Martin M. Sachs

Negative Feedback Loop of Wnt Signaling through Upregulation of Conductin/Axin2 in Colorectal and Liver Tumors

ABSTRACT Activation of Wnt signaling through β-catenin/TCF complexes is a key event in the development of various tumors, in particular colorectal and liver tumors. Wnt signaling is controlled by the negative regulator conductin/axin2/axil, which induces degradation of β-catenin by functional interaction with the tumor suppressor APC and the serine/threonine kinase GSK3β. Here we show that conductin is upregulated in human tumors that are induced by β-catenin/Wnt signaling, i.e., high levels of conductin protein and mRNA were found in colorectal and liver tumors but not in the corresponding normal tissues. In various other tumor types, conductin levels did not differ between tumor and normal tissue. Upregulation of conductin was also observed in the APC-deficient intestinal tumors of Min mice. Inhibition of Wnt signaling by a dominant-negative mutant of TCF downregulated conductin but not the related protein, axin, in DLD1 colorectal tumor cells. Conversely, activation of Wnt signaling by Wnt-1 or dishevelled increased conductin levels in MDA MB 231 and Neuro2A cells, respectively. In time course experiments, stabilization of β-catenin preceded the upregulation of conductin by Wnt-1. These results demonstrate that conductin is a target of the Wnt signaling pathway. Upregulation of conductin may constitute a negative feedback loop that controls Wnt signaling activity.

The anaerobic proteins of maize

Anaerobic treatment drastically alters the pattern of protein synthesized by maize primary roots. During the first hour of anaerobiosis, aerobic protein synthesis is halted and there is an increase in the synthesis of a class of polypeptides with approximate molecular weights of 33,000 daltons. During the second hour of anaerobic treatment, the synthesis of another small group of polypeptides is initated. This group, the anerboic polypeptides (ANPs), accounts for > 70% of total protein synthesis after 5 hr of anaerobiosis, and is synthesized in basically the same ratio until root death (approximately 70 hr). The alcohol dehydrogenase polypeptides are major ANPs. RNA isolated from roots treated anaerobically for at least 24 hr directs the translation of only the anaerobic polypeptides. However, RNA from roots treated anaerobically for only 5 hr directs translation of both anaerobic and aerobic polypeptides. Thus an early response to anaerobic treatment is the suppression of aerobic message translation. Although the anaerobic polypeptides share a formal similarity to heat-shock proteins in animals, it is probable that the anaerobic genes are an adaptation to flooding.

Elevation of cytosolic calcium precedes anoxic gene expression in maize suspension-cultured cells.

Based on pharmacological evidence, we previously proposed that intracellular Ca2+ mediates the perception of O2 deprivation in maize seedlings. Herein, using fluorescence imaging and photometry of Ca2+ in maize suspension-cultured cells, the proposal was further investigated. Two complementary approaches were taken: (1) real time analysis of anoxia-induced changes in cytosolic Ca2+ concentration ([Ca]i) and (2) experimental manipulation of [Ca]i and then assay of the resultant anoxia-specific responses. O2 depletion caused an immediate increase in [Ca2+]i, and this was reversible within a few seconds of reoxygenation. The [Ca]i elevation proceeded independent of extracellular Ca2+. The kinetics of the Ca2+ response showed that it occurred much earlier than any detectable changes in gene expression. Ruthenium red blocked the anoxic [Ca]i elevation and also the induction of adh1 (encoding alcohol dehydrogenase) and sh1 (encoding sucrose synthase) mRNA. Ca2+, when added along with ruthenium red, prevented the effects of the antagonist on the anoxic responses. Verapamil and bepridil failed to block the [Ca]i rise induced by anoxia and were equally ineffective on anoxic gene expression. Caffeine induced an elevation of [Ca]i as well as ADH activity under normoxia. The data provide direct evidence for [Ca]i elevation in maize cells as a result of anoxia-induced mobilization of Ca2+ from intracellular stores. Furthermore, any manipulation that modified the [Ca]i rise brought about a parallel change in the expression of two anoxia-inducible genes. Thus, these results corroborate our proposal that [Ca]i is a physiological transducer of anoxia signals in plants.

A flooding-induced xyloglucan endo-transglycosylase homolog in maize is responsive to ethylene and associated with aerenchyma.

