Interaction of TGFβ and BMP Signaling Pathways during Chondrogenesis

TGFβ and BMP signaling pathways exhibit antagonistic activities during the development of many tissues. Although the crosstalk between BMP and TGFβ signaling pathways is well established in bone development, the relationship between these two pathways is less well defined during cartilage development and postnatal homeostasis. We generated hypomorphic mouse models of cartilage-specific loss of BMP and TGFβ signaling to assess the interaction of these pathways in postnatal growth plate homeostasis. We further used the chondrogenic ATDC5 cell line to test effects of BMP and TGFβ signaling on each other's downstream targets. We found that conditional deletion of Smad1 in chondrocytes resulted in a shortening of the growth plate. The addition of Smad5 haploinsufficiency led to a more severe phenotype with shorter prehypertrophic and hypertrophic zones and decreased chondrocyte proliferation. The opposite growth plate phenotype was observed in a transgenic mouse model of decreased chondrocytic TGFβ signaling that was generated by expressing a dominant negative form of the TGFβ receptor I (ΔTβRI) in cartilage. Histological analysis demonstrated elongated growth plates with enhanced Ihh expression, as well as an increased proliferation rate with altered production of extracellular matrix components. In contrast, in chondrogenic ATDC5 cells, TGFβ was able to enhance BMP signaling, while BMP2 significantly reduces levels of TGF signaling. In summary, our data demonstrate that during endochondral ossification, BMP and TGFβ signaling can have antagonistic effects on chondrocyte proliferation and differentiation in vivo. We also found evidence of direct interaction between the two signaling pathways in a cell model of chondrogenesis in vitro

The BMP Antagonist Follistatin-Like 1 Is Required for Skeletal and Lung Organogenesis

Follistatin-like 1 (Fstl1) is a secreted protein of the BMP inhibitor class. During development, expression of Fstl1 is already found in cleavage stage embryos and becomes gradually restricted to mesenchymal elements of most organs during subsequent development. Knock down experiments in chicken and zebrafish demonstrated a role as a BMP antagonist in early development. To investigate the role of Fstl1 during mouse development, a conditional Fstl1 KO allele as well as a Fstl1-GFP reporter mouse were created. KO mice die at birth from respiratory distress and show multiple defects in lung development. Also, skeletal development is affected. Endochondral bone development, limb patterning as well as patterning of the axial skeleton are perturbed in the absence of Fstl1. Taken together, these observations show that Fstl1 is a crucial regulator in BMP signalling during mouse development

Intracellular iron uptake is favored in Hfe-KO mouse primary chondrocytes mimicking an osteoarthritis-related phenotype

HFE-hemochromatosis is a disease characterized by a systemic iron overload phenotype mainly associated with mutations in the HFE protein (HFE) gene. Osteoarthritis (OA) has been reported as one of the most prevalent complications in HFE-hemochromatosis patients, but the mechanisms associated with its onset and progression remain incompletely understood. In this study, we have characterized the response to high iron concentrations of a primary culture of articular chondrocytes isolated from newborn Hfe-KO mice and compared the results with that of a similar experiment developed in cells from C57BL/6 wild-type (wt) mice. Our data provide evidence that both wt- and Hfe-KO-derived chondrocytes, when exposed to 50 mu M iron, develop characteristics of an OA-related phenotype, such as an increased expression of metalloproteases, a decreased extracellular matrix production, and a lower expression level of aggrecan. In addition, Hfe-KO cells also showed an increased expression of iron metabolism markers and MMP3, indicating an increased susceptibility to intracellular iron accumulation and higher levels of chondrocyte catabolism. Accordingly, upon treatment with 50 mu M iron, these chondrocytes were found to preferentially differentiate toward hypertrophy with increased expression of collagen I and transferrin and downregulation of SRY (sex-determining region Y)-box containing gene 9 (Sox9). In conclusion, high iron exposure can compromise chondrocyte metabolism, which, when simultaneously affected by an Hfe loss of function, appears to be more susceptible to the establishment of an OA-related phenotype.European Regional Development FundEuropean Union (EU) [EMBRC.PT Alg-01-0145-FEDER-022121, Norte-01-0145-FEDER-000012]Fundacao para a Ciencia e a TecnologiaPortuguese Foundation for Science and Technology [SFRH/BD/77056/2011]Portuguese Foundation for Science and TechnologyPortuguese Foundation for Science and TechnologyPortuguese Science and Technology FoundationPortuguese Foundation for Science and Technologyinfo:eu-repo/semantics/publishedVersio

Relating the Chondrocyte Gene Network to Growth Plate Morphology: From Genes to Phenotype

During endochondral ossification, chondrocyte growth and differentiation is controlled by many local signalling pathways. Due to crosstalks and feedback mechanisms, these interwoven pathways display a network like structure. In this study, a large-scale literature based logical model of the growth plate network was developed. The network is able to capture the different states (resting, proliferating and hypertrophic) that chondrocytes go through as they progress within the growth plate. In a first corroboration step, the effect of mutations in various signalling pathways of the growth plate network was investigated

Whole-Mount Skeletal Staining

The first step in almost every investigation of skeletal phenotypes is analysis of whole-mount skeletal preparations. Whole-mount skeletal staining permits evaluation of the shapes and sizes of skeletal elements in their appropriate locations. The technique is thus the major method for detecting changes in skeletal patterning. Because cartilage and bone can be distinguished by differential staining, this technique is also a powerful means to assess the pace of skeletal maturation. This protocol covers staining of the pre- and postnatal mouse skeleton using Alcian blue and Alizarin red to identify cartilage and bone, respectively