveal that paternally expressed imprinted genes, in the absence of maternal imprinted genes, predispose fibroblasts to rapid transformation. A potent factor in their transformation is IGF2, which on increased expression results in the rapid conversion of primary cells to malignancy. These results reveal a route by which malignant choriocarcinoma may arise from molar pregnancies. They also suggest that the derivation of stem cells from parthenogenetic embryos, for the purposes of therapeutic cloning, may be ineffective.

Activation of NF-κB Signaling by Inhibitor of NF-κB Kinase β Increases Aggressiveness of Ovarian Cancer

The NF-kappaB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-kappaB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-kappaB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-kappaB in ovarian cancer using a small-molecule inhibitor of inhibitor of NF-kappaB kinase beta (IKKbeta) and confirmed with RNA interference toward IKKbeta. We developed a gene expression signature of IKKbeta signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKbeta. The expression of IKKbeta protein itself and the nine-gene ovarian cancer-specific IKKbeta signature were related to poor outcome in independently collected sets of primary ovarian cancers (P = 0.02). IKKbeta signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKbeta signaling on proliferation, invasion, and adhesion. Downregulating IKKbeta activity, either by a small-molecule kinase inhibitor or by short hairpin RNA depletion of IKKbeta, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKbeta in ovarian cancer suggests that therapeutic blockade of this pathway could be efficacious if specific IKKbeta inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKbeta activity.The NF-κB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-κB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-κB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-κB in ovarian cancer using a small molecule inhibitor of IKKβ, and confirmed with RNA interference towards IKKβ. We developed a gene expression signature of IKKβ signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKβ. The expression of IKKβ protein itself and the 9-gene ovarian cancer-specific IKKβ signature were related to poor outcome in independently collected sets of primary ovarian cancers (p=0.02). IKKβ signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKβ signaling on proliferation, invasion and adhesion. Downregulating IKKβ activity, either by a small molecule kinase inhibitor or by shRNA depletion of IKKβ, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKβ in ovarian cancer suggest that therapeutic blockade of this pathway could be efficacious if specific IKKβ inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKβ activity.

Activation of NF-kappaB signaling by inhibitor of NF-kappaB kinase beta increases aggressiveness of ovarian cancer.

The NF-kappaB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-kappaB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-kappaB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-kappaB in ovarian cancer using a small-molecule inhibitor of inhibitor of NF-kappaB kinase beta (IKKbeta) and confirmed with RNA interference toward IKKbeta. We developed a gene expression signature of IKKbeta signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKbeta. The expression of IKKbeta protein itself and the nine-gene ovarian cancer-specific IKKbeta signature were related to poor outcome in independently collected sets of primary ovarian cancers (P = 0.02). IKKbeta signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKbeta signaling on proliferation, invasion, and adhesion. Downregulating IKKbeta activity, either by a small-molecule kinase inhibitor or by short hairpin RNA depletion of IKKbeta, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKbeta in ovarian cancer suggests that therapeutic blockade of this pathway could be efficacious if specific IKKbeta inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKbeta activity.The NF-κB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-κB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-κB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-κB in ovarian cancer using a small molecule inhibitor of IKKβ, and confirmed with RNA interference towards IKKβ. We developed a gene expression signature of IKKβ signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKβ. The expression of IKKβ protein itself and the 9-gene ovarian cancer-specific IKKβ signature were related to poor outcome in independently collected sets of primary ovarian cancers (p=0.02). IKKβ signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKβ signaling on proliferation, invasion and adhesion. Downregulating IKKβ activity, either by a small molecule kinase inhibitor or by shRNA depletion of IKKβ, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKβ in ovarian cancer suggest that therapeutic blockade of this pathway could be efficacious if specific IKKβ inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKβ activity.