Endocytotic Albumin Uptake Pathways in Human Adipose Stem Cells and Connection to Intracellular Calcium Oscillations and the Neonatal Fc receptor

Introduction: Intracellular Ca2+ oscillations of unknown function occur in human adipose tissue stem cells (ASCs). In the present study, we investigated whether Ca2+ oscillations in ASCs from subcutaneous fat tissue derived from female patients were driving albumin endocytosis by involving the neonatal Fc receptor (FcRn), which is mediating the recycling of albumin and IgG.

Methods: Intracellular Ca2+ oscillations were monitored by Fluo-4 microfluorometry. Uptake of fluorescence-labeled albumin was assessed by confocal laser scanning microscopy in the presence of endocytotic pathway inhibitors. FcRn was inactivated either by use of the antagonizing antibody nipocalimab or siRNA technology.

Results: Endocytosis of albumin occurred by macropinocytosis (inhibition by cytochalasin D, wortmannin, and ethylisopropylamiloride [EIPA]), caveolae-dependent (inhibition by genistein and nystatin) and clathrin-mediated (inhibition by Dyngo-4a) pathways. In serum-free medium, Ca2+ oscillations were absent, but were induced by the addition of either fetal bovine serum, albumin, IgG or a stimulating antibody against FcRn. Consequently, FcRn expression was demonstrated in ASCs by Western blot analysis and immunohistochemistry, and colocalized with endocytosed albumin. Ca2+ oscillations were inhibited by the Ca2+ chelating agent BAPTA, the store-operated Ca2+ entry inhibitor SKF96365, the Ca2+-sensing receptor (CaSR) inhibitor NPS-2143, the macropinocytosis inhibitors cytochalasin D, wortmannin, and EIPA, the caveolae-dependent endocytosis inhibitors genistein and nystatin, and the clathrin-mediated endocytosis inhibitor Dyngo-4a. Uptake of fluorescence-labeled albumin was inhibited by agents interfering with Ca2+ oscillations and endocytosis blockers. Notably, not only intracellular albumin and IgG accumulation, but also Ca2+ oscillations were inhibited by the FcRn-blocking antibody nipocalimab, which interferes with the IgG binding site of FcRn. Moreover, siRNA-mediated downregulation of FcRn protein expression significantly reduced intracellular albumin content, the number of cells displaying Ca2+ oscillations, and the duration and amplitude of Ca2+ signals.

Conclusion: Our data suggest that Ca2+ oscillations in human ASCs regulate albumin uptake and presumably IgG recycling via FcRn.

Adipose stem cells; Adipose tissue stem cells; Albumin endocytosis; Calcium oscillations; Endocytosis; Mesenchymal stem cells; Neonatal Fc receptor; Nipocalimab.