Garamszegi, Nandor

Transforming growth factor β (TGF-β) coordinates a number of biological events important in normal and pathophysiological growth. In this study, deletion and substitution mutations were used to identify receptor motifs modulating TGF-β receptor activity. Initial experiments indicated that a COOH-terminal sequence between amino acids 482–491 in the kinase domain of the type I receptor was required for ligand-induced receptor signaling and down-regulation. These 10 amino acids are highly conserved in mammalian, Xenopus, andDrosophila type I receptors. Although mutation or deletion of the region (referred to as the NANDOR BOX, for nonactivating non–down-regulating) abolishes TGF-β–dependent mitogenesis, transcriptional activity, type I receptor phosphorylation, and down-regulation in mesenchymal cultures, adjacent mutations also within the kinase domain are without effect. Moreover, a kinase-defective type I receptor can functionally complement a mutant BOX expressing type I receptor, documenting that when the BOX mutant is activated, it has kinase activity. These results indicate that the sequence between 482 and 491 in the type I receptor provides a critical function regulating activation of the TGF-β receptor complex.

Transforming growth factor-βs (TGF-β) are multifunctional proteins capable of either stimulating or inhibiting mitosis, depending on the cell type. These diverse cellular responses are caused by stimulating a single receptor complex composed of type I and type II receptors. Using a chimeric receptor model where the granulocyte/monocyte colony-stimulating factor receptor ligand binding domains are fused to the transmembrane and cytoplasmic signaling domains of the TGF-β type I and II receptors, we wished to describe the role(s) of specific amino acid residues in regulating ligand-mediated endocytosis and signaling in fibroblasts and epithelial cells. Specific point mutations were introduced at Y182, T200, and Y249 of the type I receptor and K277 and P525 of the type II receptor. Mutation of either Y182 or Y249, residues within two putative consensus tyrosine-based internalization motifs, had no effect on endocytosis or signaling. This is in contrast to mutation of T200 to valine, which resulted in ablation of signaling in both cell types, while only abolishing receptor down-regulation in fibroblasts. Moreover, in the absence of ligand, both fibroblasts and epithelial cells constitutively internalize and recycle the TGF-β receptor complex back to the plasma membrane. The data indicate fundamental differences between mesenchymal and epithelial cells in endocytic sorting and suggest that ligand binding diverts heteromeric receptors from the default recycling pool to a pathway mediating receptor down-regulation and signaling.

Abstract Transforming growth factor β (TGF-β) signaling via Smad proteins occurs in various cell types. However, whereas the biological response to TGF-β can be as distinct as growth promoting (i.e., mesenchymal cells) versus growth inhibiting (i.e., epithelial cells), few discernible differences in TGF-β signaling have been reported. In the current study, we examined the role of Ras in the proliferative response to TGF-β and how it might interface with Smad-dependent and Smad-independent TGF-β signaling targets. TGF-β stimulated Ras activity in a subset of mesenchymal, but not epithelial, cultures and was required for extracellular signal-regulated kinase (ERK)–dependent transcriptional responses. Although dominant negative Ras had no effect on TGF-β internalization or Smad-dependent signaling (i.e., phosphorylation, nuclear translocation, or SBE-luciferase activity), it did prevent the hyperphosphorylation of the Smad transcriptional corepressor TG-interacting factor (TGIF). This was not sufficient, however, to overcome the mitogenic response stimulated by TGF-β, which was dependent on signals downstream of p21-activated kinase 2 (PAK2). Moreover, although the initial activation of Ras and PAK2 are distinctly regulated, TGF-β–stimulated PAK2 activity is required for Ras-dependent ERK phosphorylation and Elk-1 transcription. These findings show the requirement for crosstalk between two Smad-independent pathways in regulating TGF-β proliferation and indicate that the mechanism(s) by which TGF-β stimulates growth is not simply the opposite of its growth inhibitory actions. [Cancer Res 2007;67(8):3673–82]

Abstract Purpose: Increased levels of matrix metalloproteinase 1 (MMP-1) expression have been associated with poor outcome in chondrosarcoma. The existence of a single nucleotide polymorphism creating an Ets-binding site in the MMP-1 promoter may be one mechanism for elevated MMP-1 transcription. The aim of our study was to identify the prevalence of this single nucleotide polymorphism (SNP) in chondrosarcoma patients, to determine its correlation with disease outcome, and to discern whether it could serve as a prognostic marker in patients with chondrosarcoma. Experimental Design: Sixty-seven chondrosarcoma specimens were selected sequentially from an established tumor bank with a median duration of 47 months follow-up (range, 24 to 179 months). DNA was extracted, amplified with PCR, and sequenced to determine presence (GG) or absence of the Ets-binding site created by the SNP. Results: Eighteen (27%) samples were homozygous for the absence of the Ets site, 34 (51%) were heterozygous for the SNP, and 15 (22%) were homozygous for the SNP. The 5-year overall survival rate for patients was 78, 80, and 84%, respectively (P = 0.5527). The disease-free survival rate was 16, 63, and 76%, respectively (P = 0.0801). The 5-year disease-free survival rate for patients with the homozygous G/G genotype was 16%, compared with 71% for patients who were either homozygous or heterozygous for the GG allele (P = 0.0444). Conclusions: Despite a statistical correlation between MMP-1 gene expression and outcome in chondrosarcoma, this study demonstrates an absence of a correlation between the presence of the SNP and prognosis in patients with chondrosarcoma.