Characterization and functional analysis of osteoblast-derived fibulins in the human hematopoietic stem cell niche

Abstract

In the bone marrow stem cell niche, osteoblasts lining the endosteum are of major importance in supporting hematopoietic stem cell maintenance. Our objective was to analyze expression of the fibulins, highly conserved calcium-binding glycoproteins, which are components of the extracellular matrix of human osteoblasts, and to provide insights into their functional interactions with hematopoietic progenitor cells. Expression of the fibulins by human osteoblasts was determined by reverse transcription polymerase chain reaction analysis and by immunofluorescence staining and immunoblotting using fibulin-specific antisera. Recombinant fibulins were used in cell proliferation and differentiation assays with human CD34(+) hematopoietic progenitor cells. Adhesive interactions of CD34(+) cells with fibulins were investigated using cell-adhesion assays. Human osteoblasts strongly express and secrete fibulin-1 and -2. Whereas fibulin-1 is secreted in its intact form, fibulin-2 synthesized by human osteoblasts undergoes rapid proteolytic degradation. The matrix metalloproteinase-2, which is constitutively expressed by the osteoblasts, seems to be responsible for fibulin-2 degradation. Fibulin-1 showed an inhibitory effect on short-term CD34(+) hematopoietic progenitor cell proliferation. Both fibulin-1 and fibulin-2 were able to diminish erythroid and myeloid colony formation. The CD34(+) cell line KG1a strongly attached to fibulin-2, whereas magnetic-activated cell sorted CD34(+) hematopoietic progenitors did not adhere to either fibulin-1 or fibulin-2. On the other hand, fibulin-1 can strongly interfere with CD34(+) cell adhesion to fibronectin. Fibulins seem to be important components of the extracellular matrix of osteoblasts and are likely to negatively influence the proliferation rate of stem cells and the overall adhesive properties of the endosteal stem cell niche.