Arnona Gazit

Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow.

Wnt signalling has an important role in cell fate determination, tissue patterning and tumorigenesis. Secreted antagonists of Wnt include Frizzled (Fz)-related proteins (FRPs), Cerberus, Wnt inhibitory factor (WIF) and Dickkopf (Dkk). FRPs, Cerberus and WIF have all been shown to act by binding and sequestering Wnt. We report a novel mechanism of Wnt-signalling inhibition by human Dkk-1. Dkk-1 demonstrated no interaction with Wnt but bound a single cell surface site with high affinity (KD = 0.39 nM). Its receptor was detectable in a complex with a relative molecular mass of 240,000 (Mr 240K) with [125I] Dkk-1 by covalent affinity cross-linking. Wnt signalling through β-catenin is mediated by the Fz receptor and a recently identified low-density-lipoprotein-receptor-related co-receptor, LRP6/Arrow. Overproduction of the 200K LRP6 protein, but not of Fz, strikingly increased Dkk-1 binding as well as the amount of the 240K cross-linked complex, which was shown to be composed of Dkk-1 and LRP6. Moreover, Dkk-1 function was completely independent of Fz but LRP6 dramatically interfered with the Dkk-1 inhibition of Wnt signalling. Thus, unlike Wnt antagonists, which exert their effects by molecular mimicry of Fz or Wnt sequestration through other mechanisms, Dkk-1 specifically inhibits canonical Wnt signalling by binding to the LRP6 component of the receptor complex.

Expression of the normal human sis/PDGF-2 coding sequence induces cellular transformation

The human sis proto-oncogene contains the coding sequence for one of two polypeptide chains present in preparations of biologically active human platelet-derived growth factor (PDGF). A human clone, c-sis clone 8, which contains all of the v-sis-related sequences present in human DNA, was transcriptionally inactive when transfected into NIH/3T3 cells. When placed under the control of a retrovirus LTR, the clone was transcribed at levels comparable to that observed in cells transformed by SSV DNA. However, c-sis clone 8 DNA did not express detectable sis/PDGF-2 proteins and lacked biologic activity. A putative upstream exon was identified by its ability to detect the 4.2 kb sis-related transcript in certain human cells. When this sequence was inserted in the proper orientation between the LTR and c-sis clone 8, the chimeric molecule acquired high titered transforming activity, comparable to that of SSV DNA. Transformants containing this construct expressed human sis/PDGF-2 translational products. Thus the normal coding sequence for a human growth factor has transforming activity when expressed in an appropriate assay cell.

Human frizzled 1 interacts with transforming Wnts to transduce a TCF dependent transcriptional response

The human homologue of fz1 (Hfz1) was cloned from a cDNA library. Hfz1 was shown to couple to Wnt signal transduction pathways by its ability to enhance Wnt induced TCF dependent transcription in both autocrine and paracrine modes. Enhanced TCF dependent signaling was dose dependent with respect to both Wnt-3A and Hfz1. Moreover, Hfz1 deletion mutants with truncated carboxy termini showed markedly reduced capacity to enhance Wnt signal transduction. Specificity was demonstrated with respect to signal transduction by different Wnts. While Wnt-3a, -3, -1 and to a lesser extent Wnt-2 cooperated with Hfz1 in the paracrine assay for TCF dependent signaling, neither Wnt-4, -5a, -5b, -6, -7a nor -7b did so, despite similar levels of expression. However, coimmunoprecipitation of Hfz1 with both Wnt-3a and Wnt-5a indicated that TCF dependent signaling in response to Wnts is not determined solely by their ability to bind the receptor. All of these findings provide strong evidence that Hfz1 is a functional partner for certain Wnts in inducing TCF dependent transcription.

Characterization of Wnt-1 and Wnt-2 induced growth alterations and signaling pathways in NIH3T3 fibroblasts

Members of the Wnt family induce mouse mammary tumors and partially transform mammary epithelial cells in culture. However, their mechanism of transformation remains to be elucidated. In NIH3T3 mouse embryo fibroblasts, a standard transformation model, Wnt-1 and Wnt-2 were shown to induce altered properties including increased saturation density and growth in soft agar. Such cells also exhibited increased cell-cell adhesiveness. However, unlike oncogenes such as PDGFB or ras, Wnt-1 and -2 failed to induce detectable transformed foci following transfection, and stable NIH3T3 transfectants lacked tumor forming capacity. Wnt-1 and -2 transfectants exhibited increased uncomplexed, cytosolic β-catenin, which was not observed with PDGFB, ras or erbB2 transfectants. In transient transfection, Wnt-1 and -2 induced a rapid increase in cytosolic β-catenin but no detectable increase in the phosphorylated activated forms of MAP kinase. In contrast, ras was a potent activator of MAP kinase but had no effect on free β-catenin levels. These findings establish that both Wnt signaling and pattern of growth alterations differ from those of oncogenes which activate proliferative signaling pathways in NIH3T3 cells.

The Tat protein of equine infectious anemia virus is encoded by at least three types of transcripts

Nucleotide sequence analysis of a cDNA library of EIAV-infected canine cells established a complex pattern of gene expression, characterized by alternatively spliced polycistronic transcripts. The EIAV tat gene product was shown to be encoded by at least three species of mRNA which differed in their ability to trans-activate the EIAV LTR upon expression in canine cells. The most active cDNA was monocistronic, consisting of three exons. The most abundant cDNA in the library contained four exons and was identical to a polycistronic transcript previously described (Noiman et al., 1990b) which contains open frames for Tat, putative Rev, and truncated transmembrane proteins. Products consistent in size with those predicted for these last two proteins could be detected in in vitro translation experiments. The third Tat message, another four-exon form, also potentially encodes an amino terminally truncated transmembrane protein. In vitro mutagenesis experiments and analysis of subgenomic and partial cDNA clones confirmed and extended previous findings that S1 sequences are essential for trans-activation and that Tat translation initiates at a non-AUG codon either in the full-length Tat message or in the genomic S1 open reading frame. The Tat protein (8 kDa) was detected in cells transfected with a Tat cDNA construct and in canine cells persistently infected with EIAV. The Tat activity of polycistronic mRNAs was lower than that of the monocistronic form, suggesting that the expression of the EIAV trans-activator may be subject to several levels of posttranscriptional control.

