Wise regulates bone deposition through genetic interactions with Lrp5.

In this study using genetic approaches in mouse we demonstrate that the secreted protein Wise plays essential roles in regulating early bone formation through its ability to modulate Wnt signaling via interactions with the Lrp5 co-receptor. In Wise-/- mutant mice we find an increase in the rate of osteoblast proliferation and a transient increase in bone mineral density. This change in proliferation is dependent upon Lrp5, as Wise;Lrp5 double mutants have normal bone mass. This suggests that Wise serves as a negative modulator of Wnt signaling in active osteoblasts. Wise and the closely related protein Sclerostin (Sost) are expressed in osteoblast cells during temporally distinct early and late phases in a manner consistent with the temporal onset of their respective increased bone density phenotypes. These data suggest that Wise and Sost may have common roles in regulating bone development through their ability to control the balance of Wnt signaling. We find that Wise is also required to potentiate proliferation in chondrocytes, serving as a potential positive modulator of Wnt activity. Our analyses demonstrate that Wise plays a key role in processes that control the number of osteoblasts and chondrocytes during bone homeostasis and provide important insight into mechanisms regulating the Wnt pathway during skeletal development.

Altered hematopoiesis in glypican-3-deficient mice results in decreased osteoclast differentiation and a delay in endochondral ossification.

Loss of function mutations in the gene encoding the heparan sulfate proteoglycan Glypican-3 (GPC3) causes an X-linked disorder in humans known as Simpson-Golabi-Behmel Syndrome (SGBS). This disorder includes both pre- and postnatal overgrowth, a predisposition to certain childhood cancers, and a complex assortment of congenital defects including skeletal abnormalities. In this study, we have identified a previously unrecognized delay in endochondral ossification associated with the loss of Gpc3 function. Gpc3 knockout animals show a marked reduction in calcified trabecular bone, and an abnormal persistence of hypertrophic chondrocytes at embryonic day 16.5 (E16.5). These hypertrophic chondrocytes down-regulate Type X collagen mRNA expression and undergo apoptosis, suggesting a normal progression of hypertrophic chondrocyte cell fate. However, replacement of these cells by mineralized bone is delayed in association with a marked delay in the appearance of osteoclasts in the bone in vivo. This delay in vivo correlates with a significant reduction in the capacity to form osteoclasts from bone marrow macrophage precursors in vitro in response to M-CSF and RANKL, and with a reduction in the numbers of bone-marrow-derived cells expressing the markers CD11b and Gr-1. Together, these results indicate selective impairment in the development of the common hematopoietic lineage from which monocyte/macrophages and PMNs are derived. This is the first report of a requirement for heparan sulfate, and specifically Gpc3, in the lineage-specific differentiation of these cell types in vivo.

Domain-specific modification of heparan sulfate by Qsulf1 modulates the binding of the bone morphogenetic protein antagonist Noggin.

We have reported previously that Noggin is a heparin-binding protein and associates with the cell surface through heparan sulfate proteoglycans, where it remains functional for the binding of bone morphogenetic proteins (BMPs). Here we report that the binding of Noggin to the cell surface is highly selective for heparan sulfate and that specific structural features are required for the interaction. Noggin binds most efficiently to heparin sequences composed of 10 or more monosaccharides; N-, 6-O-, and 2-O-sulfates contribute to this interaction. In addition, we have shown that the developmentally regulated endosulfatase Qsulf1 selectively removes sulfate groups from the 6-O position of sugars within the most highly sulfated S domains of heparan sulfate, whereas 6-O-sulfates in the NA/NS domains are not substrates for the enzyme. The activity of Qsulf1 in cells in culture results in the release of Noggin from the cell surface and a restoration of BMP responsiveness to the cells. This shows that Noggin binds to the S domains of heparan sulfate and provides evidence that, in addition to modulating Wnt signaling in vivo by the release of heparan sulfate bound Wnt, Qsulf1 also modulates BMP signaling by the release of surface-bound Noggin.

Heparan sulfate proteoglycans retain Noggin at the cell surface: a potential mechanism for shaping bone morphogenetic protein gradients.

Bone morphogenetic proteins (BMPs) are expressed broadly and regulate a diverse array of developmental events in vivo. Essential to many of these functions is the establishment of activity gradients of BMP, which provide positional information that influences cell fates. Secreted polypeptides, such as Noggin, bind BMPs and inhibit their function by preventing interaction with receptors on the cell surface. These BMP antagonists are assumed to be diffusible and therefore potentially important in the establishment of BMP activity gradients in vivo. Nothing is known, however, about the potential interactions between Noggin and components of the cell surface or extracellular matrix that might limit its diffusion. We have found that Noggin binds strongly to heparin in vitro, and to heparan sulfate proteoglycans on the surface of cultured cells. Noggin is detected only on the surface of cells that express heparan sulfate, can be specifically displaced from cells by heparin, and can be directly cross-linked to a cell surface proteoglycan in culture. Heparan sulfate-bound Noggin remains functional and can bind BMP4 at the plasma membrane. A Noggin mutant with a deletion in a putative heparin binding domain has reduced binding to heparin and does not bind to the cell surface but has preserved BMP binding and antagonist functions. Our results imply that interactions between Noggin and heparan sulfate proteoglycans in vivo regulate diffusion and therefore the formation of gradients of BMP activity.