Tenascin-W inhibits proliferation and differentiation of preosteoblasts during endochondral bone formation

doi:10.1016/j.bbrc.2007.03.071

Biochemical and Biophysical Research Communications

Volume 356, Issue 4, 18 May 2007, Pages 935-941

https://doi.org/10.1016/j.bbrc.2007.03.071 Get rights and content

Abstract

We identified a cDNA encoding mouse Tenascin-W (TN-W) upregulated by bone morphogenetic protein (Bmp)2 in ATDC5 osteo-chondroprogenitors. In adult mice, TN-W was markedly expressed in bone. In mouse embryos, during endochondral bone formation TN-W was localized in perichondrium/periosteum, but not in trabecular and cortical bones. During bone fracture repair, cells in the newly formed perichondrium/periosteum surrounding the cartilaginous callus expressed TN-W. Furthermore, TN-W was detectable in perichondrium/periosteum of Runx2-null and Osterix-null embryos, indicating that TN-W is expressed in preosteoblasts. In CFU-F and -O cells, TN-W had no effect on initiation of osteogenesis of bone marrow cells, and in MC3T3-E1 osteoblastic cells TN-W inhibited cell proliferation and Col1a1 expression. In addition, TN-W suppressed canonical Wnt signaling which stimulates osteoblastic differentiation. Our results indicate that TN-W is a novel marker of preosteoblasts in early stage of osteogenesis, and that TN-W inhibits cell proliferation and differentiation of preosteoblasts mediated by canonical Wnt signaling.

Section snippets

Materials and methods

Cells and culture conditions. ATDC5 cells, MC3T3-E1 cells and bone marrow cells were cultured as previously described [1], [10].

RNA extraction and suppression subtractive hybridization. ATDC5 cells were cultured for a total of 5 days and were exposed to 1000 ng/ml Bmp2 or vehicle for10 h. Poly(A)⁺ RNA was isolated from Bmp2-untreated and Bmp2-treated ATDC5 cells as previously described [11] and analyzed by suppression subtractive hybridization (PCR-Select cDNA Subtractions Kit, Clontech

Identification of TN-W upregulated by Bmp2 in ATDC5 cells

To identify genes upregulated by Bmp2 in ATDC5 osteo-chondroprogenitors, we performed a two-step screening consisting of a PCR-based suppression subtractive hybridization followed by differential hybridization using the poly(A)⁺ RNA extracted from Bmp2-untreated and Bmp2-treated ATDC5 cells. We obtained a 500-bp cDNA fragment corresponding to the 3′-untranslated sequence of TN-W, and this cDNA fragment was then used as a probe to screen a mouse cDNA library generated from Bmp-treated ATDC5

Discussion

Tenascins are a family of four extracellular matrix glycoproteins [19]. In mesenchymal tissues, Tenascins contribute to matrix structure mediated by proper deposition of collagen fibers, and regulate cell morphology, growth, and migration by activating diverse intracellular signaling pathways [20]. During endochondral bone formation, Tenascin-C (TN-C) is initially expressed in condensed mesenchymal cells, and then is in both chondrocytes and osteobalsts [21]. Taking account of the lack of

Acknowledgments

We thank Janie Finch for editorial assistance. This work was supported by the National Institutes of Health, NIAMS AR49072 and P01 AR42919 (to B.d.C.).

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Cited by (21)

