Loss of Gpr68 Enhances Hematopoietic Stem Cell Function during Aging

Recent studies implicate metabolic plasticity in fine-tuning hematopoietic stem cell (HSC) homeostasis and function. HSC have been shown to rely on anaerobic glycolysis due to low energetic demand. In contrast, mitochondrial respiration is largely engaged to meet high energetic demand under certain pathophysiological processes, such as differentiation, aging and malignant transformation. The microenvironment, i.e. the HSC niche, has long been known as hypoxic. Glycolytic pathway is not only the metabolic adaptation to the hypoxic niche, but also essential for HSC function at least by providing prosurvival signals. However, glycolysis is reduced in HSC upon aging, which is associated with diminished regeneration ability of HSC. How glycolysis impacts HSC biology is unclear. G protein-coupled receptor 68 (GPR68, also known as OGR1) responds to extracellular acidosis, activating the phospholipase C (PLCb)/calcium pathway or the adenylyl cyclase (CA)/cAMP pathway. The end point product of glycolysis is lactate that leads to extracellular acidosis. This prompts us to examine the role of Gpr68 in HSC biology.

Among the hematopoietic stem and progenitor cell (HSPC) subpopulations, we found that Gpr68 expression was most enriched in mouse long-term HSC (LT-HSC, Lin^-Sca-1⁺c-Kit⁺CD34^-CD135^-). To understand the function of Gpr68 in HSC biology, we generated a genetic loss of function mouse model (i.e. the Gpr68^fl/fl;VavCre⁺ mice) where Gpr68 was specifically deleted in hematopoietic cells, including the most primitive LT-HSC. We found that the number of LT-HSC was unaltered in bone marrow (BM) from Gpr68^fl/fl;VavCre⁺ mice as compared to wild type (WT) control littermates (i.e. the Gpr68^wt/wt;VavCre⁺ mice), indicating that Gpr68 was not required for HSC homeostasis. To examine the role of Gpr68 in HSC function, we performed competitive bone marrow transplantation (cBMT). The donor-derived chimerism (i.e. the frequency of CD45.2⁺ cells) was increased in recipients with BM cells from Gpr68^fl/fl;VavCre⁺ mice (12-month old) compared to those from age-matched WT mice after primary cBMT, which was increased even more dramatically after secondary cBMT. This indicates enhanced HSC regeneration ability due to loss of Gpr68. Increased chimerism was observed in CD3⁺ T lymphocytes, B220⁺ B lymphocytes and CD11b⁺ myeloid cells, indicating that both lymphoid and myeloid lineages contributed to enhanced HSC function. Consistently, increased chimerism was also observed in BM HSPC subpopulations except the LT-HSC. Of note, increased chimerism was not observed in recipients that were transplanted with BM cells from young Gpr68^fl/fl;VavCre⁺ mice (2-month old). In addition, BM cells from Gpr68^fl/fl;VavCre⁺ mice produced slightly reduced colonies in methylcellulose, indicating that loss of Gpr68 did not lead to malignant transformation despite enhanced competitiveness of HSC.

To further understand the mechanism of Gpr68 in regulating HSC function, we studied cellular features of HSPC subpopulations. The frequency of cells in cell cycle G0 phase was unchanged in LT-HSC from Gpr68^fl/fl;VavCre⁺ mice as compared to WT mice, indicating loss of quiescence may not contribute to enhanced HSC function. Intriguingly, the frequency of Annexin V⁺ cells was largely reduced in LT-HSC from Gpr68^fl/fl;VavCre⁺ mice as compared to WT mice, suggesting that loss of Gpr68 provides a pro-survival signal in HSC. We next examined two main signaling pathways downstream of Gpr68. Cytosolic calcium levels were reduced in LT-HSC from Gpr68^fl/fl;VavCre⁺ mice as compared to WT mice, indicating Gpr68 may activate a calcium-related proapoptotic pathway. In contrast, intracellular cAMP levels were increased in HSPC, possibly from unknown compensatory mechanism. In addition, Gpr68 expression was reduced in LT-HSC from older mice (12-month old) compared to younger mice (2-month old), indicating reduced Gpr68 function at 12-month of age. This is in line with reduced glycolysis in aged HSC. Our study suggests that Gpr68 mediates a proapoptotic pathway in LT-HSC, providing a negative regulation, possibly at downstream of glycolysis, that limits HSC function. Our study also suggests that inhibiting Gpr68 function would enhance HSC function, especially in aged people or during bone marrow transplantation.