The Function of Twisted Gastrulation in Regulating Osteoclast Differentiation is Dependent on BMP Binding

Abstract

Proper regulation of osteoclast (OCL) function is critical for normal bone homeostasis. Bone morphogenetic protein (BMP) signaling and its regulation have been shown to have direct effects on OCL differentiation and activity. One of the major modulators of BMP signaling in the extracellular space is the secreted protein twisted gastrulation (TWSG1), which can inhibit BMP signaling and OCL differentiation. In this study we examine specific N-terminal regions of TWSG1 protein that have been previously proposed as BMP binding sites to determine whether TWSG1 binding to BMPs is required for its inhibitory effects on OCLs. We demonstrate that overexpression of wild type TWSG1 suppresses osteoclastogenesis, while overexpression of mutant TWSG1 proteins (W66A and N80Q/N146Q mutants), which cannot bind BMPs, leads to increased BMP signaling, enhanced osteoclastogenesis, increased resorptive activity, and expression of OCL-specific genes. Our results show that BMP binding is required for TWSG1-mediated inhibition of OCL formation and function, and validate the critical functional regions within the TWSG1 protein for these interactions.

Keywords: BMP; BONE MORPHOGENETIC PROTEIN; BONE RESORPTION; GLYCOSYLATION; OSTEOCLAST; TWISTED GASTRULATION.

Figure 1. Diagram of TWSG1 protein and expression of TWSG1 mutants

(A) Schematic of TWSG1 showing location of a single BMP binding domain, a secretory signal peptide (SP), and cysteine rich region (CR). Location of point mutations, N80Q/N146Q and W66A, are indicated. (B) Western blot against FLAG epitope of control, wild type (WT), and mutant (QQ and W66) TWSG1 adenoviruses in 293T cells.

Figure 2. Expression of TWSG1 mutants enhances osteoclast differentiation and size

(A) Images of TRAP positive mature OCLs. Cell area quantification of either control, WT-TWSG1 or mutant TWSG1 adenovirus for cells with three or more nuclei (B) or ten or more nuclei (C). Values represent average of at least three experiments run in triplicate. * indicates statistically significant, with a p value <0.05, compared to WT-TWSG1 transduced cells. Error bars represent standard deviation. Scale bar represents 0.25mm in length.

Figure 3. TWSG1 mutant expression increases osteoclast resorptive activity

(A) Binary image of calcium phosphate coated wells. (B) Quantification of percent area resorbed transduced with control, WT-TWSG1 or mutant TWSG1 adenoviruses. Values reflect average of at least three experiments run in duplicate. ** indicates statistically significant compared with a p value <0.005, *** indicates a p value <0.0005, compared to WT-TWSG1 transduced cells. Error bars represent standard deviation. Scale bar represents 0.25mm in length.

Figure 4. Increased p-Smad1/5/8 activation in osteoclasts expressing TWSG1 mutants

(A) Western blot of osteoclast transduced with control or mutant TWSG1 adenoviruses constructs. Cells were treated with M-CSF and RANKL for 3 days. Cell lysate was analyzed for the presence of p-Smad1/5/8 (Cell Signaling). Blots were striped and reblotted for Smad1/5/8 (Santa Cruz). (B) C2C12 cells starved for three hours then treated with serum free media, OCL differentiation media, or serum free media with 50 ng BMP2. Western blot of cell lysates analyzed for the expression of p-Smad1/5/8 (Cell Signaling), Smad1/5/8 (Santa Cruz).

Figure 5. TWSG1 mutant expression increases osteoclast specific gene expression

Real time RT-PCR comparing osteoclast gene expression of Nfatc1, Dc-stamp and cathepsin K (Ctsk) in OCL cultures transduced with control, WT-TWSG1 or mutant TWSG1 adenoviruses. Values were normalized to GAPDH. Values represent the average of at least three experiments run in duplicate. * indicates statistically significant, with a p value <0.05, compared to WT-TWSG1 transduced cells. A one-way ANOVA was used to determine significance. Error bars represent standard deviation.

Figure 6. WT-TWSG1 and mutant construct overexpression in Twsg1^−/− OCL

Quantification of osteoclasts from Twsg1^−/− mice transduced with control, WT-TWSG1 or mutant TWSG1 adenoviruses. Values represent average of at least three experiments run in duplicate. * indicates statistically significant, with a p value <0.05, compared to WT-TWSG1 transduced cells. Error bars represent standard deviation.