Lipid rafts integrity is essential for prolactin-induced mitogenesis in mouse embryonic stem cells

Abstract

Embryonic stem cells, ESCs, retain the capacity to self-renew, yet, the protein machinery essential in maintaining this undifferentiated status remains largely undefined. Signalling interactions are initiated and enhanced at the plasma membrane lipid rafts, within constrains and regulation applied by the actin and tubulin cytoskeleton systems. First, we undertook a comprehensive approach using twodimensional gel electrophoresis and mass spectrometry analysis combined with Western blotting and immunofluorescence analyses at the single cell level to compile the proteome profile of detergentfree preparations of lipid rafts of E14 mouse embryonic stem cells. In comparison with the proteomic profiles of other membrane fractions, recovery of actin and tubulin network proteins, including folding chaperones, was impressively high. At equally high frequency we detected annexins, pleiotropic proteins that may bind membrane lipids and actin filaments to regulate important membrane processes, and we validated their expression in lipid rafts. Next, we tested whether lipid raft integrity is required for completion of mitogenic signalling pathways. Disruption of the rafts with the cholesterol sequestering methyl-β-cyclodextrin (MCD) greatly downregulated the mitotic index of ESCs, in a dose- and time of exposure-dependent manner. Moreover, MCD greatly reduced the mitogenic actions of prolactin, a hormone known to stimulate proliferation in a great variety of stem and progenitor cells. Taken together, our data postulate that lipid rafts in ESCs are in close association with the actin and tubulin cytoskeletons to support signal compartmentalization, especially for signalling pathways pertinent to symmetric divisions for self-renewal.

Development of a chemically disclosed serum-free medium for mouse pluripotent stem cells

“…Several studies have highlighted the importance of lipid rafts in the proliferation and self-renewal of mESCs ( Lee et al, 2010 ; Karouzaki et al, 2021 ). For effective LIF signaling transduction, LIF signaling receptors, LIFR and gp130, must be localized in lipid rafts, and cholesterol level in the cell membrane influences the lipid raft-dependent signal intensity ( Nishihara, 2018 ).…”

“…Based on the results of this study, mESC cell membrane may promote the formation of stable lipid rafts if cholesterol concentrations are increased, resulting in enhanced signal transduction of the other growth factors or cytokines ( Maja and Tyteca, 2022 ). Despite being two strong candidates ( Lee et al, 2010 ; Karouzaki et al, 2021 ; Ogawa et al, 2006 ; Sheng et al, 2014 ), cholesterol did not affect LIF and Wnt signaling ( Supplementary Figures S2A,B ). Further research is needed to fully understand the precise mechanisms by which cholesterol affects mESC proliferation, but this knowledge has the potential to shed light on the molecular mechanisms underlying mESC self-renewal and the regulation of cell cycle progression.…”