Tetraspanin CD82 Regulates Hematopoietic Stem and Progenitor Cell Quiescence & Regeneration

Abstract

The significant cellular demand of the hematopoietic system is maintained by a rare pool of tissue-specific, hematopoietic stem and progenitor cells (HSPCs) that are primarily found in a quiescent state. Upon hematopoietic stress, HSPCs undergo rapid cell cycle activation, but ultimately must return to quiescence to prevent hematopoietic exhaustion. While the diverse mechanisms used by HSPCs to regulate quiescence are still being defined, it is clear that a combination of cell-intrinsic and bone marrow niche-mediated signaling pathways are involved. Tetraspanins are a unique family of membrane scaffold proteins with the capacity to modulate intrinsic and extrinsic signaling, through the formation of tetraspanin-enriched microdomains. Collectively, tetraspanins, and the microdomains they assemble, act as molecular facilitators that spatiotemporally compartmentalize membrane-associated signaling and adhesion proteins, providing a means to regulate the cell-cell communication and signal transduction associated with a breadth of cellular processes. More specifically, studies from our laboratory identified a critical role for the tetraspanin CD82 in the regulation of HSPC maintenance where we find a reduction in long term-HSPCs (LT-HSPCs) in CD82 knockout (CD82KO) mice, resulting from increased HSPC activation and a reduction of quiescent G0 cells. To further delineate how the CD82 scaffold modulates HSPC quiescence, we transplanted CD82 overexpressing (CD82OE) leukemia cell model into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Despite the fact that CD82OE cells display similar proliferation rates to control cells in culture, once localized to the bone marrow, CD82OE cells demonstrate reduced proliferation and increased quiescence, as indicated by diminished BrdU incorporation and IVIS signal. Additional mechanistic insight is provided by analysis of cells expressing a mutant form of CD82, where three N-linked glycosylation sites are inhibited (Ngly-CD82). Ngly-CD82 cells do not exhibit the same level of quiescence as CD82OE cells when injected into NSG mice, demonstrating an important role for CD82 glycosylation in the regulation of HSPC quiescence. Mechanistically, the cytokine TGF-β plays an essential role in supporting HSPC quiescence and activation, as one of the most potent inhibitors of HSPC growth. To determine if CD82 plays a role in TGF-β signaling, we stimulated WT and CD82KO HSPCs with TGF-β and measured nuclear translocation of SMAD2/3, a downstream indicator of TGF-β activation. Upon TGF-β activation, CD82KO HSPCs display decreased canonical TGF-β signaling, although our data indicate that the non-canonical TGF-β signaling pathway is not impacted. Additionally, we analyzed TGF-β signaling in primary human CD34+ HSPCs, finding that CD34+ human HSPCs with low CD82 expression also have decreased SMAD2/3 nuclear translocation suggestive of a reduced response to TGF-β stimulation. Analysis of the cell line models with differential CD82 expression suggests that cells with higher CD82 expression have increased SMAD2/3 activation following TGFβ stimulation when compared to lower expressing CD82 cells or Ngly-CD82 cells, but only when cells are engaged with fibronectin. Together, these data indicate that the CD82 scaffold has the capacity to modulate TGFb signaling through niche-mediated interactions and implicate integrins in the modulation of signal transduction.