Targeting protein degradation to promote osteogenesis in osteoporosis – PROMOTOS

Transplantation of gene-modified mesenchymal stem cells (MSCs) for bone regeneration therapy has been evaluated extensively in recent years. However, very few studies have shown significant improvement of bone mass and function in osteoporotic animals by systemic injection of MSCs ectopically expressing a foreign gene. Aside from the clearance by lung and other tissues, the surface compositions of native MSCs may not favor their bone marrow (BM) migration and engraftment. To overcome this problem, Dr Su has previously have introduced the CXCR4 gene into mouse MSCs via adenovirus infection and further demonstrated that this modification could increase BM homing and engraftment of these cells which is crucial for osteoporosis therapy. In the meantime, to maximize the osteogenic differentiation of transplanted MSCs for replenishing bone matrixes that were lost during osteoporosis, it is essential to modulate both the internal and external regulatory factors. In this regard, upregulation of Runx2 (Cbfa-1), a master transcription factor for early osteogenesis, and activation of FGFR2, one of the novel growth factor receptors for osteoblast commitment, in transplanted MSCs may enhance the therapeutic efficacy of these cells, as demonstrated by Dr Marie. Interestingly, Runx2 protein levels as well as FGFR2 signaling are controlled tightly by an ubiquitination-mediated proteasomal degradation. Taking advantage of the expertise of two laboratories, we would like to co-develop novel strategies to promote the osteogenic differentiation of human MSCs for bone regeneration and osteoporosis therapies. Herein, we proposed a 3-year joint project to address the aforementioned questions. During the first project year, we will knock down the expression of Cbl, the E3 ubiquitin ligase responsible for downregulating FGFR2, in both mouse C3H10T1/2 and C2C12 MSC-like cells and human Wharton's jelly-derived MSCs via adenovirus infection and examine whether their in vitro osteogenic differentiation will be facilitated. In the meantime, we will also analyze whether transduction of the Runx2 gene into Cbl-downregulating MSCs will further facilitate their osteogenesis. During the second year, we will examine the transactivation capacity and stability of several Runx2 mutants whose lysine residues (potential ubiquitination sites) being replaced by either arginine or alanine. Moreover, we will assess whether Cbl is involved in modifying Runx2 even though three E3 ubiquitin ligases, Smurf1, WWP1 and CHIP have already been shown to play some roles. The feasibility of using systemic transplantation of gene-modified (CXCR4-overexpressing plus Cbl and/or other aforementioned E3 ligase-knockdowned) MSCs for osteoporosis therapy will be evaluated in third year. Ovariectomized mice treated with glucocorticoid will be the osteoporotic animal model. Intravenous injection of these cells and measurements of both biochemical markers (e.g. metabolite of type I collagen and osteocalcin) and biophysical parameters (bone stiffness, strength and mineral density) will be carried out primarily as described by Dr Su's team. Our results will not only provide crucial information regarding the feasibility of using genetically-manipulated MSCs in osteoporosis therapy but also improve our understanding of how Runx2 and FGFR2 as well as its downstream effectors are regulated by proteasomal degradation. In the meantime, by constantly exchanging information between Dr Yeu Su and Dr. Pierre Marie and sending students or post-doctoral fellows to each other's laboratory to learn different concepts and methods, a long-term cooperation between two laboratories should be firmly established.