A novel mRNA binding protein complex promotes localized plasminogen activator inhibitor-1 accumulation at the myoendothelial junction

Abstract

Objective: Plasminogen activator inhibitor-1 (PAI-1) has previously been shown to be key to the formation of myoendothelial junctions (MEJs) in normal and pathological states (eg, obesity). We therefore sought to identify the mechanism whereby PAI-1 could be selectively accumulated at the MEJ.

Methods and results: We identified PAI-1 protein enrichment at the MEJ in obese mice and in response to tumor necrosis factor (TNF-α) with a vascular cell coculture. However, PAI-1 mRNA was also found at the MEJ and transfection with a PAI-1-GFP with TNF-α did not demonstrate trafficking of the protein to the MEJ. We therefore hypothesized the PAI-1 mRNA was being locally translated and identified serpine binding protein-1, which stabilizes PAI-1 mRNA, as being enriched in obese mice and after treatment with TNF-α, whereas Staufen, which degrades PAI-1 mRNA, was absent in obese mice and after TNF-α application. We identified nicotinamide phosphoribosyl transferase as a serpine binding protein-1 binding partner with a functional τ-like microtubule binding domain. Application of peptides against the microtubule binding domain significantly decreased the number of MEJs and the amount of PAI-1 at the MEJ.

Conclusions: We conclude that PAI-1 can be locally translated at the MEJ as a result of a unique mRNA binding protein complex.

Figure 1. Characterization of PAI-1 and MEJ formation in response to a high fat diet or TNF-α

The number of beads per micrometer squared for EC, VSMC and MEJs for normal (−) and high fat diet (+) is shown in A. Metamorph analysis of changes in the number of MEJs per 10 micrometers, following treatment with 10 ng/mL TNF-α to the EC monolayers is shown in (B, top). Representative images for confocal microscopy of transverse VCCC sections stained with phalloidin for control or TNF-α treated conditions are shown in (B, bottom). The fold increase for total PAI-1 (ng/mL) or active PAI-1 (units/mL) on isolated VCCC fractions, in response to TNF-α, was calculated using PAI-1 specific ELISAs (C). In D, increases in PAI-1 mRNA in response to TNF-α were quantified for isolated EC, VSMC, and MEJ fractions, using quantitative rt-PCR. Bar in B is 10 micrometers; statistical comparisons were made between each cellular compartment with *=p<0.05.

Figure 2. Effects of TNF-α or a high fat diet on SERBP1 and Staufen localization at the MEJ

Transverse sections of VCCC sections stained for SERBP1 (green) and nuclei (blue), for control (A, top) or TNF-α (A, bottom) treated conditions demonstrates SERBP1 expression at the MEJ in response to TNF-α. In B, quantified immunoblots of VSMC, EC, and MEJ fractions isolated from the VCCC treated with (+) or without (−) TNF-α were blotted for SERBP1 and GAPDH as a loading control. In C, the expression of SERBP1 was quantified as the number of beads per micrometer squared for EC, VSMC, or MEJs. In D, transverse sections of VCCCs were stained for Staufen (green) and nuclei (blue), in control (D, top) or TNF-α (D, bottom) treated conditions demonstrates a lack of Staufen expression at the MEJ in response to TNF-α. In E, quantified immunoblots of VSMC, EC, and MEJ fractions isolated from the VCCC treated with (+) or without (−) TNF-α were blotted for Staufen and GAPDH as a loading control. The expression of Staufen was quantified as the number of beads per micrometer squared for VSMC, EC or MEJs (F). Bars in A and D are 10 micrometers and for all graphs, statistical comparisons were made between each cellular compartment with *=p<0.05.

