Alba Mineo

Growth factor-like activity of gliadin, an alimentary protein: implications for coeliac disease

Background: Gliadins, a family of wheat proteins, are central to the pathogenesis of celiac disease (CD). In addition to ‘immunogenic’ effects, gliadin directly affects cultured cells and intestine preparations, and produces damage in vivo, via a separate ‘toxic’ peptide, such as A-gliadin p31–43 (P31–43). Aims: Understanding the molecular mechanisms underlying direct non T-cell mediated effects of gliadin peptides, and assessing their potential role in promoting CD. Method: Gliadin effects were tested on a number of cell lines and on cultured mucosa samples by evaluating cytoskeleton rearrangements, endocytosis, proliferation and apoptosis. Standard biochemical methods were used to assess prolonged epidermal growth factor receptor (EGFR) activation. Results: Crude gliadin peptic-tryptic peptides (PTG], or P31–43 alone, fully reproduce the effects of epidermal growth factor (EGF] on actin cytosketon, cell cycle and cell proliferation of various cell lines. Inhibitor studies demonstrate the role of EGFR in the early response to gliadin exposure, pointing to activation of the EGFR pathway. Peptide P31–43 is not similar to any EGFR ligand, but can delay inactivation of the EGFR interfering with its endocytosis. Gliadin-induced delay of EGFR endocytosis in cultured intestinal biopsies, together with S-phase entry of epithelial intestinal cells, confirm a role for EGFR activation in CD. Conclusion: The ability of gliadin peptides to delay EGFR inactivation through interference with the endocytic pathway suggests a model where gliadin fragments amplify the effects of trace amounts of EGF, and possibly of other growth factors, by prolonging receptor activation. The results, using cultures of coeliac intestinal biopsies, highlight the role of the EGF pathway in establishing and maintaining the typical atrophic and proliferative alterations of the small intestine in CD.

The RFG oligomerization domain mediates kinase activation and re-localization of the RET/PTC3 oncoprotein to the plasma membrane.

The RET/PTC3 oncogene arises from the fusion between the N-terminal encoding domain of the RFG gene and the tyrosine kinase encoding domain of RET receptor. RET/PTC3 is very frequent in papillary thyroid carcinomas, especially in children exposed to the Chernobyl accident. We have studied the functional consequences of the RFG–RET fusion. Here we show that the N-terminal coiled-coil domain of RGF mediates oligomerization and activation of the kinase and of the transforming capability of RET/PTC3. In addition, the RFG coiled-coil domain mediates a physical association between RET/PTC3 and RGF proteins, rendering RFG a bona fide substrate of RET/PTC3 kinase. Finally, we show that the coiled-coil domain of RGF is essential for the distribution of the RET/PTC3 protein at the membrane/particulate cell compartment level, where also most of the RFG protein is localized. We propose that fusion to the RFG coiled-coil domain provides RET kinase with a scaffold that mediates oligomerization and re-localization of the RET/PTC3 protein, a process that may be crucial for the signalling of this specific RET/PTC variant.

RET/PTC1 oncogene signaling in PC Cl 3 thyroid cells requires the small GTP-binding protein Rho

Thyroid papillary carcinomas are characterized by RET/PTC rearrangements that cause the tyrosine kinase domain of the RET receptor to fuse with N-terminal sequences encoded by heterologous genes. This results in the aberrant expression of a ligand-independent and constitutively active RET kinase. We analysed actin reorganization induced by the RET/PTC1 oncogene in PC Cl 3 rat thyroid epithelial cells. Differently from oncogenes Src, Ras and Raf, RET/PTC1 caused actin filaments to form prominent stress fibers. Moreover, stress fibers were identified in human thyroid papillary carcinoma cell lines harboring RET/PTC1 rearrangements but not in thyroid carcinoma cells negative for RET/PTC rearrangements. RET/MEN 2A, a constitutively active but unrearranged membrane-bound RET oncoprotein, did not induce stress fibers in PC Cl 3 cells. Induction of stress fibers by RET/PTC1 was restricted to thyroid cells; it did not occur in NIH3T3 fibroblasts or MCF7 mammary cells. RET/PTC1-mediated stress fiber formation depended on Rho but not Rac small GTPase activity. In addition, inhibition of Rho, but not of Rac, caused apoptosis of RET/PTC1-expressing thyroid cells. We conclude that Rho is implicated in the actin reorganization and cell survival mediated by the chimeric RET/PTC1 oncogene in thyroid epithelial cells, both phenotypes being cell type- and oncogene type-specific.

Thymosin β-10 Protein Synthesis Suppression Reduces the Growth of Human Thyroid Carcinoma Cells in Semisolid Medium

Beta-thymosins are structurally related, highly conserved acidic polypeptides, originally isolated from calf thymus. We have recently shown that the TB10 gene is overexpressed in human thyroid carcinoma cell lines and tissues, particularly in undifferentiated thyroid carcinomas, but expressed at an undetectable level in normal thyroid cells. The precise role of thymosin beta-10 (TB10) activity in maintaining the malignant phenotype of thyroid cell lines is unknown. To investigate TB10 function and relevance in a model system we used an antisense methodology to suppress TB10 protein synthesis in two human thyroid carcinoma cell lines (NPA and ARO). The growth in soft agar of NPA and ARO cells carrying a TB10 construct in an antisense orientation was significantly reduced. Conversely, anchorage-dependent growth was unchanged in NPA and ARO cells carrying the TB10 construct in a sense orientation or carrying the backbone vector. TB10 expression also affected actin organization. In fact, stress fibers were long and thick in ARO cells in which TB10 expression was suppressed by the antisense construct. Conversely, they were scarce and short in the vector-transfected ARO cells. These data suggest that TB10 plays a critical role in the regulation of anchorage-independent growth and assembly of actin filaments.