Development of aerenchyma (soft cortical tissue with large intercellular air spaces) in flooded plants results from cell-wall hydrolysis and eventual cell lysis and is promoted by endogenous ethylene. Despite its adaptive significance, the molecular mechanisms behind aerenchyma development remain unknown. We recently isolated a flooding-induced maize (Zea mays L.) gene (wusl1005[gfu]; abbreviated as 1005) encoding a homolog of xyloglucan endo-transglycosylase (XET), a putative cell-wall-loosening enzyme active during germination, expansion, and fruit softening. XET and related enzymes may also be involved in cell-wall metabolism during flooding-induced aerenchyma development. Under flooding, 1005 mRNA accumulated in root and mesocotyl locations that subsequently exhibited aerenchyma development and reached maximum levels within 12 h of treatment. Aerenchyma development was observed in the same locations by 48 h of treatment. Treatment with the ethylene synthesis inhibitor (aminooxy)acetic acid (AOA), which prevented cortical air space formation under flooding, almost completely inhibited 1005 mRNA accumulation in both organs. AOA treatment had little effect on the accumulation of mRNA encoded by adh1, indicating that it did not cause general suppression of flooding-responsive genes. Additionally, ethylene treatment under aerobic conditions resulted in aerenchyma development as well as induction of 1005 in both organs. These results indicate that 1005 is responsive to ethylene. Treatment with anoxia, which suppresses ethylene accumulation and aerenchyma development, also resulted in 1005 induction. However, in contrast to flooding, AOA treatment under anoxia did not affect 1005 mRNA accumulation, indicating that 1005 is induced via different mechanisms under flooding (hypoxia) and anoxia.

Involvement of Intracellular Calcium in Anaerobic Gene Expression and Survival of Maize Seedlings

Ca-mediated processes are known to be involved in transducing many developmental, hormonal, and environmental cues in plant cells. In this study, the role of Ca in the perception of anoxic stress signals by maize (Zea mays L. cv B73) roots was assessed by studying the effect of various Ca antagonists on the induction of alcohol dehydrogenase (ADH) and sucrose synthase mRNA as well as ADH activity under anoxia. The effect of these compounds on the poststress recovery of the seedlings was also monitored. Ruthenium red (RR), an inhibitor of organellar Ca fluxes, repressed the anoxic activation of the alcohol dehydrogenase1 and shrunken1 genes as measured by their transcript levels as well as ADH activity. Furthermore, RR-treated seedlings could not recover even after only 2 h of flooding, in contrast to untreated B73 seedlings that survived 72 h of submergence. Ca, when supplied along with RR, allowed normal anoxic gene expression and also prevented the RR-imposed death of the seedlings from short-term anoxia. Ca (45Ca) fluxes were measured in maize roots to elucidate the mode of action of RR. RR abolished anoxia-stimulated 45Ca influx into maize roots but did not affect aerobic Ca2+ uptake, unlike a few other antagonists that blocked both the aerobic and anoxic fluxes. However, Ca uptake across the plasma membrane was not necessary for the adaptive response to anoxia, since chelation of extracellular Ca by ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime] -tetraacetic acid or 1,2-bis(2-aminophenoxy)ethane N,N,N[prime],N[prime] -tetraacetic acid did not affect the induction of ADH activity or poststress survival of flooded seedlings. The data suggest that RR may act on one of the intracellular stores of Ca and the Ca mobilized from this source is a physiological transducer of anoxic stress signals in maize roots.

Novel p62dok family members, dok-4 and dok-5, are substrates of the c-Ret receptor tyrosine kinase and mediate neuronal differentiation

Docking proteins are substrates of tyrosine kinases and function in the recruitment and assembly of specific signal transduction molecules. Here we found that p62dok family members act as substrates for the c-Ret receptor tyrosine kinase. In addition to dok-1, dok-2, and dok-3, we identified two new family members, dok-4 and dok-5, that can directly associate with Y1062 of c-Ret. Dok-4 and dok-5 constitute a subgroup of dok family members that is coexpressed with c-Ret in various neuronal tissues. Activated c-Ret promotes neurite outgrowth of PC12 cells; for this activity, Y1062 in c-Ret is essential. c-Ret/dok fusion proteins, in which Y1062 of c-Ret is deleted and replaced by the sequences of dok-4 or dok-5, induce ligand-dependent axonal outgrowth of PC12 cells, whereas a c-Ret fusion containing dok-2 sequences does not elicit this response. Dok-4 and dok-5 do not associate with rasGAP or Nck, in contrast to p62dok and dok-2. Moreover, dok-4 and dok-5 enhance c-Ret–dependent activation of mitogen-activated protein kinase. Thus, we have identified a subclass of p62dok proteins that are putative links with downstream effectors of c-Ret in neuronal differentiation.

