The coding sequence for the TGFBIp C-terminal protein encoding amino acid residues 615 to 683 (TGFBIp615C683) was amplified using the forward primer (5-GACGACGACAAGATGAGCCTGACATCATGGCC-3)

The coding sequence for the TGFBIp C-terminal protein encoding amino acid residues 615 to 683 (TGFBIp615C683) was amplified using the forward primer (5-GACGACGACAAGATGAGCCTGACATCATGGCC-3). At this time point, apoptosis was significantly increased (p 0.001) and was prevented by an anti-TGFBIp, polyclonal antibody (p 0.05). Overexpression of TGFBIp by transient transfection produced a 2-fold increase in apoptosis (p 0.01). Exogenous purified TGFBIp at concentrations of 37 to 150 nM produced a dose dependent increase in apoptosis (p 0.001). Mass spectrometry analysis of TGFBIp isolated from conditioned medium of cells treated with TGF-1 revealed truncated forms of TGFBIp that lacked integrin-binding sequences in the C-terminus. Recombinant TGFBIp truncated, similarly, at amino acid 614 failed to induce apoptosis. A recombinant fragment encoding the final 69 amino acids of the TGFBIp C-terminus produced significant apoptosis. This apoptosis level was comparable to that induced by TGF-1 upregulation of endogenous TGFBIp. Mutation of the integrin-binding sequence EPDIM, but not RGD, blocked apoptosis (p 0.001). These pro-apoptotic actions are dependent TMS on the C-terminus most likely to interact with integrins. are C-terminally fragmented. TGFBIp fragmentation might be progressive, yielding TGFBIp exhibiting a relative mobility of 60 kDa as the lowest mass form detected in this study. We report that the minimal TGFBIp that includes both integrin-binding sites causes apoptosis. The work of others suggests that further fragmentation of TGFBIp614C683 to at least an RGD hexapeptide can also lead to apoptosis (Kim et al., 2003). The ultimate fate of TGFBIp fragments is unknown, but the process exists and recombinant TGFBIp expression reliably and predictably resulted in osteosarcoma cell death, and may evoke a similar outcome when Eng upregulated by TGF-1 and on various transformed cell types. Conversely, a mechanism that blocks TGFBIps influence on the progression of tumor growth has been linked to hypermethylation of the TGFBIp promoter, effectively inactivating the gene (Shah et al., 2008; Shao et al., 2006). The RGD and EPDIM motifs implicate integrin actions in apoptosis. In addition to TGFBIp fragmentation, TMS TGFBIp-mediated apoptosis involves executioner caspase-3 activation (Kim et al., 2003; Morand et al., 2003; Nam et al., 2005) and is likely regulated by the expression of particular integrin types, suggesting characteristically anoikis (Frisch and Screaton, 2001). Ours is the first report to demonstrate that the apoptotic mechanism in osteosarcoma cells involves TGFBIp, particularly EPDIM, and in addition, corroborates actions of C-terminal integrin-binding sequences in apoptosis (Kim et al., 2003; Morand et al., 2003). Though activation of various signaling pathways TGF-1 affects a considerable complement of cellular processes during embryogenesis and in adult tissues including an increase in apoptosis (Rahimi and Leof, 2007). These data implicate TGFBIp expression and C-terminal fragmentation as an apoptotic switch affecting tumor biology, embryonic development, tissue remodeling and morphogenesis. Natural steps in our studies are to determine the particular integrin receptor that mediates TGFBIp apoptosis and further elucidate the apoptotic pathways activated by TGFBIp, in comparison to pathways activated by TGF-1. 4. Experimental Procedures 4.1 Materials A549 lung adenocarcinoma cells (CCL-185), MG-63 (CCL-1427) and Saos-2 osteosarcoma cells (HTB-85) were purchased from the American Type Culture Collection (Rockville, MD). Dr. Gary Sunter (UTSA) provided Spodoptera frugiperda (Sf9) insect cells. NovaBlue GigaSingles? competent cells and Origami (DE3) pLacI cells were from Novagen (San Diego, CA). Ni-NTA agarose and RNeasy were purchased from Qiagen (Valencia, CA). TaqMan was purchased from Applied Biosystems (Foster City, CA), and reagents to detect ssDNA were from Chemicon International (Temecula, CA). Bicinchoninic acid protein reagents were from Pierce Biochemicals. Dulbeccos Modified Eagle Medium (DMEM) was from Gibco BRL. Vectors pTriEX-4 Ek/LIC and pIEX-3 Ek/LIC, and Insect GeneJuice? Transfection Reagent were from Novagen (Novagen, WI). CHO-VGS cell growth TMS media was purchased from Irvine Scientific (Santa Ana, CA), and SF900 II medium and annexin staining reagents were from Invitrogen (Carlsbad, CA). 4.2 cDNA Constructs The cDNA that encodes full-length TGFBIp was obtained by reverse transcription-PCR from RNA isolated from human A549 cells. Coding sequences of TGFBIpwt were amplified using the forward primer 5-GACGACGACAAGATGTCGCCCTACCAGCTG-3 and reverse primer 5-GAGGAGAAGCCCGGTCTAATGCTTCATCC-3. Coding sequences for TGFBIp truncated at amino acid 614 (TGFBIp27C614) were obtained using the wt forward primer above and the reverse primer 5-GAGGAGAAGCCCGGTCTAGGCAACAGGCTCC-3. Amplicons cloned into the expression vector pIEX-3 Ek/LIC were expressed with GST and 6His-tag sequences at the N-terminus. The coding sequence for a TGFBIp C-terminal protein encoding amino acid residues 615 to 683 (TGFBIp615C683) was amplified using the forward primer (5-GACGACGACAAGATGAGCCTGACATCATGGCC-3). The reverse primer was the same as wt. The amplified product was cloned into pTriEx-4 EK/LIC. Non-tagged.