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 up-regulated 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.
Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, D1-100, PO Box 19024, Seattle, WA 98109-1024, USA.
Mineo
M
Iwata
Norihiro
N
Awaya
Lynn
L
Graf
Christoph
C
Kahl
Beverly
B
Torok-Storb
D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D017931:DNA Primers; D005786:Gene Expression Regulation / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D006801:Humans; D008297:Male; D009000:Monocytes / Q000378:metabolism / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D053495:Osteopontin; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface / Q000235:genetics*; D012016:Reference Values; D012795:Sialoglycoproteins / Q000378:metabolism* / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D017154:Stromal Cells / Q000502:physiology*; D014157:Transcription Factors
D015703:Antigens, CD; D018952:Antigens, CD34; D017077:Culture Media, Conditioned; D017931:DNA Primers; C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D014157:Transcription Factors; D053495:Osteopontin; D019000:Phosphotyrosine