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Results for “ectopic gene conversion”

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PubMed2001

Nuclear export of human beta-catenin can occur independent of CRM1 and the adenomatous polyposis coli tumor suppressor.

beta-Catenin is a mediator of the Wnt-signaling pathway. In many cancers, beta-catenin is stabilized and accumulates in the nucleus where it associates with lymphoid-enhancing factor 1/ T-cell transcription factors to activate genes involved in cell transformation. Previously, we showed that adenomatous polyposis coli (APC) protein can regulate beta-catenin localization by nuclear export. In this study, we used in vitro transport assays to test whether cellular beta-catenin can exit the nucleus independent of APC and the CRM1 export receptor. In digitonin-permeabilized SW480 (APC(mut/mut)) tumor cells, nuclear beta-catenin decreased >60% in export reactions in the absence of exogenous factors. Under similar conditions, nuclear c-ABL was only exported after the addition of cytosolic extract, and the export was blocked by the CRM1-specific inhibitor, leptomycin B. The nuclear export of beta-catenin was not blocked by leptomycin B treatment, revealing a CRM1- and APC-independent pathway. The export of beta-catenin was sensitive to lower temperatures and the removal of ATP, indicating an active process. Ectopically expressed yellow fluorescent protein-beta-catenin also displayed CRM1-independent export. Conversely, the overexpression of the CRM1 transporter moderately stimulated export of nuclear beta-catenin, confirming that beta-catenin exits the nucleus by at least two distinct pathways. The shuttling ability of tumor cell beta-catenin has implications for its regulation and its role in transferring signals between the nucleus and plasma membrane.

Adenomatous Polyposis Coli Protein↗

PubMed2005

Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis.

Chondrocytes and osteoblasts are two primary cell types in the skeletal system that are differentiated from common mesenchymal progenitors. It is believed that osteoblast differentiation is controlled by distinct mechanisms in intramembranous and endochondral ossification. We have found that ectopic canonical Wnt signaling leads to enhanced ossification and suppression of chondrocyte formation. Conversely, genetic inactivation of beta-catenin, an essential component transducing the canonical Wnt signaling, causes ectopic formation of chondrocytes at the expense of osteoblast differentiation during both intramembranous and endochondral ossification. Moreover, inactivation of beta-catenin in mesenchymal progenitor cells in vitro causes chondrocyte differentiation under conditions allowing only osteoblasts to form. Our results demonstrate that beta-catenin is essential in determining whether mesenchymal progenitors will become osteoblasts or chondrocytes regardless of regional locations or ossification mechanisms. Controlling Wnt/beta-catenin signaling is a common molecular mechanism underlying chondrocyte and osteoblast differentiation and specification of intramembranous and endochondral ossification.

Animals↗

PubMed2002

Dorsoventral patterning is established in the telencephalon of mutants lacking both Gli3 and Hedgehog signaling.

Considerable data suggest that sonic hedgehog (Shh) is both necessary and sufficient for the specification of ventral pattern throughout the nervous system, including the telencephalon. We show that the regional markers induced by Shh in the E9.0 telencephalon are dependent on the dorsoventral and anteroposterior position of ectopic Shh expression. This suggests that by this point in development regional character in the telencephalon is established. To determine whether this prepattern is dependent on earlier Shh signaling, we examined the telencephalon in mice carrying either Shh- or Gli3-null mutant alleles. This analysis revealed that the expression of a subset of ventral telencephalic markers, including Dlx2 and Gsh2, although greatly diminished, persist in Shh(-/-) mutants, and that these same markers were expanded in Gli3(-/-) mutants. To understand further the genetic interaction between Shh and Gli3, we examined Shh/Gli3 and Smoothened/Gli3 double homozygous mutants. Notably, in animals carrying either of these genetic backgrounds, genes such as Gsh2 and Dlx2, which are expressed pan-ventrally, as well as Nkx2.1, which demarcates the ventral most aspect of the telencephalon, appear to be largely restored to their wild-type patterns of expression. These results suggest that normal patterning in the telencephalon depends on the ventral repression of Gli3 function by Shh and, conversely, on the dorsal repression of Shh signaling by Gli3. In addition these results support the idea that, in addition to hedgehog signaling, a Shh-independent pathways must act during development to pattern the telencephalon.

