Role of GATA2 in the Maintenance of the Bone Marrow Microenvironment

(Background) The bone marrow (BM) microenvironment comprises multiple stem cell niches derived from BM mesenchymal stem cells (BM-MSCs). Previous in vitro analyses have suggested that transcription factor GATA2 plays an important role in adipocyte differentiation and hematopoietic supporting activity of BM-MSCs (Kamata et al. Haematologica 2014). However, the role of GATA2 in BM-MSCs in vivo remains uncharacterized.

(Method) Mice harboring a Gata2 exon 5 flanked by loxP sites ( Gata2 -floxed mice, designated Gata2^flox) were a kind gift from Dr. Camper (Charles et al. Molecular Endocrinology 2006). Gata2 was deletedin under the MSC-related promoters ( Nestin , Prx1 , and Lepr ) (Matsuzaki et al. Cell Stem Cell 2014) based on the Cre-loxP system ( Gata2^f/f/ Nes -ER-Cre, Gata2^f/f/ Prx1 -Cre, and Gata2^f/f/ Lepr -Cre mice) (The Jackson Laboratory). For transplantation analysis, 2x10⁵ BM cells from SJL (CD45.1⁺; The Jackson Laboratory) mice were intravenously injected into lethally irradiated CD45.2⁺Gata2^flox/flox/ER-Cre or CD45.2⁺Gata2^flox/flox mice (a total of 10 Gy). The recipient mice were injected intraperitoneally with 1 mg of tamoxifen (Sigma, USA) on days 29-31 and 36-38 after transplantation and subsequently analyzed on days 50-51. For the analysis, we focused on the CD45.1 donor cell fraction. To isolate MSCs, the compact bones were crushed with a pestle and treated with collagenase (Wako, Japan), after which Ter119^-CD45^- MSC fraction was separated using EasySep Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (StemCell Technologies, Canada). BM hematopoietic progenitors were separated and evaluated with both MACS (Miltenyi Biotech) and BD FACSAria II (BD Biosciences). For transcription profiling, a GeneChip Mouse Gene 2.0 ST Array (Affymetrix, USA) was used and the data was subsequently analyzed with the DAVID Bioinformatics Program (http://david.abcc.ncifcrf.gov/).

(Results) After confirming Gata2 deletion under each BM-MSC-related promoter ( Nestin , Prx1 , and Lepr ), we evaluated the hematological profile, total BM cell counts, BM progenitor frequency (LSK, CMP, GMP, MEP, and CLP), colony assay with sorted LSK fraction, BM histology, and colony-forming cell assay of BM-MSCs. Whereas we found a significant decrease in the colony number of Gata2^flox/flox/ Prx1 -Cre mice, the phenotypic alterations in BM-MSC-specific conditional Gata2 knockout mice are somewhat weak, implying that Gata2 deletion with a single marker might be insufficient. Thus, we next transplanted normal BM cells into irradiated Gata2^flox/flox/ER-Cre mice, after which Gata2 was knocked out by tamoxifen administration. In this case, Gata2 would be deleted in all cell types constituting the BM microenvironment but not the hematopoietic cells. Based on the analyses of the CD45.1⁺ donor cell fraction, we found a significant decrease in the percentage of the CMP fraction, whereas that of CLP was marginally increased (p = 0.081) by Gata2 knockout, suggesting that GATA2 expression in BM-MSCs might function in the differentiation of LSKs to CMPs.

To elucidate the underlying molecular mechanisms, we performed expression profiling with the MSCs obtained from Gata2^flox/flox/ER-Cre mice. Prior to the profiling analysis, RT-PCR analysis confirmed that the separated MSCs abundantly expressed MSC-related genes, including Nestin , Prx1 , Lepr , Cd29 , and Sca-1 . Based on the average of 2 independent datasets, we demonstrated that GATA2 knockout activated 110 and repressed 141 genes (> 2-fold). The repressed gene ensemble included adhesion molecules ( Itga11 , Itgb3 ), extracellular matrix ( Prg3 ), and genes involved in hematopoietic progenitor mobilization ( Mmp9 , Elane , Ctsg ). Activated genes included Fabp5 , involved in fatty acid metabolism, suggesting that GATA2 knockout might accelerate adipocyte differentiation in vivo . Gene Ontology analysis revealed significant enrichment of genes related to cell adhesion ( P = 4.8E−05) and chemotaxis ( P = 2.7E−08), suggesting that the expressions for extracellular signals in BM-MSCs might be regulated by GATA2.

(Conclusion) Besides its critical role in hematopoietic stem cells, GATA2 could also affect the function of BM-MSCs in vivo . Our data might offer a better understanding of the pathophysiology of bone marrow failure syndrome.