In conclusion our results demonstrated that TGF

In conclusion, our results demonstrated that TGF-β1 promotes linear invadosome formation in hepatocellular carcinoma cells, through the regulation of collagen I, its receptor and its cross-linker (Fig. 6). The key role of TGF-β1 in HCC development allow us to suggest that linear invadosomes and their receptor DDR1 could be an interesting target to stop HCC progression.
Acknowledgements
Introduction Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase (RTK) superfamily and is distinguished by a discoidin-homology region in its extracellular portion. Uniquely, DDR1 is not a receptor for soluble growth factors but for native, triple-helical collagens, in particular type I to type IV collagens [28], [35], [38]. DDR1 is expressed in a number of tissues, including the mammary gland epithelium [40]. The knockout of DDR1 showed an essential role in postnatal mammary gland development and in the formation of lactogenic tissue. In DDR1-null females, pubertal mammary gland outgrowth is delayed and matrix deposition enhanced [40]. At parturition, knockout females failed to nourish their litter due to the absence of alveolar opening and milk protein secretion. Transplantation experiments of knockout mammary tissue into the cleared fat pad of a wild type recipient revealed that the defect in DDR1-null mice is cell-autonomous [9]. While the absence of lactation is the most striking defect in DDR1-knockout mice, we also documented aberrant formation of the renal basement membrane and reduced signaling capacity of knockout mesangial cells [8], [11]. Additionally, smooth-muscle cells isolated from DDR1-null mice show reduced migration and chemotaxis compared to control cells [12], [13]. A more complete understanding of the DDR1 signaling pathway is complicated by: (i) shedding of the extracellular domain; (ii) by alternative splicing of the DDR1 gene leading to at least 5 distinct isoforms [1], [2], [39]. The DDR1c isoform is the longest, while DDR1b lacks six URB597 in the kinase region, and exon 11 coding for 37 amino acids in the juxtamembrane region is absent in DDR1a. DDR1d and DDR1e are truncated receptors missing the entire kinase domain or parts of it, respectively. Previous work showed that the sequence encoded by the alternatively spliced exon 11 present in DDR1b/c isoforms contains an LLXNPXpY-motif, which functions as docking site for the ShcA adaptor molecule [35]. In human breast cancer cells, ShcA was found to bind to DDR1b upon activation by collagen; while in colon carcinoma cells, signaling between activated DDR1 and p53 was found [25], [35]. Furthermore, in macrophages and in bronchoalveolar fluid cells, the recruitment of ShcA by DDR1b resulted in p38 MAPK and NFκB activation through the TRAF6 pathway [20], [21]. However, a detailed knowledge of other substrates and their mode of interaction downstream of DDR1 are still missing. In particular, the role of individual phosphotyrosines in recruiting specific DDR1 substrates is largely unexplored.
Materials and methods
Results Activation of the DDR1 tyrosine kinase function by collagen causes robust and sustained autophosphorylation [35]. In order to determine the extent of tyrosine phosphorylation of the two most common isoforms, DDR1a and DDR1b, we labeled human embryonic kidney fibroblast 293 cells expressing DDR1a or DDR1b with [32P]-orthophosphate. Upon receptor activation, DDR1 protein was isolated by immunoprecipitation and subjected to trypsin digestion. Tryptic peptides of DDR1a and DDR1b were resolved by two-dimensional thin-layer chromatography. Seven [32P]-phosphate-labeled peptides were found for DDR1a, and an additional three spots for the b-isoform (Fig. 1A). A mix of equal amounts of both isoforms allowed us to confirm the identity of each spot in the two samples. We found peptides numbered as 1, 3 and 9 to be major sites of autophosphorylation. Because peptides 8–10 were only found in the b-isoform and not in the a-isoform, they are most likely located within the alternatively spliced insert. Peptide 1 is more strongly phosphorylated in DDR1a than DDR1b suggesting an isoform-specific mechanism of kinase function. These results indicate that DDR1 undergoes multiple tyrosine phosphorylation events upon kinase activation thereby opening up several potential substrate-docking sites.