Abstract
In a previous report we identified gene products that may be associated with stem cell maintenance by comparative transcriptome analysis of 2 functionally distinct stromal cell lines: HS-27a which supports primitive hematopoietic progenitor cells and HS-5 which stimulates differentiation. Since the ability of stromal cells to maintain stem cells is lost as the percentage of monocytes in stromal cultures increases, monokine-induced changes in HS-27a gene expression were also determined. An algorithm that combined these datasets was developed and used to identify factors produced by stroma that could be hypothesized to influence hematopoietic stem cell fate. Bone Morphogenetic protein 4 (BMP4) was identified and selected for study. Real time quantitative PCR confirmed that BMP4 gene expression was 9 fold higher in HS-27a than HS-5 and suppressed 6-fold by IL-1β. BMP4 protein secretion followed a similar pattern: HS-27a cells secreted 70 pg/ml BMP4 protein and treatment with IL-1β resulted in a 3 fold suppression; no BMP-4 secretion was detected from HS-5 cells. BMP4 is a critical factor for regulating hematopoietic development during embryogenesis and is involved in the regulation of T-cell differentiation by thymic stroma. However, relatively little is known about the role of bone marrow stromal derived BMP4 in adult hematopoiesis. BMP4 has been implicated in Notch signaling in muscle development. Since the Notch pathway is a key determinant of stem cell fate in hematopoiesis and the Notch ligand, Jagged 1, is differentially expressed in HS-5 and HS-27a cells, we investigated the effect of BMP4 on stromal expression of Jagged 1. We exposed HS-5 cells to BMP4 and assayed for Jagged 1 expression by western blot analysis. BMP4 induced both expression and modification of Jagged 1 in HS-5 cells. To determine if changes in Jagged 1 expression altered signaling between stroma and CD34+ cells, we exposed HS-5 cells to BMP4 for 24 hours. The medium was subsequently removed and replaced with fresh medium that did not contain BMP4. CD34+ cells were then added to the HS-5 cells and incubated at 37° for 2 to 24 hours. CD34+ cells were collected for RNA extraction and whole cell protein extracts were made from the HS-5 cells to verify changes in Jagged 1 expression. Pre-incubation of HS-5 cells with BMP4 prior to co-culture with CD34+ cells resulted in a consistent increase (1.4 to 2.0 fold) in gene expression of the notch regulated genes, Hey1 and Hes1. Although other, as yet undefined, BMP4 induced changes in marrow stroma may be responsible for this induction, we hypothesize that BMP4-induced changes in stromal Jagged 1 expression increases Hey 1 and Hes 1 gene expression via ligand engagement and activation of Notch signaling. Taken together, these studies suggest that BMP4 acts indirectly on progenitor cells via bone marrow stroma through a previously undescribed mechanism whereby BMP4 induces changes in stromal cell expression of the Notch ligand, Jagged1.
MLL-rearranged acute lymphoblastic leukaemia stem cell interactions with bone marrow stroma promote survival and therapeutic resistance that can be overcome with CXCR4 antagonism