Overexpression of Twisted Gastrulation Inhibits Bone Morphogenetic Protein Action and Prevents Osteoblast Cell Differentiation in Vitro

Abstract

Twisted gastrulation (Tsg) is a secreted glycoprotein that binds bone morphogenetic protein-2 (BMP-2) and BMP-4 and can display both BMP agonist and antagonist functions. Tsg acts as a BMP agonist in chondrocytes, but its expression and actions on the differentiation of cells of the osteoblastic lineage are not known. We investigated the effects of Tsg overexpression by transducing murine ST-2 stromal and MC3T3 cells with a retroviral vector where Tsg is under control of the cytomegalovirus promoter and compared them to cells transduced with the parental vector alone. ST-2 cells were cultured in osteoblastic differentiating conditions in the presence or absence of BMP-2. Tsg overexpression precluded the appearance of mineralized nodules induced by BMP-2, led to a delay in the expression of osteoblastic gene markers, and decreased the responsiveness of ST-2 differentiating cells to PTH. BMP-2 induced the phosphorylation of signaling mothers against decapentaplegic-1/5/8, but not ERK, c-Jun N-terminal kinase, and p38. ST-2 cells overexpressing Tsg displayed an inhibition of BMP/signaling mother against decapentaplegic signaling. Tsg action was specific to BMP, because Tsg overexpression did not affect TGF-beta or Wnt/beta-catenin signaling pathways. Tsg also opposed MC3T3 cell differentiation and the expression of a mature osteoblast phenotype. In conclusion, Tsg overexpression inhibits BMP action in stromal and preosteoblastic cells and, accordingly, arrests their differentiation toward the osteoblastic pathway.

Twisted Gastrulation Modulates Bone Morphogenetic Protein-induced Collagen II and X Expression in Chondrocytesin Vitroandin Vivo

“…This is in line with a recent report showing the expression of TSG in osteogenic MC3T3 cells (53) and interference of TSG with BMP-induced osteoblast differentiation (54).…”

CCAAT/Enhancer-binding protein homologous protein (CHOP) decreases bone formation and causes osteopenia

“…It is not unusual for intracellular and extracellular proteins to be necessary for a biological effect, but when expressed in excess to be inhibitory. For example, Hes-1 is necessary for adipogenesis, but its overexpression inhibits adipogenesis, twisted gastrulation and connective tissue growth factor are necessary for BMP effects on osteoblastogenesis, but when in excess they can be inhibitory [48][49][50][51]. Similarly, CHOP is necessary for normal osteoblastic function, but when in excess, and under specific conditions in vivo, it may interact with intracellular proteins necessary for osteoblastic function and become inhibitory.…”

von Willebrand Factor Type C Domain-containing Proteins Regulate Bone Morphogenetic Protein Signaling through Different Recognition Mechanisms

“…Binding-It has been shown that CHL2, CV2, chordin, and Tsg inhibited BMP activity in a competitive manner in vitro (21,25,33,36). Co-precipitation experiment showed that CHL2 and chordin competed with type I receptors (BMPR-IA or BMPR- , 1000 nM chordin-VWC1 (f), 3000 nM CHL2-VWC3 (g), 4000 nM chordin-VWC3 (h), and 4000 nM CHL2-VWC1 (i), respectively, and 50 nM BMP-2 alone (j), were injected over the chip surface.…”