Tenascin-C signaling through induction of 14-3-3 tau

We searched by a cDNA subtraction screen for differentially expressed transcripts in MCF-7 mammary carcinoma cells grown on tenascin-C versus fibronectin. On tenascin-C, cells had irregular shapes with many processes, whereas on fibronectin they were flat with a cobble stone–like appearance. We found elevated levels of 14-3-3 tau transcripts and protein in cells grown on tenascin-C. To investigate the consequences of an increased level of this phospho-serine/threonine–binding adaptor protein, we transfected MCF-7 cells with a construct encoding full-length 14-3-3 tau protein and selected clones with the highest expression levels. The morphology of these cells on tenascin-C was flat, resembling that of cells on fibronectin. This was reflected by a similar pattern of F-actin staining on either substratum. Furthermore, the growth rate on tenascin-C was increased compared with the parental cells. After transient transfection of HT1080 fibrosarcoma and T98G glioblastoma cells with 14-3-3 tau, only the 14-3-3 tau–expressing cells were able to adhere and survive on tenascin-C, whereas all cells adhered well on fibronectin. Therefore, we postulate that tenascin-C promotes the growth of tumor cells by causing an increase in the expression of 14-3-3 tau, which in turn has a positive effect on tumor cell adhesion and growth

C. elegans Agrin Is Expressed in Pharynx, IL1 Neurons and Distal Tip Cells and Does Not Genetically Interact with Genes Involved in Synaptogenesis or Muscle Function

Agrin is a basement membrane protein crucial for development and maintenance of the neuromuscular junction in vertebrates. The C. elegans genome harbors a putative agrin gene agr-1. We have cloned the corresponding cDNA to determine the primary structure of the protein and expressed its recombinant fragments to raise specific antibodies. The domain organization of AGR-1 is very similar to the vertebrate orthologues. C. elegans agrin contains a signal sequence for secretion, seven follistatin domains, three EGF-like repeats and two laminin G domains. AGR-1 loss of function mutants did not exhibit any overt phenotypes and did not acquire resistance to the acetylcholine receptor agonist levamisole. Furthermore, crossing them with various mutants for components of the dystrophin-glycoprotein complex with impaired muscle function did not lead to an aggravation of the phenotypes. Promoter-GFP translational fusion as well as immunostaining of worms revealed expression of agrin in buccal epithelium and the protein deposition in the basal lamina of the pharynx. Furthermore, dorsal and ventral IL1 head neurons and distal tip cells of the gonad arms are sources of agrin production, but no expression was detectable in body muscles or in the motoneurons innervating them. Recombinant worm AGR-1 fragment is able to cluster vertebrate dystroglycan in cultured cells, implying a conservation of this interaction, but since neither of these proteins is expressed in muscle of C. elegans, this interaction may be required in different tissues. The connections between muscle cells and the basement membrane, as well as neuromuscular junctions, are structurally distinct between vertebrates and nematodes

Tenascin-W is found in malignant mammary tumors, promotes alpha8 integrin-dependent motility and requires p38MAPK activity for BMP-2 and TNF-alpha induced expression in vitro

Tenascins represent a family of extracellular matrix glycoproteins with distinctive expression patterns. Here we have analyzed the most recently described member, tenascin-W, in breast cancer. Mammary tumors isolated from transgenic mice expressing hormone-induced oncogenes reveal tenascin-W in the stroma around lesions with a high likelihood of metastasis. The presence of tenascin-W was correlated with the expression of its putative receptor, alpha8 integrin. HC11 cells derived from normal mammary epithelium do not express alpha8 integrin and fail to cross tenascin-W-coated filters. However, 4T1 mammary carcinoma cells do express alpha8 integrin and their migration is stimulated by tenascin-W. The expression of tenascin-W is induced by BMP-2 but not by TGF-beta1, though the latter is a potent inducer of tenascin-C. The expression of tenascin-W is dependent on p38MAPK and JNK signaling pathways. Since preinflammatory cytokines also act through p38MAPK and JNK signaling pathways, the possible role of TNF-alpha in tenascin-W expression was also examined. TNF-alpha induced the expression of both tenascin-W and tenascin-C, and this induction was p38MAPK- and cyclooxygenase-dependent. Our results show that tenascin-W may be a useful diagnostic marker for breast malignancies, and that the induction of tenascin-W in the tumor stroma may contribute to the invasive behavior of tumor cells

Detection of endogenous <i>C. elegans</i> agrin by western blot and immunofluorescence.

<p>Lysates of wild type (N₂) and agrin mutant worms (<i>eg1770</i>, <i>eg153</i>, <i>tm2051</i>) were analysed on western blots (A). Two prominent bands of about 160 kDa and 75 kDa were present exclusively in the wild type (Wt) worms and not the mutants. The larger band corresponds to the calculated size of the full length AGR-1 protein and the smaller band may represent an agrin degradation product. Asterisks denote two additional background bands present in all the strains. B, Worms were immunostained with the monoclonal antibody pool against <i>C. elegans</i> agrin (green) and Rim, a synaptic marker prominent in nerve ring (red). Agrin was detected in the basal lamina around the pharynx procorpus (arrow) and anterior bulb (asterisk). Posterior bulb staining was weaker possibly due to poor antibody penetration (dashed arrow). (C–H) Polyclonal antiserum staining resulted in the same pattern in the pharynx of wild type worms (C and D, asterisk for anterior bulb) whereas it was clearly absent in agrin mutants (F–H). Prominent background staining of the gut was present in all strains (C–H, arrowhead). Preimmune serum of the same rabbit was used as negative control on wild type worms (E) where both pharyngeal and gut staining was clearly missing.</p

<i> In vitro</i> interaction between <i>C. elegans</i> agrin and vertebrate α-dystroglycan.

<p>Purified chicken α-DG (lanes 1–3 and 5) or crude COS cell extract (lane 4) was transferred to the membrane after separation by SDS-PAGE and membrane strips were incubated with different samples of agrin: lane 1, chicken muscle agrin isoform; lane 2, chicken neuronal isoform; lanes 3 and 4, <i>C. elegans</i> agrin in conditioned medium of transfected COS cells; lane 5, conditioned medium of non-transfected COS cells. Binding of the respective agrins was detected by anti-chick agrin antibody (lanes 1 and 2) or with the Tn60 antibody recognizing the short tenascin C fragment which was fused to the <i>C. elegans</i> agrin fragment (lanes 3, 4 and 5). Binding of <i>C. elegans</i> agrin to α-DG was detected in lane 3, but not in the negative controls (lanes 4 and 5).</p

Primer sequences.

<p>Primer sequences.</p

C. elegans agrin DNA and protein sequence with predicted domain architecture.

<p>The <i>C. elegans</i> agrin coding sequence was assembled from overlapping cDNA fragments, amplified by RT-PCR. The positions of the primers are shown by black arrows, where the corresponding pairs are depicted with the same line pattern (full line, dotted line, “dash-dot-dash” line). The three nucleotides missing in the genomic sequence of the database entry are framed with red rectangles. Based on the nucleotide numbering in cosmid F41G3, their positions are: C after 30028, A after 29776 and C after position 28351. The coding region of the gene is 4422 bp long with 5′ and 3′ untranslated regions of 212 and 160 bp, respectively (dark gray boxes; EMBL/GeneBank Accession AM773423). The predicted protein sequence is 1473 amino acids long and the domain architecture is shown in different colors. A putative signal sequence (purple box) is followed by seven follistatin domains (blue), two epidermal growth factor domains of the laminin-type (light gray), a follistatin domain (blue), an EGF-like domain (orange) and two laminin G domains (yellow).</p