Cripto Regulates Hematopoietic Stem Cells As a Hypoxic Niche Related Factor Through the Cell Surface Receptor GRP78

Abstract 2332

Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of embryonic stem (ES) and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells (HSCs) has been unknown. We have reported that Cripto increases colony formation and maintains reconstitution ability of HSCs after growth in vitro and that this signaling is mediated through association of Cripto with cell surface receptor GRP78 which is expressed on a subset of HSCs (Miharada et al, Blood abstract, 2010). Here we show that Cripto/GRP78 signaling is important to sustain HSCs in the hypoxic niche. In order to clarify what pathway(s) are activated by Cripto/GRP78 signaling, we performed proteomics analysis using the HSC-like Lhx2 cell line. Two dimentional electrophoresis (2D-DIGE) of proteins was used to analyze Cripto stimulated- and unstimulated-Lhx2 cells. The findings show that several glycolytic metabolism-related proteins were up-regulated and/or phosphorylated by Cripto treatment, e.g. Pyruvate kinase, Phosphoglycerate kinase 1, and Triosephosphate isomerase. Glycolysis is active under hypoxia and it is known that HSC located in the endosteal area of bones are in a hypoxic environment. We therefore separately analyzed central HSCs (cHSCs) and endosteal HSCs (eHSCs) and their hypoxic/metabolic properties. The findings show that eHSCs exhibit a larger proportion of GRP78⁺HSCs than cHSC and are more dormant (18.9±4.3% of eHSCs and 5.2±3.2% of cHSCs). GRP78⁺HSCs exhibit a higher content of Pimonidazole high positive cells indicating more hypoxic cells (37.4±13.2% in GRP78⁺ and 10.2±6.4% in GRP78⁻, p=0.001), and they also have a higher proportion of MitoTracker (MT) low cells which indirectly represents higher glycolytic activity (0.29±0.02 in GRP78⁺ and 0.42±0.07 in GRP78⁻, as relative MFI, p=0.011). It is noteworthy that after 3 days culture with Cripto, GRP78⁺HSCs showed significantly lower increase in MT intensity relative to the control condition (7,993±3,223 with Cripto and 14,952±2,857 without Cripto, as MFI, p=0.018). To see how Cripto/GRP78 signaling is important in vivo, we analyzed endosteal niche cells. An endosteal cell analysis revealed that part of ALCAM⁺ and Sca-I⁺ cells which have been shown to have supportive ability for HSCs, expressed cell membrane associated Cripto on their cell surface. Moreover, injection of anti-GRP78 blocking antibody led to displacement of GRP78⁺HSCs from the endosteal area to the central marrow (16.9±5.2% with control IgG and 9.6±3.8% with blocking antibody, in eHSCs, p=0.023). Hypoxia-inducible factor 1α (HIF-1α) null mice, have been shown to exhibit failure in the maintenance of functional HSCs even though the immunophenotypic percentage of HSCs is normal. Since the Cripto promoter region has HIF-1 complex binding sites, we analyzed whether HIF-1α null mice exhibit alterations in the Cripto/GRP78 pathway. Analysis of HIF-1α null mice demonstrated a reduced number of GRP78⁺HSCs within the eHSC compartment (13.8±1.6% in Mx-Cre control and 5.3±2.5% in HIF-1α null mice, p=0.001) and decreased number of cell surface Cripto⁺ endosteal niche cells. Taken together, our study strongly suggests that the Cripto/GRP78 pathway is an important signal, as an intermediary of HIF-1 regulation, to maintain HSCs in the hypoxic environment of the endosteal niche, by inducing glycolytic metabolism in dormant HSCs.