Early Growth Response (EGR)-1 Is a Key Regulator of Human Primary Bone Marrow Stroma Cells with a Dual Role in Proliferation and Hematopoietic Stroma Support Function

Hematopoietic stem cell (HSC) niches are specialized local tissue microenvironments that maintain HSCs and regulate HSC functions. Bone marrow mesenchymal stroma cells (BMSCs) are essential constituents of the HSC niche and they give rise to skeletal lineage cells as well as the hematopoietic stroma. Despite recent progress in the identification of the primary bone fide BMSC in human bone marrow, knowledge regarding the regulation of BMSC proliferation, differentiation and hematopoietic support function is incomplete.

We have previously shown that the expression of Early Growth Response 1 (EGR1) in primary BMSCs is substantially higher compared to the non-colony-forming cells and cultured stromal cells, respectively, indicating a possible role of EGR1 in BMSC regulation (Li et al., Stem Cell Reports, 3(6):965-74, 2014). EGR1 encodes a zinc finger transcription factor that exemplifies a group of immediate early response genes, which have diverse functions in cell growth, development and stress response. EGR1 regulates murine HSC proliferation and localization (Min et al., Cell Stem Cell, 2(4):380-91, 2008), but its role in stroma cell regulation has thus far not been investigated. This study therefore aimed to investigate the functional role of EGR1 in human primary BMSCs.

Quantitative gene expression analysis demonstrated that EGR1 expression was 126.9 ± 9.1-fold and 2.8 ± 0.2-fold higher in highly CFU-F (colony-forming unit, fibroblast) enriched human primary lin^neg/CD45^neg/CD271^pos/CD140a^neg bone marrow cells compared to the non-colony forming CD45^neg/CD271^neg cell population and CD45^neg/CD271^pos/CD140a^pos cells with minimum CFU-F activity. Furthermore, we confirmed that EGR1 expression in cultured cells was down-regulated in comparison with primary BMSCs.

Multi-lineage differentiation capacity and surface marker expression profiles of BMSCs were not affected by shRNA-induced EGR1 down-regulation. However, colony-forming capacity was enhanced (1.9 ± 0.1-fold) and population doubling times were reduced by EGR1 knockdown compared to scramble controls. Furthermore, the HSC stroma supporting function as measured by production of transplantable HSC in co-culture experiments (4-day serum-free culture supplemented with SCF 25 ng/ml, TPO 25 ng/ml, and FLT3L 25 ng/ml, 1×10⁴ CD34^pos seeded cord blood cells) was reduced when EGR1 knockdown cells were used as stroma layer.

On the other hand, whereas CFU-F formation was virtually absent in EGR1 overexpressing cells and population doubling times increased by 2.4 ± 0.3-fold, the hematopoietic stroma support function was considerably improved by EGR1 overexpression as indicated by increased percentages and absolute numbers of transplantable CD34^posCD90^pos cells produced in co-cultures with EGR1 overexpressing BMSCs as stromal cells (12,496 ± 887 for EGR1 overexpression vs 6,867 ± 1242 for GFP control). Accordingly, CD34^pos cells expanded on EGR1 overexpressing stroma exhibited improved in-vivo long-term reconstitution levels in NSG mice compared with cells grown on control stroma.

To investigate how EGR1 regulates proliferation and hematopoiesis support in BMSCs, transwell experiments were performed showing that both soluble factors and cell-to-cell contact were important for EGR1-mediated hematopoiesis support. Furthermore, proteomic analysis revealed that a group of proteins related to oxidative-reduction processes was downregulated whereas reactive oxygen species (ROS) levels were elevated in EGR1 knockdown cells. In addition, gene expression profiling analysis identified a number of hematopoietic support genes that were upregulated in EGR1 overexpressing cells compared with control cells.

In summary, our data indicate that EGR1 is an important BMSC regulator with a dual role on proliferation and hematopoietic stroma support function. EGR1 expression negatively regulated BMSC proliferation while positively regulating hematopoietic support function, which points to its possible role in coordinating the specific functions of BMSC in their different biological contexts.