Abstract
We investigated intracellular mechanisms involved in the up-regulation of plasminogen activator inhibitor I (PAI-1) synthesis by human recombinant tumor necrosis factor-alpha (TNF) during monocyte differentiation of HL-60 cells triggered by the transforming growth factor-beta1/vitamin D(3) (TGF/D3) mixture. TGF/D3-treated cells expressed surface monocytic markers and produced noticeable amounts of PAI-1 but stopped to proliferate. A reduced p70 S6 kinase (p70(S6K)) phosphorylation was also observed and, in this situation, TNF dramatically enhanced PAI-1 synthesis. Similarly, TNF significantly up-regulated PAI-1 synthesis when p70(S6K) phosphorylation was inhibited by rapamycin. This phenomenon was not due to a general decrease in protein synthesis but involved the activation of gene transcription rather than PAI-1 mRNA stabilization. The level of the transcriptional regulator factor E2F1, a repressor of PAI-1 gene expression, was shown to be down-modulated in TGF/D3- as well as in rapamycin-treated cells. Furthermore, the apoptotic effect of TNF in HL-60 cells appeared to be prevented by the addition of either TGF/D3 or rapamycin. In conclusion, these results indicate that inhibition of p70(S6K) phosphorylation during TGF/D3-induced monocyte differentiation of HL-60 cells is a determinant factor that allows TNF to exert its up-regulating effect on PAI-1 synthesis while protecting cells from apoptosis.
Highlights
We investigated intracellular mechanisms involved in the up-regulation of plasminogen activator inhibitor I (PAI-1) synthesis by human recombinant tumor necrosis factor-␣ (TNF) during monocyte differentiation of HL-60 cells triggered by the transforming growth factor-1/ vitamin D3 (TGF/D3) mixture
PAI-1 Synthesis Is Up-regulated by TNF during TGF/D3induced Monocyte Differentiation of HL-60 Cells—We reported that HL-60 cells treated with a mixture of TGF/D3 increased PAI-1 synthesis, which was largely potentiated by the addition of TNF [28], whereas others showed that this mixture triggers HL-60 cell differentiation toward the monocytic lineage [31,32,33]
To clarify if PAI-1 production correlated with monocytic progression, we analyzed the effect of TGF/D3, associated or not with TNF on some parameters characteristic of monocyte differentiation
Summary
Reagents—Monoclonal antibodies (12A4 and 15H12) specific for human PAI-1 enzyme-linked immunosorbent assay were generously given by Dr P. Proteins of the supernatant were separated using an 8% SDS-PAGE according to Laemmli [30], electrotransferred onto a polyvinylidene difluoride membrane, which was hybridized with the antibody. Total Protein Synthesis Analysis—Cells were treated for 12 h with TNF or TNF ϩ RAP, 1 mCi of [35S] methionine/106 cells was added to the media for 30 min. The amount of precipitated counts was measured by scintillation counting, and proteins were assayed according to specifications of the bicinchoninic acid protein assay kit (Sigma). Semiquantitative RT-PCR and Real Time Semiquantitative RT-PCR Analysis—Total RNA extraction, cDNA synthesis, PAI-1, and eEF1␣ PCR were performed as described previously [28]. The mRNA of PAI-1 was measured by real time semiquantitative RT-PCR using a PE Applied Biosystems Prism, model 7700 sequence detection instrument. Comparisons were analyzed by analysis of variance, and significance was calculated at p Ͻ 0.01 using the Scheffe F-test
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