Animals↗

PubMed1991

Ectopic expression of an A-type lamin does not interfere with differentiation of lamin A-negative embryonal carcinoma cells.

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. It is believed to be important for nuclear envelope integrity and the organization of interphase chromatin. On the basis of biochemical properties and sequence criteria, vertebrate lamin proteins are classified as either A- or B-type. While B-type lamins are expressed in almost all cell types, no A-type lamins are present in early vertebrate embryos or undifferentiated embryonal carcinoma cell lines. Intriguingly, expression of A-type lamins occurs concomitant with cell differentiation and embryonic development. These findings have led to the hypothesis that A-type lamins might play a role in establishing or stabilizing cell-type specific differences in nuclear organization, which in turn might relate to the developmental potential of a cell. To test this hypothesis, we have stably expressed chicken lamin A in undifferentiated murine embryonal carcinoma (P19) cells, and examined the consequences of ectopic lamin A expression for the differentiation state and potential of these cells. Our results demonstrate that the P19 cells, although normally devoid of lamin A, properly incorporate and process chicken lamin A. Moreover, the stably transfected cell lines maintain the properties of undifferentiated cells, demonstrating that expression of lamin A does not directly induce differentiation. Conversely, when exposed to retinoic acid, an inducer of differentiation, lamin A-expressing P19 cells are able to differentiate normally. Taken together, our results suggest that unscheduled expression of A-type lamins is not sufficient to deregulate cell differentiation programs. The implications of these findings for the possible role for lamin A expression during development are discussed.

Animals↗

PubMed2006

Combined ectopic expression of Pdx1 and Ptf1a/p48 results in the stable conversion of posterior endoderm into endocrine and exocrine pancreatic tissue.

Patterning of the embryonic endoderm into distinct sets of precursor cells involves the precisely regulated activities of key transcription regulators. Ectopic, pan-endodermal activation of XPtf1a/p48 during pancreas precursor cell stages of Xenopus embryogenesis results in an expansion of the pancreatic territory, precisely within the borders of XlHbox8 expression. A combination of both activities is sufficient to expand the pancreatic precursor cell population also into more posterior portions of the endoderm. Both treatments result in the formation of a giant pancreas that persists up to late tadpole stages of development and carries both supernumerary endocrine and exocrine cells. A combination of XPtf1a/p48 and XlHbox8 is thus sufficient to convert nonpancreatic endodermal cells into pancreatic precursor cells.

Animals↗

PubMed1994

Cyclin E controls S phase progression and its down-regulation during Drosophila embryogenesis is required for the arrest of cell proliferation.

Most cells of the dorsal epidermis exit from the mitotic cycle after division 16 in Drosophila embryogenesis. This exit is dependent on the down-regulation of Drosophila cyclin E (DmcycE) during the final mitotic cycle. Ectopic expression of DmcycE after the final mitosis induces entry into S phase and reaccumulation of G2 cyclins and results in progression through a complete additional cell cycle. Conversely, analyses in DmcycE mutant embryos indicate that cyclin E is required for progression through S phase of the mitotic cycle. Moreover, endoreplication, which occurs in late wild-type embryos in the same pattern as DmcycE expression, is not observed in the mutant embryos. Therefore, Drosophila cyclin E, which forms a complex with the Dmcdc2c kinase, controls progression through S phase and its down-regulation limits embryonic proliferation.

Animals↗

PubMed2001

The cell adhesion molecule Echinoid defines a new pathway that antagonizes the Drosophila EGF receptor signaling pathway.