Figure 3. Characterization of NAMPT as a microtubule binding protein

In A, using Quantitative DeCyder analysis, a 3D visualization from a 2D-DIGE analysis compares a single protein expression, identified by a magenta tracer, between isolated VSMC (S), MEJ (M), or EC (E) VCCC fractions, with a minimum 2.5 fold increase in protein expression in the MEJ fractions compared to EC or VSMC. All three spots were identified as NAMPT using mass spectroscopy (A, bottom). Quantified immunoblots of VSMC, EC, and MEJ fractions isolated from VCCCs treated with (+) or without (−) TNF-α, probed for NAMPT are shown in B (top). Transverse sections of VCCCs stained for NAMPT (green) and nuclei (blue) for control (B, bottom left) and TNF-α (B, bottom right) treated conditions demonstrates changes in NAMPT expression in response to TNF-α. In C, the number of gold beads corresponding to NAMPT was quantified against each cellular part of the coronary vessel wall. Immunoblots probed for NAMPT on supernatant (S) and pellet (P) fractions from a co-sedimentation microtubule binding assay microtubules are shown in D. For D, experimental paradigms include maintaining the microtubule (MT) length at 3 micrometers with increasing amounts of purified rNAMPT at 5, 20 and 50 micrograms (top) and maintaining the amount of purified rNAMPT at 15 micrograms and increasing the length of microtubules at 2, 6.5 and 16 micrometers. The bar in B is 10 micrometers and in all graphs, statistical comparisons were made between each cellular compartment with *=p<0.05.

Figure 4. NAMPT and SERBP1 interact at the MEJ in vitro and colocalize at the MEJ, in vivo

In A, co-immunstainings for SERBP1 (green) and NAMPT (red) on transverse sections of the VCCC are shown for control and TNF-α treated conditions. Interactions between SERBP1 and NAMPT were shown for isolated VCCC fractions by immunoprecipitating (IP) with SERPB1 (B, top) or NAMPT (B, bottom) and probing (WB) for NAMPT or SERBP1 respectively, with (+) or without (−) TNF-α. In C, representative TEM images of mouse coronaries from mice fed a normal (top) or high fat diet (bottom) are co-labeled for NAMPT using 10 nm gold beads (top enlargement, green arrowhead) and SERBP1 using 15 nm gold beads (top enlargement, red arrowhead). Colocalization of SERBP1 and NAMPT is shown in C (bottom enlargement, yellow arrowhead). In D, the distance between the 10 nm and 15 nm gold beads at the MEJ is averaged and compared between control and high fat conditions. In A, large arrows indicate single protein staining for SERBP1 (top arrow) or NAMPT (bottom arrow), white arrow heads indicate colocalization of SERBP1 and NAMPT. In C, “E” indicates endothelial cells, “S” indicates vascular smooth muscle cells. Bar in A is 5 micrometers, bars in C are 0.5 micrometers. In graph, statistical comparisons were made between each cellular compartment with *=p<0.05.

Figure 5. NAMPT anchoring is crucial for TNF-α induced increase in PAI-1 and MEJ formation, in vitro

A demonstrates quantitative immunoblot analysis of PAI-1 protein expression on isolated MEJ fractions for the following experimental paradigms, control (untreated), TNF-a (10 ng/mL), TNF-α + Scrambled peptide or TNF-α + MBD peptide. In B (left), the same experimental condition is shown for transverse sections of VCCCs stained with phalloidin. The number of MEJs for each paradigm was quantified and shown on right. Bar in B is 10 micrometers, *p<0.05.

Figure 6. Localization of PAI-1 mRNA by NAMPT anchoring is crucial for PAI-1 expression at the MEJ and MEJ formation

Summary of PAI-1 mRNA localization. Application of inflammatory stimulation (TNF-α or high fat diet, 1) induces a global increase in PAI-1 mRNA (2a), decreases Staufen expression at the MEJ (2b) and increases NAMPT and SERBP1 expression at the MEJ (2c). This subcellular organization promotes the formation of a PAI-1 RBP-complex, comprised of PAI-1 mRNA, SERBP1 and NAMPT (3). The PAI-1 RBP complex is stabilized at the MEJ, via microtubule binding by NAMPT (4), which allows for the localized translation of PAI-1 protein at the MEJ (5) and subsequent increase in MEJ formation (6).