Stem Cells Int. 2016;2016:3865315. doi: 10.1155/2016/3865315. 

PS1/γ-secretase-mediated cadherin cleavage induces β-catenin nuclear translocation and osteogenic differentiation of human Bone Marrow Stromal Cells

Danielle C. Bonfim1,2,a, Rhayra B. Dias1,2,a, Anneliese Fortuna-Costa2,3, Leonardo Chicaybam4,5, Daiana V. Lopes1,2,a, Hélio S. Dutra1,2, Radovan Borojevic1,2, Martin Bonamino4, Claudia Mermelstein1, and Maria Isabel D. Rossi1,2 

1Institute of Biomedical Sciences, 2Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Brazil; 3Institute of Medical Biochemistry, 4Molecular Carcinogenesis Program, National Institute of Cancer, Rio de Janeiro, Brazil; 5Evandro Chagas Clinical Research Institute, Oswaldo Cruz Institute (FIOCRUZ), Rio de Janeiro, Brazil



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 indicate 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.

KEYWORDS: Bone Marrow Stromal Cells (BMSCs), Osteogenic differentiation, β-catenin, Cadherin, Wnt3a, PS1/γ-secretase

PMID: 28053606




Proper canonical Wnt signaling activation is required for normal osteogenesis (1). Evidence of this notion initially came from the observation that many pathologies that affect bone development resulted from alterations in the function of Wnt members. For example, the loss-of-function mutation of the Wnt co-receptor LRP5 (Low density lipoprotein receptor-related protein 5) causes “Osteoporosis-Pseudoglioma (OPPG)”, a condition that promotes a reduction in bone mass and predisposition to fractures (2). Similarly, mutations in the Wnt inhibitors SOST (Sclerostin) (3), DKK-1 (Dickoppf-1) (4) and sFRP-1 (secreted frizzeld related-protein -1) (5) all result in altered bone mass. Since, the role of Wnt members and the mechanisms that regulate their function became central themes in bone research. Specially, β-catenin – the intracellular transducer of the canonical pathway – is one of the most explored (1).

What is interesting about β-catenin is that it is a multifunctional protein. Under canonical Wnt activation, β-catenin translocates to the nucleus and acts as a transcription factor, in association with TCF/LEF (T Cell Factor/ Lymphoid Enhancer Factor) (6). Besides, β-catenin is a structural component of cell-cell adhesion complexes, connecting the C-terminal portion of cadherins to the actin cytoskeleton (7, 8). At first, these two functions of β-catenin were considered to be independent and carried out by two structurally different pools of the protein (9). Later it became clear that the intracellular pool of β-catenin was actually one, and the adhesion set was found to be unphosphorylated, which is exactly the form it works as a transcription factor (10). Soon enough, the idea that cadherins and Wnt signaling were tightly coordinated to regulate cellular functions was strengthened, but how this integration worked was an intriguing puzzle (11-13). One possibility was that cadherin complexes would act as β-catenin reservoirs, and their disassembly result in a fast release of β-catenin to the cytosol, to engage in signaling (11).

Aware that cadherins were specifically modulated during osteogenic differentiation of mesenchymal progenitors (14, 15), we therefore wondered whether Wnt signaling and cadherins were integrated to drive BMSCs to the osteogenic pathway. By then, the cleavage of cadherins by PS1/γ-secretase – an enzymatic complex responsible for the proteolysis of several transmembrane proteins – had been described (16, 17), and several studies showed that β-catenin release and translocation to nucleus were a direct result of it (18-21). In light of this knowledge, we asked whether cadherin cleavage occurred during osteogenic commitment of BMSCs, in association with Wnt signaling, to promote β-catenin-mediated transcription of target genes.

As expected, we confirmed the requirement of β-catenin signaling activation for BMSCs osteoinduction in vitro. But interestingly, this event was not promoted by Wn3a, a canonical Wnt protein. β-catenin-mediated transcription was only observed when cells were treated with dexamethasone, a known osteogenic inducer. Under this condition, we found that cadherins were cleaved, and nuclear translocation of β-catenin was enhanced. Since the pharmacological inhibition of PS1/γ-secretase inhibited β-catenin signaling and, consequently, osteogenic differentiation, we concluded that cadherin proteolysis is a fundamental event to trigger BMSCs differentiation towards the osteoblastic pathway. However, considering that β-catenin only translocates to nucleus when its degradation complex – whose activity is controlled by the activation of Wnt receptors – is inhibited, it is reasonable to speculate that signaling through Wnt proteins is simultaneously required.

Therefore, we propose a synergistic model (Figure 1) in which the triggering of osteogenic differentiation depends equally upon cadherin cleavage and canonical Wnt signaling. From its part, cadherin proteolysis would account for β-catenin release into the cytoplasm. Then, the activation of Wnt receptors would allow β-catenin stabilization and translocation to the nucleus. Following this rationale, if cadherin cleavage is not simultaneously stimulated, the activation of Wnt receptors alone would lead to alternative signaling cascades, such as the PDGFR transactivation axis reported by Caverzasio and colleagues (22), resulting in cell proliferation. In summary, the findings of our study strengthened the cadherin/Wnt integration notion and added a new piece in the challenging puzzle of BMSCs cell fate determination.




Figure 1. Proposed model of cadherin cleavage/Wnt signaling integration for BMSC cell fate determination. (A) When both cadherin cleavage and canonical Wnt signaling are inactive, the GSK3β/Axin/APC complex phosphorylates and marks free β-catenin for degradation. (B) Once an osteogenic stimuli is triggered, cadherins are cleaved by PS1/γ-secretase, releasing β-catenin from the membrane. In this state, the simultaneous Wnt signaling activation favors β-catenin translocation to nucleus and the subsequent activation of osteoblastic genes. (C) On the other hand, when only Wnt signaling is activated, that is, without cadherin cleavage, PDGF-R transactivation directs BMSCs to a proliferative state.



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