Photoreceptor and cone cells in the Drosophila eye are recruited following activation of the epidermal growth factor receptor (EGFR) pathway. We have identified echinoid (ed) as a novel putative cell adhesion molecule that negatively regulates EGFR signaling. The ed mutant phenotype is associated with extra photoreceptor and cone cells. Conversely, ectopic expression of ed in the eye leads to a reduction in the number of photoreceptor cells. ed expression is independent of EGFR signaling and ED is localized to the plasma membrane of every cells throughout the eye disc. We present evidence that ed acts nonautonomously to generate extra R7 cells by a mechanism that is sina-independent but upstream of Tramtrack (TTK88). Together, our results support a model whereby ED defines an independent pathway that antagonizes EGFR signaling by regulating the activity, but not the level, of the TTK88 transcriptional repressor.

Alleles↗

PubMed2002

BMP signaling positively regulates Nodal expression during left right specification in the chick embryo.

Exogenous application of BMP to the lateral plate mesoderm (LPM) of chick embryos at the early somite stage had a positive effect on Nodal expression. BMP applications into the right LPM were followed by a rapid activation of Nodal, while applications into the left LPM resulted in expansion of the normal domain of Nodal expression. Conversely, blocking of BMP signaling by Noggin in the left LPM interfered with the activation of Nodal expression. These results support a positive role for endogenous BMP on Nodal expression in the LPM. We also report that BMP positively regulates the expression of Caronte, Snail and Cfc in both the left and right LPM. BMP-treated embryos had molecular impairment of the midline with downregulation of Lefty1, Brachyury and Shh but we also show that the midline defect was not sufficient to induce ectopic Nodal expression. We discuss our findings in the context of the known molecular control of the specification of left-right asymmetry.

Animals↗

PubMed1999

Primary human CD4+ T cells contain heterogeneous I kappa B kinase complexes: role in activation of the IL-2 promoter.

NF-kappa B transcription factors play an important role in the activation of the IL-2 gene in response to TCR ligation. The release of NF-kappa B factors to the nucleus requires phosphorylation and degradation of the inhibitory kappa-B proteins (I kappa Bs). I kappa B alpha and I kappa B beta phosphorylation is dependent on dual signaling by the TCR and the CD28 accessory receptor. This pathway involves a multisubunit I kappa B kinase (IKK) complex, which includes the IKK alpha (IKK-1) and IKK beta (IKK-2) kinases. We demonstrate that stimulation of primary human CD4+ T cells by CD3/CD28 activates two distinct endogenous IKK complexes, a heterodimeric IKK alpha/beta and a homodimeric IKK beta complex. IKK beta overexpression in a Jurkat cell line resulted in the formation of a constitutively active IKK complex, which was CD3/CD28 inducible. In contrast, ectopic expression of IKK alpha assembled into a complex with negligible I kappa B kinase activity. Moreover, IKK beta, but not IKK alpha, overexpression enhanced transcriptional activation of the CD28 response element in the IL-2 promoter. Conversely, only kinase-inactive IKK beta interfered in the activation of the IL-2 promoter. Sodium salicylate, an inhibitor of IKK beta, but not IKK alpha, activity, inhibited IL-2 promoter activation as well as IL-2 secretion and interfered in activation of both the heterodimeric as well as the homodimeric IKK complexes in primary CD4+ T cells. Taken together, these data demonstrate the presence of an IKK beta-mediated signaling pathway that is activated by TCR and CD28 coligation and regulates IL-2 promoter activity.

Antibodies, Monoclonal↗

PubMed1999

Role of transmembrane 4 superfamily (TM4SF) proteins CD9 and CD81 in muscle cell fusion and myotube maintenance.

