Alternatively spliced tissue factor and full-length tissue factor protect cardiomyocytes against TNF-α-induced apoptosis.
Tissue Factor (TF) is expressed in various cell types of the heart, such as cardiomyocytes. In addition to its role in the initiation of blood coagulation, the TF:FVIIa complex protects cells from apoptosis. There are two isoforms of Tissue Factor (TF): "full length" (fl)TF--an integral membrane protein, and alternatively spliced (as)TF--a protein that lacks a transmembrane domain and can thus be secreted in a soluble form. Whether asTF or flTF affects apoptosis of cardiomyocytes is unknown. In this study, we examined whether asTF or flTF protects murine cardiomyocytes from TNF-α-induced apoptosis. We used murine cardiomyocytic HL-1 cells and primary murine embryonic cardiomyocytes that overexpressed either murine asTF or murine flTF, and stimulated them with TNF-α to initiate cell death. Apoptosis was assessed by annexin-V assay, propidium iodide assay, as well as activation of caspase-3 and -9. In addition, signaling via integrins, Akt, NFκB and Erk1/2, and gene-expression of Bcl-2 family members were analyzed. We here report that overexpression of asTF reduced phosphatidylserine exposure upon TNF-α-stimulation. asTF overexpression led to an increased expression and phosphorylation of Akt, as well as up-regulation of the anti-apoptotic protein Bcl-x(L). The anti-apoptotic effects of asTF overexpression were mediated via α(V)β(3)/Akt/NFκB signaling and were dependent on Bcl-x(L) expression in HL-1 cells. The anti-apoptotic activity of asTF was also observed using primary cardiomyocytes. Analogous yet less pronounced anti-apoptotic sequelae were observed due to overexpression of flTF. Importantly, cardiomyocytes deficient in TF exhibited increased apoptosis compared to wild type cells. We propose that asTF and flTF protect cardiomyocytes against TNF-α-induced apoptosis via activation of specific signaling pathways, and up-regulation of anti-apoptotic members of the Bcl-2 protein family.
Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Zentrum für Herz und Kreislaufmedizin, D-12200 Berlin, Germany.
U
U
Boltzen
A
A
Eisenreich
S
S
Antoniak
A
A
Weithaeuser
H
H
Fechner
W
W
Poller
H P
HP
Schultheiss
N
N
Mackman
U
U
Rauch
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D015870:Gene Expression; D020935:MAP Kinase Signaling System; D051379:Mice; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D061251:Primary Cell Culture; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D015854:Up-Regulation; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
C494812:Bcl2l1 protein, mouse; D016328:NF-kappa B; D020033:Protein Isoforms; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D051020:bcl-X Protein; D013925:Thromboplastin