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, 17 (4), 670-680

A Unique Protein Kinase C-dependent Pathway for Tissue Factor Downregulation in Pericytes

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A Unique Protein Kinase C-dependent Pathway for Tissue Factor Downregulation in Pericytes

Laura J Sommerville et al. J Thromb Haemost.

Abstract

Essentials Many mediators increase tissue factor (TF) expression in a wide variety of cell types. The only known example of TF downregulation is by pericytes during wound healing angiogenesis. Downregulation of TF mRNA and protein in cultured pericytes is Protein Kinase C (PKC) dependent. Pericyte TF regulation is unique, since PKC mediates increased TF in all other cell types tested. SUMMARY: Background Embryonic and tumor-associated angiogenesis are linked to elevated expression of the procoagulant transmembrane receptor tissue factor (TF). In contrast, we have reported that high baseline TF expression by perivascular cells (pericytes) is dramatically reduced during angiogenesis at sites of wound healing. This is the only setting in which active TF downregulation has been reported, thus revealing a novel mechanism of TF regulation. Objectives To define the mechanisms underlying the unique pattern of TF expression in pericytes. Methods TF expression in primary cultures of human pericytes is not altered by angiogenic cytokines or growth factors, but is actively downregulated by phorbol 12-myristate 13-acetate (PMA). We characterized TF transcription, protein stability and trafficking in response to PMA. Results Exposure to PMA reduced TF mRNA synthesis and shortened the half-life of TF protein from 11 h to 4.5 h. Addition of PMA rapidly triggered endocytosis of cell surface TF, followed by degradation in lysosomes. Cell surface TF coagulant activity was maintained until internal stores were depleted. Reduction of TF transcription, TF endocytosis and enhanced degradation of TF protein were all blocked by broad-spectrum inhibitors of protein kinase C (PKC). This was a surprising finding, because PKC activation increases TF expression in other cell types that have been tested. Conclusions The unique PKC-dependent pathway of TF downregulation in pericytes suggests that TF downregulation may play a functional role in angiogenesis. Distinct pathways regulating pathological and physiological TF expression could be utilized to modulate TF expression for therapeutic purposes.

Keywords: microvessels; neovascularization, physiological; pericytes; protein kinase C; thromboplastin.

Conflict of interest statement

Disclosure of Conflicts of Interests

The authors state that they have no conflict of interest.

Figures

Figure 1.
Figure 1.
PMA actively downregulates TF in primary human pericytes. Pericyte cultures were treated with PMA for the indicated times. The mean +/− standard deviation (SD) of 3 independent experiments is shown. *p<0.05, **p<0.01, ***p<0.001. A. Expression of TF protein was assessed by western blot. B. For each time point the relative amount of TF protein expressed by PMA-treated cells is shown as a percentage of TF expressed by vehicle-treated cells. C. Activity of total TF as measured in lysed pericytes was determined by measuring cleavage of a FXa-specific chromogenic substrate as described in “Methods”. TF activity is presented as the rate of FXa generation. D. Expression of TF mRNA was quantitated by qRT-PCR. For each time point the amount of TF mRNA expressed by PMA-treated cells is presented as the fold change relative to TF mRNA expressed by vehicle-treated control cells.
Figure 2.
Figure 2.
PMA shortens the half-life of TF protein while leaving degradation of TF mRNA unaffected. The mean +/− standard deviation (SD) of 3 independent experiments is shown. **p<0.01, ***p<0.001. A. Pericytes were pre-treated with Cyclohexamide (CHX) prior to addition of vehicle or PMA for the indicated times. Degradation of TF protein was assessed by western blot. B. Relative amounts of TF protein expressed by CHX-treated cells are given as a percentage of TF expressed by non-CHX-treated control cells. C. Pericytes were pre-treated with Actinomycin D (ActD) prior to receiving vehicle or PMA for the indicated times. Degradation of TF mRNA was analyzed by qRT-PCR. The amount of TF mRNA expressed by ActD-treated pericytes relative to non-ActD-treated control cells is shown.
Figure 3.
Figure 3.
Inhibition of Protein Kinase C attenuates PMA-mediated downregulation of TF. Pericytes were pre-treated with Go 6983 or GFX prior to PMA for the indicated times. The mean + SD of 3 independent experiments is shown. *p<0.5, **p<0.1, ***p<0.001. A. TF protein expression was analyzed by western blot. B. For each time point the amount of TF protein is shown as a percentage of the TF expressed by control cells. C. Expression of TF mRNA was quantified by qRT-PCR. The amount of expressed TF mRNA is presented as the fold change relative to TF mRNA expressed by control cells. D. Activity of total TF in lysed pericytes is shown as the rate of FXa generation.
Figure 4.
Figure 4.
PMA increases internalization of TF from the pericyte surface. Where specified, pericytes were pre-treated with either Go 6983 or GFX prior to receiving PMA for the times indicated. The mean +SD of 3 independent experiments is shown. **p<0.01, ***p<0.001. A. Pericyte cultures were fixed and stained for TF (green). Prominent punctate staining for TF was seen 4 hours after PMA while TF was virtually absent 8 hours after PMA. B. The total TF content of pericytes was determined by western blotting of whole cell lysates. Internalized surface TF was determined by labeling cell surface proteins with biotin, isolating biotin-labeled proteins from cell lysates, and assessing their TF content by western blotting. C. Relative amounts of internalized TF are shown as a percentage of the total surface TF. D. Relative amounts of total TF are presented as a percentage of the total surface TF expressed by vehicle-treated control cells. E. Activity of surface TF was determined on whole cells by measuring the rate of FXa generation.
Figure 5.
Figure 5.
Internalized TF is degraded primarily in lysosomes. Pericytes were treated with MG132 or chloroquine prior to PMA for the indicated times. The mean + SD of 3 independent experiments is shown. **p<0.01, ***p<0.001. A. Relative amounts of internalized TF and expression of total TF were assessed by biotin labeling of cell surface proteins and western blotting as in “Methods”. B. The amounts of internalized TF are expressed as percentages relative to the total amount of TF expressed on the cell surface. C. Relative amounts of total TF expressed by PMA-treated pericytes are shown as percentages of TF expressed by control cells. D. Pericytes were stained for TF (green), the lysosomal marker LAMP-1 (red), and nuclei (blue, DAPI). E. Co-localization of TF and LAMP-1 was determined by calculating Pearson’s Correlation Coefficient with Image J, Fiji Plugin.

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