The role of transmembrane 4 superfamily (TM4SF) proteins during muscle cell fusion has not been investigated previously. Here we show that the appearance of TM4SF protein, CD9, and the formation of CD9-beta1 integrin complexes were both regulated in coordination with murine C2C12 myoblast cell differentiation. Also, anti-CD9 and anti-CD81 monoclonal antibodies substantially inhibited and delayed conversion of C2C12 cells to elongated myotubes, without affecting muscle-specific protein expression. Studies of the human myoblast-derived RD sarcoma cell line further demonstrated that TM4SF proteins have a role during muscle cell fusion. Ectopic expression of CD9 caused a four- to eightfold increase in RD cell syncytia formation, whereas anti-CD9 and anti-CD81 antibodies markedly delayed RD syncytia formation. Finally, anti-CD9 and anti-CD81 monoclonal antibodies triggered apoptotic degeneration of C2C12 cell myotubes after they were formed. In summary, TM4SF proteins such as CD9 and CD81 appear to promote muscle cell fusion and support myotube maintenance.

Animals↗

PubMed2003

Cyclic strain inhibits switching of smooth muscle cells to an osteoblast-like phenotype.

Ectopic calcification is commonly associated with cardiovascular disease, injury, aging, and biomaterial implantation. We hypothesized that the normal mechanical environment of smooth muscle cells (SMCs) inhibits a phenotypic switch to a pattern of gene expression more typical for bone and inducive for calcification. This hypothesis was tested using a 3-D engineered smooth muscle tissue model subjected to cyclic mechanical strain. This simplified model maintained a 3-D tissue architecture while eliminating systemic effects as can be seen with in vivo models. All engineered tissues were found to express bone-associated genes (osteopontin, matrix gla protein, alkaline phosphatase, and the transcription factor CBFA-1). Strikingly, however, expression of these genes was down-regulated in tissues exposed to cyclic strain at all time points ranging from 5 to 150 days. Furthermore, long-term strain played a protective role in regard to calcification, as unstrained tissues exhibited increased calcium deposition with respect to strained tissues. The results of this study suggest that without an appropriate mechanical environment, SMCs in 3-D culture undergo a phenotypic conversion to an osteoblast-like pattern of gene expression. This finding has significant implications for the mechanisms underlying a variety of cardiovascular diseases and indicates the broad utility of engineered tissue models in basic biology studies.

Animals↗

PubMed2004

Loss of negative regulation by Numb over Notch is relevant to human breast carcinogenesis.

The biological antagonism between Notch and Numb controls the proliferative/differentiative balance in development and homeostasis. Although altered Notch signaling has been linked to human diseases, including cancer, evidence for a substantial involvement of Notch in human tumors has remained elusive. Here, we show that Numb-mediated control on Notch signaling is lost in approximately 50% of human mammary carcinomas, due to specific Numb ubiquitination and proteasomal degradation. Mechanistically, Numb operates as an oncosuppressor, as its ectopic expression in Numb-negative, but not in Numb-positive, tumor cells inhibits proliferation. Increased Notch signaling is observed in Numb-negative tumors, but reverts to basal levels after enforced expression of Numb. Conversely, Numb silencing increases Notch signaling in normal breast cells and in Numb-positive breast tumors. Finally, growth suppression of Numb-negative, but not Numb-positive, breast tumors can be achieved by pharmacological inhibition of Notch. Thus, the Numb/Notch biological antagonism is relevant to the homeostasis of the normal mammary parenchyma and its subversion contributes to human mammary carcinogenesis.

Breast Neoplasms↗

PubMed2000

The Phox2b transcription factor coordinately regulates neuronal cell cycle exit and identity.

In the vertebrate neural tube, cell cycle exit of neuronal progenitors is accompanied by the expression of transcription factors that define their generic and sub-type specific properties, but how the regulation of cell cycle withdrawal intersects with that of cell fate determination is poorly understood. Here we show by both loss- and gain-of-function experiments that the neuronal-subtype-specific homeodomain transcription factor Phox2b drives progenitor cells to become post-mitotic. In the absence of Phox2b, post-mitotic neuronal precursors are not generated in proper numbers. Conversely, forced expression of Phox2b in the embryonic chick spinal cord drives ventricular zone progenitors to become post-mitotic neurons and to relocate to the mantle layer. In the neurons thus generated, ectopic expression of Phox2b is sufficient to initiate a programme of motor neuronal differentiation characterised by expression of Islet1 and of the cholinergic transmitter phenotype, in line with our previous results showing that Phox2b is an essential determinant of cranial motor neurons. These results suggest that Phox2b coordinates quantitative and qualitative aspects of neurogenesis, thus ensuring that neurons of the correct phenotype are generated in proper numbers at the appropriate times and locations.

