Differential Effect of Extracellular Acidosis on the Release and Dispersal of Soluble and Membrane Proteins Secreted from the Weibel-Palade Body

Abstract

Proteins secreted from Weibel-Palade bodies (WPBs) play important roles in regulating inflammatory and hemostatic responses. Inflammation is associated with the extracellular acidification of tissues and blood, conditions that can alter the behavior of secreted proteins. The effect of extracellular pH (pH o ) on the release of von Willebrand factor (VWF), the VWFpropolypeptide (Proregion), interleukin-8, eotaxin-3, P-selectin, and CD63 from WPBs was investigated using biochemical approaches and by direct optical analysis of individual WPB fusion events in human endothelial cells expressing green or red fluorescent fusions of these different cargo proteins. Between pH o 7.4 and 7.0, ionomycin-evoked WPB exocytosis was characterized by the adhesion of VWF to the cell surface and the formation of long filamentous strands. The rapid dispersal of Proregion, interleukin-8, and eotaxin-3 into solution, and of P-selectin and CD63 into the plasma membrane, was unaltered over this pH o range. At pH o 6.8 or lower, Proregion remained associated with VWF, in many cases WPB failed to collapse fully and VWF failed to form filamentous strands. At pH o 6.5 dispersal of interleukin-8, eotaxin-3, and the membrane protein CD63 remained unaltered compared with that at pH o 7.4; however, P-selectin dispersal into the plasma membrane was significantly slowed. Thus, extracellular acidification to levels of pH o 6.8 or lower significantly alters the behavior of secreted VWF, Proregion, and P-selectin while rapid release of the small pro-inflammatory mediators IL-8 and eotaxin-3 is essentially unaltered. Together, these data suggest that WPB exocytosis during extracellular acidosis may favor the control of inflammatory processes.Local acidosis is associated with inflammation and ischemia and can have significant effects on the normal function of cells, tissues, cellular, and blood components, in particularly those associated with the immune, vascular, and hemostatic systems (1-8). Endothelial cells regulate inflammatory, vascular and hemostatic responses through the secretion of a wide range of bioactive molecules from specialized secretory organelles, the Weibel-Palade bodies (WPBs). 3The major WPB core proteins are von Willebrand factor (VWF) and the VWF-propolypeptide (Proregion). VWF is synthesized as a pre-proprotein comprising an N-terminal signal peptide (pre-), and several distinct repeating structural domains (termed A, B, C, and D) arranged as D1-D2-DЈ-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK (9). During translation, the signal peptide is removed to yield proVWF, which then undergoes disulfide-linked dimerization to produce proVWF dimers (10). The Proregion domains (D1-D2) are cleaved from the main peptide in the Golgi apparatus, and further disulfide bond formation produces VWF multimers. The two resulting proteins, VWF and Proregion are co-packaged into the WPB where they noncovalently associate to form ordered tubules in a pH-and Ca 2ϩ -dependent fashion (11). Other physiologically important WPB proteins include P-selectin, interle...

Protein mobilities and P-selectin storage in Weibel–Palade bodies

“…ssEGFP mobilities were the same in both cell types (P0.19). Coexpression of ProVWF-mRFP did not change D (P0.31; (Erent et al, 2007) it will not interact strongly with ProVWF (Babich et al, 2009;Hannah et al, 2005). Consistent with this, Proregion-EGFP was significantly more mobile than ProVWF-EGFP (P<10 -4 ).…”

“…These data suggest that immobilisation is unlikely to be due to a specific interaction between P-selectin and Proregion or VWF per se, but rather to the presence of the Proregion-VWF paracrystal. Consistent with this is the dramatic slowing of P-selectin release during WPB exocytosis under conditions that prevent complete collapse of the Proregion-VWF paracrystal, as indicated by retention of the rod-like WPB morphology (Babich et al, 2009). Analysis of P-selectin-EGFP mutants in WPBs of HUVECs and HEK293 cells showed that immobilisation depends on the size of the extracellular domain.…”

“…The large extracellular domain of P-selectin-EGFP could mediate immobilisation either through a specific interaction with Proregion-VWF tubules or by simply becoming entrapped as the tubules condense into a paracrystal (Berriman et al, 2009). Although the soluble extracellular domain of P-selectin alone is reported to bind the DЈ-D3 region of VWF (Michaux et al, 2006b), 2972 Journal of Cell Science 123 (17) endogenous P-selectin does not bind to VWF in resting HUVECs (Padilla et al, 2004), and does not associate with secreted VWF at the extracellular surface (Babich et al, 2009). Unlike acute exposure to NH 4 Cl, where the rod-like WPB morphology (resulting from the Proregion-VWF paracrystal) is retained, prolonged exposure to NH 4 Cl causes WPBs to round up and lose their luminal structure (Michaux et al, 2006a), reflecting the disruption of the Proregion-VWF paracrystal.…”

“…During WPB exocytosis intra-WPB pH rises, and the organelle hydrates (Erent et al, 2007). The reversible effect of pH on mobilities of small stored molecules would contribute, together with the formation of a fusion pore of restricted dimensions, to the regulation of their selective release during transient or lingering kiss fusion events (Babich et al, 2009;Babich et al, 2008).…”

“…Specific molecular shapes of the two molecules or weak interactions between eotaxin-3-EGFP and other ER components may account for the slower eotaxin-3 mobility. Strong interactions of this protein with endogenous ProVWF seem unlikely because eotaxin-3 does not associate with VWF or Proregion under neutral conditions (pH 7.4) (Babich et al, 2009), similar to those that exist in the HUVEC ER (pH 7.2) (Erent et al, 2007). Large differences in apparent D of ProVWF-EGFP relative to ssEGFP alone (~50-fold) is unlikely to be due entirely to their different molecular masses (the predicted ratio of D assuming globular shapes is ~3), but rather to other factors such as the highly prolate molecular shape of soluble ProVWF (Singh et al, 2006) or interactions of ProVWF-EGFP with ER-resident components, possibly through its extensive sugar modifications.…”