Identification of sequences encoding the equine infectious anemia virus tat gene.

Equine infectious anemia virus (EIAV), a lentivirus, encodes a trans-activator (tat) which stimulates gene expression directed by the viral long terminal repeat (LTR). This function has been previously shown by us and others to be encoded by sequences within the middle region of the EIAV genome in which two short open reading frames, S1 and S2, reside. In the present study, by using in vitro mutagenesis, we show that disruption of S1, but not S2, completely abolished trans-activation. Addition of oligonucleotides complementary to S1 to cells transfected with a tat expression vector resulted in inhibition of trans-activation. EIAV cDNAs were isolated from a library of EIAV-infected cells constructed by using a eukaryotic expression vector. One cDNA clone which contained S1 sequences was able to trans-activate the EIAV LTR. Sequence analysis of this cDNA clone revealed that, in addition to S1, two other open reading frames were present. The cDNA still retained its activity when the latter two sequences were deleted.

The caprine arthritis-encephalitis virus is a distinct virus within the lentivirus group

The genetic relatedness among viral genomes of caprine arthritis-encephalitis virus, visna virus, and progressive pneumonia virus, was determined. Whereas the genomic RNAs of two strains of visna virus are indistinguishable as reflected both by their annealing kinetics as well as by the thermal stability of the hybrids, the caprine arthritis-encephalitis virus and visna virus have only 30% of their nucleic acid sequences in common. Furthermore, within the homologous regions of the two viral genomes, there is a significant level (approximately 10%) of mismatched base pairs. This limited homology that exists between caprine arthritis-encephalitis virus and visna virus was lower than the sequence homology observed between the genomes of visna virus and progressive pneumonia virus, or between the genomes of caprine arthritis-encephalitis virus and progressive pneumonia virus. All this indicates that caprine arthritis-encephalitis virus is an additional distinct member of the Lentivirus group of the Retroviridae family.

Nucleotide sequence analysis of the long terminal repeat of integrated caprine arthritis encephalitis virus

The nucleotide sequence of the long terminal repeat (LTR) of caprine arthritis encephalitis virus (CAEV), a prototype lentivirus was determined. 6-bp directly repeated host cell sequences flank the 376-bp proviral LTRs. By comparison with other retroviral sequences, the CAEV LTR likely contains U3, R and U5 regions 207, 86 and 83 base-pairs in length, respectively. Sequences conforming to consensus transcriptional promoter sites were identified in the U3 region upstream of a potential transcription initiation site. A consensus polyadenylation signal is present 20 bases upstream of the putative R-U5 border and a potential poly(A) addition site. Sequence comparisons of the CAEV LTR with those of other retroviruses uncovered significant similarities with that of visna virus. No other global homologies with other retrovirus LTRs could be detected. CAEV utilizes a primer binding site complementary to lysine tRNA as does visna, AIDS associated retroviruses, and mouse mammary tumor virus. The putative primer for positive-strand DNA synthesis identified in the CAEV sequence is identical to that of visna virus and very similar to those of AIDS retroviruses and MMTV. In addition, a stretch that includes the TATA box of the CAEV LTR resembles closely the corresponding region in the AIDS retrovirus. These and other findings further strengthen the classification of AIDS retrovirus as a lentivirus.

Isolation and characterization of a novel retrovirus from sheep affected by pulmonary carcinoma

Sheep pulmonary carcinoma (SPC) has been shown to be associated in nature with a retrovirus, by electron microscopic, biochemical, and epidemiological criteria and by experimental transmission. In this study, a retrovirus has been isolated from SPC tumors which were experimentally induced by inoculation with a cell-free, reverse transcriptase containing fraction from a spontaneous field case of SPC, and propagated in culture. This novel virus was shown to be unrelated to murine, avian, and bovine leukemia viruses, to be exogenous to the ovine species, and to have only limited genetic relatedness to the lentiviridae (maedi-visna and caprine arthritis encephalitis virus).

The Tat protein of the caprine arthritis encephalitis virus interacts with the Notch2 EGF-like repeats and the epithelin/granulin precursor.

Using the yeast two-hybrid system, we screened a human placenta cDNA library and identified two proteins that interacted with the Tat protein of the caprine arthritis encephalitis virus (CAEV): the EGF-like repeats 1–6 of the extracellular domain of the human Notch2 receptor and the epithelin/granulin growth factor precursor. This interaction was also confirmed in mammalian cells. Using in vitro mutagenesis assays, we showed that each one of the three cysteine residues located within the cysteine-rich domain of the CAEV Tat protein is essential for the binding of Tat to both the Notch2 and the epithelin/granulin protein. It is thus suggested that the cysteine-rich domain of Tat plays a role in the interaction between the Tat and either Notch2 or the epithelin/granulin domains, both of which exhibit EGF-like-repeat-imposed spatial conformation. It is assumed that such interactions might modulate the physiological functions of Notch2 and epithelin/granulin, thereby affecting various pathologies associated with CAEV.