The Plant Ontology Database: a community resource for plant structure and developmental stages controlled vocabulary and annotations

The Plant Ontology Consortium (POC, http://www.plantontology.org) is a collaborative effort among model plant genome databases and plant researchers that aims to create, maintain and facilitate the use of a controlled vocabulary (ontology) for plants. The ontology allows users to ascribe attributes of plant structure (anatomy and morphology) and developmental stages to data types, such as genes and phenotypes, to provide a semantic framework to make meaningful cross-species and database comparisons. The POC builds upon groundbreaking work by the Gene Ontology Consortium (GOC) by adopting and extending the GOCs principles, existing software and database structure. Over the past year, POC has added hundreds of ontology terms to associate with thousands of genes and gene products from Arabidopsis, rice and maize, which are available through a newly updated web-based browser (http://www.plantontology.org/amigo/go.cgi) for viewing, searching and querying. The Consortium has also implemented new functionalities to facilitate the application of PO in genomic research and updated the website to keep the contents current. In this report, we present a brief description of resources available from the website, changes to the interfaces, data updates, community activities and future enhancement.

Selective synthesis of alcohol dehydrogenase during anaerobic treatment of maize

SummaryAnaerobic treatment of a maize seedling mediates the synthesis of five major, native proteins and seven polypeptide size-classes; slab polyacrylamide and autoradiographic techniques were used to analyze extracts from single primary roots. The alcohol dehydrogenase-1 polypeptide is most dramatically synthesized and accumulated during anaerobiosis, as compared to aerobic control data. Allozymes were used to identify alcohol dehydrogenase unequivocally. Our results pertain to interpretations of previous studies on the alcohol dehydrogenase gene system in maize, and to work on the stress proteins of Drosophila.

Plant Ontology (PO) : a controlled vocabulary of plant structures and growth stages

The Plant Ontology Consortium (POC) (www.plantontology.org) is a collaborative effort among several plant databases and experts in plant systematics, botany and genomics. A primary goal of the POC is to develop simple yet robust and extensible controlled vocabularies that accurately reflect the biology of plant structures and developmental stages. These provide a network of vocabularies linked by relationships (ontology) to facilitate queries that cut across datasets within a database or between multiple databases. The current version of the ontology integrates diverse vocabularies used to describe Arabidopsis, maize and rice (Oryza sp.) anatomy, morphology and growth stages. Using the ontology browser, over 3500 gene annotations from three species-specific databases, The Arabidopsis Information Resource (TAIR) for Arabidopsis, Gramene for rice and MaizeGDB for maize, can now be queried and retrieved.

Differential expression and sequence analysis of the maize glyceraldehyde-3-phosphate dehydrogenase gene family.

Two cDNA clones for maize cytosolic glyceraldehyde-3-phosphate dehydrogenase are described. One is about 97% similar in coding capacity to a previously published clone [Brinkmann et al. (1987). J. Mol. Evol. 26, 320-328], while the other shows only 88% similarity. Evidence points toward the three cDNAs being the products of three genes, to be called Gpc1, Gpc2, and Gpc3. When the least similar clone, corresponding to Gpc3, was used to analyze RNA gel blots, anaerobic treatment for 6 hours induced RNA accumulation in the shoots 15.6-fold, while a 1-hour shift from 28 degrees C to 40 degrees C increased accumulation 5.1-fold. Roots had a higher basal level of expression, leading to a 6.0-fold anaerobic induction, and a 2.4-fold heat stress induction. RNA gel blot analysis using the clone corresponding to Gpc2 showed decreased RNA accumulation within 6 hours of anaerobiosis, while analysis with the previously published clone, corresponding to Gpc1, showed a decrease within 24 hours. Neither Gpc1 nor Gpc2 showed heat stress induction, while some other known anaerobic genes did. Through the use of hybrid selection, in vitro translation, and immune precipitation, the relative expression of the three genes is shown. The role of the observed changes in gene expression is discussed in relation to stress physiology.