Colocalization of serum amyloid a with microtubules in human coronary artery endothelial cells

Abstract

Serum amyloid A (SAA) acts as a major acute phase protein and represents a sensitive and accurate marker of inflammation. Besides its hepatic origin, as the main source of serum SAA, this protein is also produced extrahepatically. The mRNA levels of SAA become significantly elevated following proinflammatory stimuli, as well as, are induced through their own positive feedback in human primary coronary artery endothelial cells. However, the intracellular functions of SAA are so far unknown. Colocalization of SAA with cytoskeletal filaments has previously been proposed, so we analyzed the colocalization of SAA with all three cytoskeletal elements: actin filaments, vimentin filaments, and microtubules. Immunofluorescent double-labeling analyses confirmed by PLA method revealed a strict colocalization of SAA with microtubules and a very infrequent attachment to vimentin while the distribution of actin filaments appeared clearly separated from SAA staining. Also, no significant colocalization was found between SAA and endomembranes labeled with the fluorescent lipid stain DiO₆. However, SAA appears to be located also unbound in the cytosol, as well as inside the nucleus and within nanotubes extending from the cells or bridging neighboring cells. These different locations of SAA in endothelial cells strongly indicate multiple potential functions of this protein.

Figure 1

SAA localizes with microtubules in untreated HCAEC, but not with actin filaments or vimentin. In HCAEC Von Willebrand factor (vWB) is demonstrated (red) in all cells (a). In (b) labeling of actin filaments, SAA, and nuclei is presented. In (c) (stack of optical sections) and (e) (larger magnification) SAA labeling can be detected attached to microtubules MT (arrowheads). Vimentin labeling together with SAA does not show any specific colocalization ((d)—stack of optical sections and (f) larger magnification). In (b, c, d, e and f) the labeling of SAA is red, of cytoskeletal elements is green, nuclei is blue. With PLA method colocalization of SAA with tubulin ((g)-TUB) or with vimentin ((h)-VIM) resulted in red reaction product strongly expressed only for tubulin and SAA. Anti-SAA polyclonal antibodies 3, 5, and 6, which target SAA peptides with amino acid sequences 27–44, 59–72, and 68–84, respectively, were used in the designated SAA panels. Bar = 10 μm.

Figure 2

Staining for SAA is found in the nucleus, nanotubes, and budding vesicles; however, its association with endomembranes remains unclear. SAA in HCAEC shows a predominantly nuclear localization, with a strong signal also concentrated in the juxta-nuclear cytoplasm (a′). In (a) and (a′) the cut views above and on the right of each panel represents the SAA mainly inside nuclei and in the perinuclear space. In HCAEC nuclei were stained with DAPI (blue), endomembranes were stained with 3,3′-dihexyloxacarbocyanin iodide (DiO₆) (green) and SAA was labeled with anti-SAA antibodies (red) (c). Merged labeling indicates SAA staining distinct from the endomembranes (b) and (c). Endoplasmic reticulum is shown by the purple arrows, mitochondrion with the green arrow. HCAEC were stained with DiO₆ and show colocalization of SAA with nanotubes (d). SAA was clearly detected at the tips of the filopodial protrusions (white arrows), and in vesicles dispersed outside the cells (white arrowheads). The association of SAA with endomembranes remains unclear. Anti-SAA polyclonal antibodies 3, 5, and 6, which target SAA peptides with amino acid sequences 27–44, 59–72, and 68–84, respectively, were used in the designated SAA panels.

Figure 3

Multimerization of SAA. Human recombinant SAA was blotted onto nitrocellulose membranes and overlayed with polyclonal antibodies against synthetic SAA human peptides ranging from the indicated amino acid sequences: lane 1: SAA1–17, lane 2: SAA14–30, lane 3: SAA27–44, lane 4: SAA40–63, lane 5: SAA59–72, lane 6: SAA68–84, lane 7: SAA79–94, and lane 8: SAA 89–104. HRP-labeled secondary goat antirabbit antibody was used for secondary incubation followed by luminol and chemiluminescent detection. The SAA monomer at around 12 kD is indicated.