Abstract

Angiogenesis is a hallmark of a variety of malignancies including multiple myeloma (MM). We have shown that MM patients express pleiotrophin (PTN), and this protein stimulates MM growth and prevents apoptosis. We have also demonstrated that PTN when combined with mCSF stimulates angiogenesis through the transdifferentiation (TD) of monocytes into endothelial cells. Our pathway-specific microarray results have shown that the Wnt pathway was up-regulated when monocytes were induced by mCSF and PTN. This signaling pathway plays an important role in cell growth, differentiation, function, and death. To further explore the role of Wnt/β-catenin signaling in the TD of monocytes into endothelial cells by PTN and mCSF, we examined whether monocytes induced to TD with this combination involved induction of the Wnt/β-catenin signal transduction pathway using immunohistochemical (IHC) staining and RT-PCR. Normal human blood monocytes were purified using density centrifugation followed by anti-CD14+ micro-bead affinity column selection. After one week of culture in the presence of mCSF and PTN, 5-10% of purified monocytes showed TD into endothelial cells as quantified by RT-PCR whereas cells treated with mCSF or PTN alone or without these factors did not. The cells also formed tube-like structures with positive staining for the endothelial cell markers Tie2, FLK-1, and vWF. The results showed β-catenin gene expression was markedly increased when CD14+ cells were treated with mCSF and PTN as demonstrated by RT-PCR. IHC studies showed CD14+ cells induced with mCSF and PTN expressed increased amounts of β-catenin in nuclear extracts but not cells treated with mCSF or PTN alone or without treatment. The nuclear gene or protein expression of β-catenin was blocked by adding casein kinase (CK)-1 inhibitor, a Wnt pathway inhibitor. We further examined whether CK-1 inhibitor can prevent TD of monocytes into endothelial cells by mCSF and PTN. CD14+ cells were exposed to mCSF and PTN or co-cultured with cells from fresh MM bone marrow or MM cell lines (U266 or MM1s). The Wnt inhibitor markedly decreased the formation of tube-like structures and endothelial cell gene expression. We also examined Smad7 gene expression. Smad7 is highly expressed in human hematopoietic stem cells (HSCs) and required for TGF-β-induced β-catenin expression. Our RT-PCR results showed that Smad7 gene expression in CD14+ cells induced with mCSF and PTN was not changed compared with untreated cells. These results suggest that the signaling pathways involved in TD of monocytes into endothelial cells are different from pathways involved in stem cell differentiation. We also examined Runx2 gene expression and this protein is a target of β-catenin /TCF1 involved in the stimulation of bone formation. Runx2 gene expression was up-regulated when monocytes were stimulated with mCSF and PTN. Runx2 gene expression was markedly increased by adding CK-1 inhibitor. Our findings further our understanding of the mechanisms involved in the TD of monocytes in endothelial cells and suggest the importance of the Wnt/β-catenin pathway in this critical early step in tumoral angiogenesis.

Author notes

Disclosure:Research Funding: Kramer, Annenberg & Skirball Foundation Grants.