Adaptor Proteins, Signal Transducing↗

PubMed1993

The 5'-flanking region of the human dopamine beta-hydroxylase gene promotes neuron subtype-specific gene expression in the central nervous system of transgenic mice.

Dopamine beta-hydroxylase (DBH, EC 1.14.17.1) catalyzes the conversion of dopamine to norepinephrine, the third step of catecholamine biosynthesis. We have previously created transgenic mice harboring a chimeric gene consisting of the 4-kb DNA fragment of the human DBH gene promoter and the human phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) cDNA, to express PNMT in norepinephrine- and epinephrine-producing cells in the brain, sympathetic ganglia, and adrenal medullary chromaffin cells (Kobayashi et al., Proc. Natl. Acad. Sci. U.S.A., 89 (1992) 1631-1635). In this paper, we produced for the first time the antibody that specifically detects human PNMT, but not mouse PNMT, with the synthetic oligopeptide characteristic of the human PNMT sequence, and used this antibody to investigate the cells expressing human PNMT in transgenic mice. Immunohistochemical analysis of transgenic mice showed typical expression of human PNMT immunoreactivity in norepinephrinergic and epinephrinergic neurons in brain, as well as norepinephrine- and epinephrine-producing cells in the adrenal gland, indicating that the 4-kb 5'-flanking region is essential for the tissue-specific expression of the DBH gene. We also detected the ectopic expression in some DBH-immunonegative cells in the olfactory bulb of transgenic mice.

Amino Acid Sequence↗

PubMed1995

Distinct modes of cyclin E/cdc2c kinase regulation and S-phase control in mitotic and endoreduplication cycles of Drosophila embryogenesis.

Drosophila cyclin E (DmcycE) is required in embryos for S phase of mitotic and endoreduplication cycles. Here, we describe regulatory differences characteristic for these two cell cycle types. While DmcycE transcript levels decline in DmcycE mutant cells programmed for mitotic proliferation, they are maintained and no longer restricted to transient pulses in DmcycE mutant cells programmed for endoreduplication. Moreover, DmcycE expression in endoreduplicating cells is down-regulated by ectopic expression of a heat-inducible cyclin E transgene. DmcycE expression in endoreduplicating tissues, therefore, is restricted by a negative feedback to the transient pulse triggering entry into S-phase. Conversely, during mitotic cycles, where S phase entry is not only dependent on cyclin E but also on progression through M phase, cyclin E and associated Dmcdc2c kinase activity are present throughout the cell cycle. Reinitiation of DNA replication during the G2 phase of the mitotic cell cycle, therefore, is prevented by cyclin E/Dmcdc2c kinase-independent regulation. Observations in cyclin A mutants implicate G2 cyclins in this regulation. Our results suggest molecular explanations for the different rules governing S phase during mitotic and endoreduplication cycles.

Animals↗

PubMed1998

Akt, a target of phosphatidylinositol 3-kinase, inhibits apoptosis in a differentiating neuronal cell line.

