Expression of glypican-4 in haematopoietic-progenitor and bone-marrow-stromal cells

Abstract

Heparan sulphate proteoglycans and the extracellular matrix of bone-marrow-stromal cells are important components of the microenvironment of haematopoietic tissues and are involved in the interaction of haematopoietic stem and stromal cells. Previous studies have emphasized the role of heparan sulphate proteoglycan synthesis by bone-marrow-stromal cells. In the present study we describe the expression of glypican-4 (GPC-4), belonging to the glypican family, in bone-marrow-stromal cells and haematopoietic-progenitor cells of human and murine origin. Expression of GPC-4 was shown on the mRNA-level by reverse transcription-PCR and Northern blot analysis. Amplification products were cloned and sequenced, to confirm these results. To analyze the expression of GPC-4 on the protein level, polyclonal antibodies against selected peptides were raised in rabbits. Western blot analysis showed expression of GPC-4 as a heparan sulphate proteoglycan in the human haematopoietic-progenitor cell line TF-1 and normal human bone marrow. These results were confirmed by FACS analysis of TF-1 cells. Furthermore, GPC-4-positive progenitor cells and stromal cells were enriched from normal human bone marrow by magnetic-cell sorting and analysed by confocal laser-scanning microscopy.

Use of a Phage Display Technique to Identify Potential Osteoblast Binding Sites Within Osteoclast Lacunae

“…(19,20) These receptors have no identifiable function as yet; however, they have been implicated in bone morphogenetic protein (BMP) signaling in Drosophila and as a cytokine presenting receptors in bone marrow cells. Perhaps of great interest, mutations in the GPC family lead to a syndrome known as Simpson-GolabiBehmel syndrome.…”

“…It does not have a cytosolic domain and is believed to function solely in cell attachment. (19,20) The Wnt family of genes is known to play a key role in cell differentiation and has been associated with skeletal development. (21,22) To show that GPC4 exists in osteoblasts and that its full-length sequence also has affinity for TRAP, we performed a mammalian two-hybrid study with a human TRAP cDNA and GPC4 obtained from a human osteoblast-like FIG.…”

Linking Hematopoiesis to Endochondral Skeletogenesis through Analysis of Mice Transgenic for Collagen X

“…For example, heparan sulfate PGs (HSPGs) have been proposed to orchestrate hematopoietic niches by sequestering cytokines and juxtaposing them with the marrow stroma to which they bind, and to hematopoietic progenitor cells. [17][18][19][20] Below, four mechanistic scenarios are summarized that could explain how a physical alteration in collagen X and hypertrophic cartilage may ultimately affect either one or both the spatial and chemical components within the chondro-osseous junction, and thus contribute toward the observed hematopoietic abnormalities in the collagen X mice.…”

“…13 Both of these molecules have been implicated as key components of marrow niches required for hematopoiesis, and likely mediate their affects by regulating cytokine bioactivity and availability. [17][18][19][20] It remains to be established whether collagen X supramolecular aggregates in the hypertrophic chondrocyte pericellular matrix require associations with specific classes of GAGs/PGs for stability and function, and if these interactions are needed for hematopoiesis.…”

Skeletogenesis and the Hematopoietic Niche

“…However, the contribution of various matrix components, in particular the heparan sulfate proteoglycans (HSPG), in establishing reservoirs of soluble factors for cell signaling and/or retention of HSPCs has been well established (as reviewed in (Rodgers et al 2008) also see (Gordon et al 1988;Roberts et al 1988;Siczkowski et al 1992;Verfaillie 1993;Allouche and Bikfalvi 1995;Bruno et al 1995;Klein et al 1995;Gupta et al 1996;Gupta et al 1998;Borghesi et al 1999;Siebertz et al 1999;Zweegman et al 2004;Rodgers et al 2008;. In support, our laboratory has shown altered localization of both hyaluronan and HSPGs within the hypertrophic cartilage zone of the growth plates in the collagen X mouse models that display altered hematopoiesis (Jacenko et al 2001), which directly links EO and the hypertrophic cartilage matrix to hematopoiesis (Jacenko et al 1993;Gress and Jacenko 2000;Jacenko et al 2001;Jacenko et al 2002;Sweeney et al 2008;Sweeney et al 2010).…”