Abstract
Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair.
Highlights
Human bone marrow stromal cells (BMSCs) constitute a heterogeneous population of clonogenic progenitors [1], characterized in vitro by the expression of CD90, CD73, CD105, CD146, and the ability to differentiate into osteoblasts, chondrocytes, and adipocytes [2,3,4]
These results indicate that cadherins are cleaved during osteogenic differentiation of Bone marrow stromal cells (BMSCs) by a PS1/γ-secretase-dependent mechanism, generating soluble fragments that are translocated to the nucleus
We evaluated the levels of luciferase activity in BMSCs transfected with the TCF reporter system and observed a significant impairment of β-catenin/TCF signaling after 5 days of osteoinduction in the presence of Dapt (Figure 5(e))
Summary
Human bone marrow stromal cells (BMSCs) constitute a heterogeneous population of clonogenic progenitors [1], characterized in vitro by the expression of CD90, CD73, CD105, CD146, and the ability to differentiate into osteoblasts, chondrocytes, and adipocytes [2,3,4]. Developmental studies using mice models showed that the differentiation of mesenchymal progenitors into the osteoblastic lineage requires the upregulation of Runx-2 [7, 8] downstream of β-catenin signaling [9, 10] This pathway is classically known to be activated by receptormediated canonical Wnt signaling, which turns off the β-catenin destruction complex composed by GSK3β (Glycogen synthase kinase), Axin, and APC (Adenomatous Polyposis Coli) [11, 12]. A possible explanation to these findings came from the observation that the Wnt coreceptor LRP5/6 (low-density lipoprotein receptor-related protein) is frequently associated with the adhesion protein Ncadherin in osteoprogenitor cells, which prevents its activation and the transduction of Wnt signaling [19] In this way, a more complex transduction signaling pathway, involving the regulation of cadherins, would be required to induce βcatenin signaling in these cells [20]. Cells were harvested by enzymatic digestion with 0.125% trypsin and 0.78 mM EDTA (both from Sigma-Aldrich) and expanded in DMEM with 10% FBS and antibiotics (expansion medium, EM)
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