Phosphatidylinositol (PI) 3-kinase has been suggested to mediate cell survival. Consistent with this possibility, apoptosis of conditionally (simian virus 40 Tts) immortalized rat hippocampal H19-7 neuronal cells was increased in response to wortmannin, an inhibitor of PI 3-kinase. Downstream effectors of PI 3-kinase include Rac1, protein kinase C, and the serine-threonine kinase Akt (protein kinase B). Here, we show that activation of Akt is one mechanism by which PI 3-kinase can mediate survival of H19-7 cells during serum deprivation or differentiation. While ectopic expression of wild-type Akt (c-Akt) does not significantly enhance survival in H19-7 cells, expression of activated forms of Akt (v-Akt or myristoylated Akt) results in enhanced survival which can be comparable to that conferred by Bcl-2. Conversely, expression of a dominant-negative mutant of Akt accelerates cell death upon serum deprivation or differentiation. Finally, the results indicate that Akt can transduce a survival signal for differentiating neuronal cells through a mechanism that is independent of induction of Bcl-2 or Bcl-XL or inhibition of Jun kinase activity.

Androstadienes↗

PubMed1998

Differential expression of Wnt genes in normal and flat variants of PC12 cells, a cell line responsive to ectopic Wnt1 expression.

Wnt genes encode secreted growth factor-like proteins that participate in growth regulation, differentiation and tumorigenesis. Ectopic expression of Wnt1 converts the PC12 neural crest-derived rat pheochromocytoma cell line from a round phenotype that express chromaffin markers to flat adherent cells (termed PC12/Wnt1) that do not express them. A pool of spontaneously flat variants of PC12 cells (PC12/flat) is phenotypically similar to the PC12/Wnt1 cells, but does not express Wnt1. Here we describe the expression of 13 Wnt genes in wild type PC12, PC12/flat and PC12/Wnt1 cells. Wild type PC12 expressed Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt6, Wnt10a and Wnt11. Compared with expression in wild type cells, both PC12/flat and PC12/Wnt1 cells lost most or all expression of Wnt3a and Wnt4 and gained expression of Wnt7b. Wnt5a and Wnt6 expression was higher in PC12/Wnt1 cells than in PC12 or PC12/flat. Wnt3 was expressed at low levels in both PC12 and PC12/flat, but was absent in PC12/Wnt1 cells. Wnt10a and Wnt11 were approximately equally expressed in the three groups, and Wnt2, Wnt5b, Wnt7a, Wnt10b and endogenous Wnt1 mRNAs were not detected. These results demonstrate that the expression of some Wnt genes changes in PC12 cells upon conversion to the flat phenotype, and suggest that Wnt1 may modulate expression of several other Wnt genes in these cells.

Animals↗

PubMed2006

Regulation of wingless signaling by the CKI family in Drosophila limb development.

The Wingless (Wg)/Wnt signaling pathway regulates a myriad of developmental processes and its malfunction leads to human disorders including cancer. Recent studies suggest that casein kinase I (CKI) family members play pivotal roles in the Wg/Wnt pathway. However, genetic evidence for the involvement of CKI family members in physiological Wg/Wnt signaling events is lacking. In addition, there are conflicting reports regarding whether a given CKI family member functions as a positive or negative regulator of the pathway. Here we examine the roles of seven CKI family members in Wg signaling during Drosophila limb development. We find that increased CKIepsilon stimulates whereas dominant-negative or a null CKIepsilon mutation inhibits Wg signaling. In contrast, inactivation of CKIalpha by RNA interference (RNAi) leads to ectopic Wg signaling. Interestingly, hypomorphic CKIepsilon mutations synergize with CKIalpha RNAi to induce ectopic Wg signaling, revealing a negative role for CKIepsilon. Conversely, CKIalpha RNAi enhances the loss-of-Wg phenotypes caused by CKIepsilon null mutation, suggesting a positive role for CKIalpha. While none of the other five CKI isoforms can substitute for CKIalpha in its inhibitory role in the Wg pathway, several CKI isoforms including CG12147 exhibit a positive role based on overexpression. Moreover, loss of Gilgamesh (Gish)/CKIgamma attenuates Wg signaling activity. Finally, we provide evidence that several CKI isoforms including CKIalpha and Gish/CKIgamma can phosphorylate the Wg coreceptor Arrow (Arr), which may account, at least in part, for their positive roles in the Wg pathway.

Animals↗