C-terminal fragment of transforming growth factor beta-induced protein (TGFBIp) is required for apoptosis in human osteosarcoma cells.
Transforming growth factor beta-induced protein (TGFBIp), is secreted into the extracellular space. When fragmentation of C-terminal portions is blocked, apoptosis is low, even when the protein is overexpressed. If fragmentation occurs, apoptosis is observed. Whether full-length TGFBIp or integrin-binding fragments released from its C-terminus is necessary for apoptosis remains equivocal. More importantly, the exact portion of the C-terminus that conveys the pro-apoptotic property of TGFBIp is uncertain. It is reportedly within the final 166 amino acids. We sought to determine if this property is dependent upon the final 69 amino acids containing the integrin-binding, EPDIM and RGD, sequences. With MG-63 osteosarcoma cells, transforming growth factor (TGF)-beta1 treatment increased expression of TGFBIp over 72 h (p<0.001). 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 37-150 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-beta1 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-beta1 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 on the C-terminus most likely to interact with integrins.
Zamilpa
Rogelio
R
Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA.
Rupaimoole
Rajesha
R
Phelix
Clyde F
CF
Somaraki-Cormier
Maria
M
Haskins
William
W
Asmis
Reto
R
LeBaron
Richard G
RG
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology*; D001859:Bone Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016023:Integrins / Q000378:metabolism; D012516:Osteosarcoma; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D000099222:betaIG-H3 Protein
D016326:Extracellular Matrix Proteins; D016023:Integrins; D010446:Peptide Fragments; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D000099222:betaIG-H3 Protein