Only the Co-Transcriptional Activity of ß-Catenin Is Required for the Local Regulatory Effects in Hypertrophic Chondrocytes on Developmental Bone Modeling.

In hypertrophic chondrocytes, ß-catenin has two roles. First, it locally suppresses the differentiation of osteoclasts at the chondro-osseous junction by maintaining the pro-osteoclastic factor receptor activator of NF-κB ligand (RANKL) at low levels. Second, it promotes the differentiation of osteoblast-precursors from chondrocytes. Yet, ß-catenin is a dual-function protein, which can either participate in cell-cell adherens junctions or serve as a transcriptional co-activator in canonical Wnt signaling interacting with T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors. Hence, whenever studying tissue-specific requirements of ß-catenin using a conventional conditional knockout approach, the functional mechanisms underlying the defects in the conditional mutants remain ambiguous. To decipher mechanistically which of the two molecular functions of ß-catenin is required in hypertrophic chondrocytes, we used different approaches. We analyzed the long bones of newborn mice carrying either the null-alleles of Lef1 or Tcf7, or mice in which Tcf7l2 was conditionally deleted in the hypertrophic chondrocytes, as well as double mutants for Lef1 and Tcf7l2, and Tcf7 and Tcf7l2. Furthermore, we analyzed Ctnnb1 mutant newborns expressing a signaling-defective allele that retains the cell adhesion function in hypertrophic chondrocytes. None of the analyzed Tcf/Lef single or double mutants recapitulated the previously published phenotype upon loss of ß-catenin in hypertrophic chondrocytes. However, using this particular Ctnnb1 allele, maintaining cell adhesion function, we show that it is the co-transcriptional activity of ß-catenin, which is required in hypertrophic chondrocytes to suppress osteoclastogenesis and to promote chondrocyte-derived osteoblast differentiation. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Loss of the WNT9a ligand aggravates the rheumatoid arthritis-like symptoms in hTNF transgenic mice.

Agonists and antagonists of the canonical Wnt signaling pathway are modulators of pathological aspects of rheumatoid arthritis (RA). Their activity is primarily modifying bone loss and bone formation, as shown in animal models of RA. More recently, modulation of Wnt signaling by the antagonist Sclerostin has also been shown to influence soft-tissue-associated inflammatory aspects of the disease pointing towards a role of Wnt signaling in soft-tissue inflammation as well. Yet, nothing is known experimentally about the role of Wnt ligands in RA. Here we provide evidence that altering Wnt signaling at the level of a ligand affects all aspects of the rheumatoid arthritic disease. WNT9a levels are increased in the pannus tissue of RA patients, and stimulation of synovial fibroblasts (SFB) with tumor necrosis factor (TNF) leads to increased transcription of Wnt9a. Loss of Wnt9a in a chronic TNF-dependent RA mouse model results in an aggravation of disease progression with enhanced pannus formation and joint destruction. Yet, loss of its activity in the acute K/BxN serum-transfer induced arthritis (STIA) mouse model, which is independent of TNF signaling, has no effect on disease severity or progression. Thus, suggesting a specific role for WNT9a in TNF-triggered RA. In synovial fibroblasts, WNT9a can activate the canonical Wnt/ß-catenin pathway, but it can also activate P38- and downregulate NFκB signaling. Based on in vitro data, we propose that loss of Wnt9a creates a slight proinflammatory and procatabolic environment that boosts the TNF-mediated inflammatory response.

Wnt signaling and Loxl2 promote aggressive osteosarcoma.

Osteosarcoma (OS) is the most frequent primary malignant bone tumor in urgent need of better therapies. Using genetically modified mouse models (GEMMs), we demonstrate that Wnt signaling promotes c-Fos-induced OS formation via the actions of the collagen-modifying enzyme Loxl2. c-Fos/AP-1 directly regulates the expression of the Wnt ligands Wnt7b and Wnt9a in OS cells through promoter binding, and Wnt7b and Wnt9a in turn promote Loxl2 expression in murine and human OS cells through the transcription factors Zeb1 and Zeb2. Concordantly, inhibition of Wnt ligand secretion by inactivating the Wnt-less (Wls) gene in osteoblasts in c-Fos GEMMs either early or in a therapeutic setting reduces Loxl2 expression and progression of OS. Wls-deficient osteosarcomas proliferate less, are less mineralized and are enriched in fibroblastic cells surrounded by collagen fibers. Importantly, Loxl2 inhibition using either the pan-Lox inhibitor BAPN or a specific inducible shRNA reduces OS cell proliferation in vitro and decreases tumor growth and lung colonization in murine and human orthotopic OS transplantation models. Finally, OS development is delayed in c-Fos GEMMs treated with BAPN or with specific Loxl2 blocking antibodies. Congruently, a strong correlation between c-FOS, LOXL2 and WNT7B/WNT9A expression is observed in human OS samples, and c-FOS/LOXL2 co-expression correlates with OS aggressiveness and decreased patient survival. Therefore, therapeutic targeting of Wnt and/or Loxl2 should be considered to potentiate the inadequate current treatments for pediatric, recurrent, and metastatic OS.

CaMKII Signaling Stimulates Mef2c Activity In Vitro but Only Minimally Affects Murine Long Bone Development in vivo.

The long bones of vertebrate limbs form by endochondral ossification, whereby mesenchymal cells differentiate into chondrogenic progenitors, which then differentiate into chondrocytes. Chondrocytes undergo further differentiation from proliferating to prehypertrophic, and finally to hypertrophic chondrocytes. Several signaling pathways and transcription factors regulate this process. Previously, we and others have shown in chicken that overexpression of an activated form of Calcium/calmodulin-dependent kinase II (CaMKII) results in ectopic chondrocyte maturation. Here, we show that this is not the case in the mouse. Although, in vitro Mef2c activity was upregulated by about 55-fold in response to expression of an activated form of CaMKII (DACaMKII), transgenic mice that expressed a dominant-active form of CaMKII under the control of the Col2a1 regulatory elements display only a very transient and mild phenotype. Here, only the onset of chondrocyte hypertrophy at E12.5 is accelerated. It is also this early step in chondrocyte differentiation that is temporarily delayed around E13.5 in transgenic mice expressing the peptide inhibitor CaM-KIIN from rat (rKIIN) under the control of the Col2a1 regulatory elements. Yet, ultimately DACaMKII, as well as rKIIN transgenic mice are born with completely normal skeletal elements with regard to their length and growth plate organization. Hence, our in vivo analysis suggests that CaMKII signaling plays a minor role in chondrocyte maturation in mice.