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.
Iwata
Mineo
M
Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, D1-100, PO Box 19024, Seattle, WA 98109-1024, USA.
Awaya
Norihiro
N
Graf
Lynn
L
Kahl
Christoph
C
Torok-Storb
Beverly
B
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