Wnt11 Promotes Osteoblast Maturation and Mineralization through R-spondin 2

Abstract

Wnt11 signals through both canonical (␤-catenin) and noncanonical pathways and is up-regulated during osteoblast differentiation and fracture healing. In these studies, we evaluated the role of Wnt11 during osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases ␤-catenin accumulation and promotes bone morphogenetic protein (BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11 dramatically increases expression of the osteoblast-associated genes Dmp1 (dentin matrix protein 1), Phex (phosphateregulating endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also increases expression of Rspo2 (R-spondin 2), a secreted factor known to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing Dmp1, Phex, and Bsp expression and promotes bone morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor (Tcf)/ ␤-catenin signaling with dominant negative Tcf blocks Wnt11-mediated expression of Dmp1, Phex, and Rspo2 and decreases mineralization. However, dominant negative Tcf fails to block the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11 signals through ␤-catenin, activating Rspo2 expression, which is then required for Wnt11-mediated osteoblast maturation.Wnt signaling is a key regulator of osteoblast differentiation and maturation. In mesenchymal stem cell lines, canonical Wnt signaling by Wnt10b enhances osteoblast differentiation (1). Canonical Wnt signaling through ␤-catenin has also been shown to enhance the chondroinductive and osteoinductive properties of BMP2 2 (2, 3). During BMP2-induced osteoblast differentiation of mesenchymal stem cell lines, cross-talk between BMP and Wnt pathways converges through the interaction of Smad4 with ␤-catenin (2).Canonical Wnt signaling is also critical for skeletal development and homeostasis. During limb development, expression of Wnt3a in the apical ectodermal ridge of limb buds maintains cells in a highly proliferative and undifferentiated state (4, 5). Disruption of canonical Wnt signaling in Lrp5/Lrp6 compound knock-out mice results in limb-and digit-patterning defects (6). Wnt signaling is also involved in the maintenance of postnatal bone mass. Gain of function in the Wnt co-receptor Lrp5 leads to increased bone mass, whereas loss of Lrp5 function is associated with decreased bone mass and osteoporosis pseudoglioma syndrome (7,8). Mice with increased Wnt10b expression have increased trabecular bone, whereas Wnt10b-deficient mice have reduced trabecular bone (9). Similarly, mice nullizygous for the Wnt antagonist sFrp1 have increased trabecular bone accrual throughout adulthood (10).Although canonical Wnt signaling regulates osteoblastogenesis and bone formation, the profile of endogenous Wnts that play a role in osteoblast differentiation and maturation is not well described. During development, Wnt11 is expressed in the perichondrium and in the axial skeleton and sternum (11). Wnt11 expression is incr...

Aberrantly expressed LGR4 empowers Wnt signaling in multiple myeloma by hijacking osteoblast-derived R-spondins

“…Whereas the presence of Wnt ligands in the BM niche is well established, expression of R-spondins in the BM microenvironment thus far has remained largely unexplored. Because autocrine R-spondins have been suggested to regulate differentiation of osteoblasts (38), which are important constituents of the MM niche (39,40), we hypothesized that osteoblasts could be a major source of R-spondins driving oncogenic Wnt signaling. Corroborating this hypothesis, we found expression of several R-spondins in (pre)osteoblasts and demonstrated that (pre)osteoblast-conditioned media robustly synergized with Wnt ligands in activating Wnt signaling.…”

“…However, the source of R-spondins remains to be defined. Prime candidates are osteoblasts, which have recently been suggested to use autocrine R-spondin for their differentiation (38) and, moreover, are key constituents of the MM niche (39,40). We therefore analyzed R-spondin mRNA expression in primary human osteoblasts (hOB) by qPCR.…”

Atherosclerotic Calcification: Wnt Is the Hint

“…Activation of Wnt11 through β‐catenin–independent Wnt signaling is essential for embryonic cardiac development 142. In terms of its role in osteogenic development, Wnt11 activation in MC3T3E1 preosteoblasts increases β‐catenin accumulation, promoting BMP‐induced osteoblast maturation and mineralization through canonical Wnt signaling 143. Wnt11 induces the expression of R‐spondin 2, a secreted enhancer of Wnt signaling, which stimulates osteoblastogenesis through increasing the expression of osteogenic factors, such as Dmp1 (dentin matrix protein 1), Phex (phosphate‐regulating endopeptidase homolog), and Bsp (bone sialoprotein) 143…”

Determining the Role of Sost and Sostdc1 During Fracture Healing

“…The behavior of Sostdc1-positive cells is similar to osteochondroprogenitor cells in the periosteum that, using lineage tracing, make a major contribution to the soft callus [194]. The role of Sostdc1 as a WNT antagonist, and the known role of WNT/β-catenin signaling to promote osteogenic [198] and chondrogenic [199,200] differentiation and maturation, suggests that Sostdc1 may act as the rate-limiting inhibitor of the differentiation of pluripotent periosteal cells during fracture repair. Indeed, we observe a temporal shift in the frequency of MSC and OB subpopulations during fracture repair in Sostdc1 -/-mice, as well as greater area-under-curve of OBs, but not of MSCs, indicating more rapid activation of migration, proliferation and differentiation of MSCs and OBs, and further supporting the role of Sostdc1 as an osteochondral progenitor gatekeeper.…”