Human marrow stromal cells activate monocytes to secrete osteopontin, which down-regulates Notch1 gene expression in CD34+ cells

The hematopoietic microenvironment, approximated in vitro by long-term marrow cultures (LTCs), consists of both nonhematopoietic-derived stromal elements and hematopoietic-derived monocyte/macrophages. To better understand the consequences of monocyte-stroma interactions, we compared gene expression profiles of CD14(+) peripheral blood monocytes and HS-27a stromal cells cultured alone and together in cocultures. Results from 7 separate experiments revealed 22 genes were significantly up- or down-regulated in the cocultures, with osteopontin (OPN) up-regulated more than 15-fold. The microarray OPN data were confirmed by Northern blot, real-time polymerase chain reaction (PCR), and by detection of OPN protein. High levels of OPN gene expression were also detected in 2- to 3-week-old primary LTCs. Using Transwells we determined that stromal cells were secreting a factor that upregulated OPN gene expression in CD14(+) cells. When CD34(+) cells were cultured in the presence of purified OPN, tyrosine phosphorylation of a 34-kDa molecule was increased 2- to 3-fold, an effect that was diminished in the presence of an OPN neutralizing monoclonal antibody. In addition, Notch1 gene expression was decreased 5-fold in OPN-treated CD34(+) cells. We conclude that interactions between stroma and monocytes can result in activities that limit the role of Notch signaling in hematopoietic regulation. (C) 2004 by The American Society of Hematology.