Refining the identity of mesenchymal cell types associated with murine periosteal and endosteal bone
2024, Journal of Biological Chemistry
Single-cell RNA-seq has led to novel designations for mesenchymal cells associated with bone as well as multiple designations for what appear to be the same cell type. The main goals of this study were to increase the amount of single-cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. We created an atlas of murine bone–associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-Cxcl12 abundant reticular (CAR), osteo-CAR, preosteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by periostin expression. Osteo-X, osteo-CAR, and preosteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in preosteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single-cell RNA-seq datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone.
Asporin stably expressed in the surface layer of mandibular condylar cartilage and augmented in the deeper layer with age
2017, Bone Reports
Citation Excerpt :
Among them, Sema3e inhibits osteoblast migration and decrease osteoclast formation (Hughes et al., 2012). Tenascin-N was reported to inhibit proliferation and differentiation of preosteoblasts during endochondral bone formation (Kimura et al., 2007). These also indicate that MCC is different from the other growth cartilage.
Mandibular condylar cartilage (MCC) exhibits dual roles both articular cartilage and growth center. Of many growth factors, TGF-β has been implicated in the growth of articular cartilage including MCC. Recently, Asporin, decoy to TGF-β, was discovered and it blocks TGF-β signaling. Asporin is expressed in a variety of tissues including osteoarthritic articular cartilage, though there was no report of Asporin expression in MCC. In the present study, we investigated the temporal and spatial expression of Asporin in MCC.
Gene expression profile of MCC and epiphyseal cartilage in tibia of 5 weeks old ICR mice were firstly compared with microarray analysis using the laser capture microdissected samples. Variance of gene expression was further confirmed by real-time RT-PCR and immunohistochemical staining at 1,3,10, and 20 weeks old. TGF-β and its signaling molecule, phosphorylated Smad-2/3 (p-Smad2/3), were also examined by immunohistochemical staining.
Microarray analysis revealed that Asporin was highly expressed in MCC. Real-time RT-PCR analysis confirmed that the fibrous layer of MCC exhibited stable higher Asporin expression at any time points as compared to epiphyseal cartilage. This was also observed in immunohistochemical staining. Deeper layer in MCC augmented Asporin expression with age. Whereas, TGF-β was stably highly observed in the layer. The fibrous layer of MCC exhibited weak staining of p-Smad2/3, though the proliferating layer of MCC was strongly stained as compared to epiphyseal cartilage of tibia at early time point. Consistent with the increase of Asporin expression in the deeper layer of MCC, the intensity of p-Smad-2/3 staining was decreased with age.
In conclusion, we discovered that Asporin was stably expressed at the fibrous layer of MCC, which makes it possible to manage both articular cartilage and growth center at the same time.
Tenascin-C is required for normal Wnt/β-catenin signaling in the whisker follicle stem cell niche
2014, Matrix Biology
Citation Excerpt :
For example, tenascin-W is highly expressed in the periosteum, which is the stem cell compartment of osteoblasts (Scherberich et al., 2004). The addition of tenascin-W to osteoblast cultures suppresses Wnt/β-catenin signaling accompanied by an inhibition of cell proliferation (Kimura et al., 2007). Co-localization of tenascin-C and β-catenin activation has also been observed in a specialized stem cell niche that enables repetitive tooth renewal in the alligator (Wu et al., 2013).
Whisker follicles have multiple stem cell niches, including epidermal stem cells in the bulge as well as neural crest-derived stem cells and mast cell progenitors in the trabecular region. The neural crest-derived stem cells are a pool of melanocyte precursors. Previously, we found that the extracellular matrix glycoproteins tenascin-C and tenascin-W are expressed near CD34-positive cells in the trabecular stem cell niche of mouse whisker follicles. Here, we analyzed whiskers from tenascin-C knockout mice and found intrafollicular adipocytes and supernumerary mast cells. As Wnt/β-catenin signaling promotes melanogenesis and suppresses the differentiation of adipocytes and mast cells, we analyzed β-catenin subcellular localization in the trabecular niche. We found cytoplasmic and nuclear β-catenin in wild-type mice reflecting active Wnt/β-catenin signaling, whereas β-catenin in tenascin-C knockout mice was mostly cell membrane-associated and thus transcriptionally inactive. Furthermore, cells expressing the Wnt/β-catenin target gene cyclin D1 were enriched in the CD34-positive niches of wild-type compared to tenascin-C knockout mice. We then tested the effects of tenascins on this signaling pathway. We found that tenascin-C and tenascin-W can be co-precipitated with Wnt3a. In vitro, substrate bound tenascins promoted β-catenin-mediated transcription in the presence of Wnt3a, presumably due to the sequestration and concentration of Wnt3a near the cell surface. We conclude that the presence of tenascin-C in whiskers assures active Wnt/β-catenin signaling in the niche thereby maintaining the stem cell pool and suppressing aberrant differentiation, while in the knockout mice with reduced Wnt/β-catenin signaling, stem cells from the trabecular niche can differentiate into ectopic adipocytes and mast cells.
Tenascins in stem cell niches
2014, Matrix Biology
Citation Excerpt :
One important mechanism by which tenascin-C promotes tumor angiogenesis involves downregulation of DKK1 and activation of Wnt signaling. Given the pleiotropic effects of DKK1, it is likely that multiple pro-angiogenic LRP5/6-dependent pathways (blocked by DKK1) are de-repressed due to DKK1 downregulation by tenascin-C. Interestingly, effects of tenascin-W on Wnt-signaling have been observed as well (Kimura et al., 2007). Similarly to tenascin-C, tenascin-W has been found in many stem cell niches as summarized in Section 2.
Tenascins are extracellular matrix proteins with distinct spatial and temporal expression during development, tissue homeostasis and disease. Based on their expression patterns and knockout phenotypes an important role of tenascins in tissue formation, cell adhesion modulation, regulation of proliferation and differentiation has been demonstrated. All of these features are of importance in stem cell niches where a precise regulation of growth versus differentiation has to be guaranteed. In this review we summarize the expression and possible functions of tenascins in neural, epithelial and osteogenic stem cell niches during normal development and organ turnover, in the hematopoietic and pro-inflammatory niche as well as in the metastatic niche during cancer progression.
Tenascin-W: An extracellular matrix protein associated with osteogenesis and cancer
2010, International Journal of Biochemistry and Cell Biology
Tenascin-W was the last member of a family of four large extracellular matrix glycoproteins to be discovered. The original member of the tenascin family, tenascin-C, has been widely studied due to its association with asthma, fibrosis, infection, inflammation and cancer. Recent studies report multiple common features between tenascin-W and tenascin-C in terms of structure, expression and function, especially in the context of tumorigenesis. Furthermore, specific functions for tenascin-W in osteogenesis have been revealed. This review presents an update on our current knowledge concerning tenascin-W and discusses potential medical applications of this cancer-enriched extracellular matrix protein.
Revisiting the Tenascins: Exploitable as Cancer Targets?
2022, Frontiers in Oncology

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Tenascin-W inhibits proliferation and differentiation of preosteoblasts during endochondral bone formation

Abstract

Section snippets

Materials and methods

Identification of TN-W upregulated by Bmp2 in ATDC5 cells

Discussion

Acknowledgments

Biochem. Biophys. Res. Commun.

Cell

Cell

Cell

FEBS Lett

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

Mol Cell Neurosci.

Dev. Cell

Dev. Cell

Int. J. Biochem. Cell Biol.

Differential expressions of BMP family genes during chondrogenic differentiation of mouse ATDC5 cells

Cell Struct. Funct.