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)ß(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.

Regulation of cardiomyocyte full-length tissue factor expression and microparticle release under inflammatory conditions in vitro.

SUMMARY BACKGROUND: Myocardial inflammation is associated with an increase in circulating microparticles (MPs) and procoagulability. OBJECTIVES: We determined whether acute inflammation was associated with altered full-length tissue factor (flTF) expression and increased procoagulability in cardiomyocytic cells. METHODS: This study examined the transcriptional regulation of flTF expression in murine cardiomyocytic (HL-1) cells. Also, the generation of MPs by HL-1 cells and their ability to diffuse through an artificial endothelium was evaluated. RESULTS: Constitutive and tumor necrosis factor-alpha (TNF-alpha)-induced flTF expression of HL-1 was reduced when c-Jun N-terminal kinase (JNK) was inhibited. Tissue factor (TF)-positive procoagulant MPs were released from HL-1 cells in response to TNF-alpha. JNK inhibition potentiated the release of MPs from HL-1 cells without affecting MP-associated TF activity. MP generation was dependent on RhoA activation and associated with a reorganization of the actin cytoskeleton. Increased diffusion of HL-1-derived MPs through an endothelial monolayer was found after TNF-alpha treatment. The increased diffusion was dependent not only on TNF-alpha but also on HL-1-released mediators. CONCLUSIONS: Full-length TF expression in HL-1 cells was regulated through JNK. The TNF-alpha-induced increase in procoagulability was mediated through RhoA-dependent release of flTF-bearing MPs. These MPs were able to diffuse through an endothelial barrier adjacent to HL-1 cells and increased the procoagulability of the extracellular endothelial space. Cardiomyocytes seem to be a likely source of flTF-bearing procoagulant MPs.

SR proteins ASF/SF2 and SRp55 participate in tissue factor biosynthesis in human monocytic cells.

BACKGROUND: Human monocytes express two naturally occurring forms of circulating tissue factor (TF) - full-length TF, a membrane-spanning protein, and alternatively spliced TF, a soluble molecule. Presence of the variable exon 5 in TF mRNA determines whether the encoded TF protein is transmembrane, or soluble. Recently, an essential SR protein ASF/SF2 was implicated in TF pre-mRNA processing in human platelets. OBJECTIVE: To examine molecular mechanisms governing regulated processing of TF pre-mRNA in human monocytic cells. METHODS AND RESULTS: In silico analysis of the human TF exon 5, present only in full-length TF mRNA, revealed putative binding motifs termed exonic splicing enhancers (ESE) for the SR proteins ASF/SF2 and SRp55, which were found to be abundantly expressed in monocytic cell lines THP-1 and SC, as well as monocyte-enriched peripheral blood mononuclear cells (PBMC). Using a splice competent mini-gene reporter system transiently expressed in monocytic cells, it was determined that weakening of either five closely positioned ASF/SF2 ESE (bases 87-117) or a single conserved SRp55 ESE (base 39) results in severe skipping of exon 5. ASF/SF2 and SRp55 were found to physically associate with the identified ESE. CONCLUSIONS: SR proteins ASF/SF2 and SRp55 appear to interact with the variable TF exon 5 through ESE at bases 39 and 87-117. Weakening of the above ESE modulates splicing of TF exon 5. This study is the first to identify and experimentally characterize cis-acting splicing elements involved in regulated biosynthesis of human TF.