Knockdown of SMOX and SAT1 to elucidate polyamine function within bone formation and proliferation in MSCs

Patients suffering from Snyder-Robinson Syndrome (SRS) exhibit debilitating features such as low muscle mass, dysmorphism, kyphoscoliosis, and osteoporosis. The known cause of this X-linked intellectual disorder is due to a loss of function mutation within the gene that encodes Spermine Synthase (SMS). The loss of SMS enzymatic activity, which catalyzes the conversion of spermidine into spermine, leads to an increase in the spermidine/spermine ratio in all documented cases of this disorder. SRS patients display decreased osteoblasts and osteoclasts, low cortical bone volume, and reduced trabecular meshwork. This project aims to clarify the role of polyamines within bone formation, and in turn, uncover the molecular mechanisms leading to osteoporosis observed in SRS patients. Due to deficient therapeutic intervention, it is imperative to continue researching the molecular mechanisms in which the polyamine pathway regulates bone formation. We have previously modelled SRS in vitro by silencing SMS expression in human bone marrow derived mesenchymal stromal cells (MSCs). As expected, impaired SMS expression in MSCs inhibits cell proliferation and blocks osteogenic differentiation. However, many questions remain uncovered. For example, it is still unclear whether SRS is due to the aggregation of spermidine, a decrease in spermine, or both. Here we suggest investigating the polyamine pathway in the context of bone formation in vitro. This will be done through both exogenous supplementation of polyamines throughout osteogenic differentiation, as well as downregulation of key enzymes involved in regulating spermidine and spermine levels. First, direct supplementation of polyamines will elucidate both the ability to transport polyamines within MSCs, as well as the effects throughout osteogenesis. Second, utilizing shRNAs, we will knockdown the genes encoding the enzymes spermine oxidase (SMOX) and spermine/spermidine N1-acetlytransferase (SAT1), which act as inverse reactions seen with SMS, catalyzing the conversion of spermine into either spermidine or N1-acetlyspermine, respectively. Following these experiments, we will observe the outcomes of polyamine modulation within osteogenic differentiation, proliferation, and gene expression. We hypothesize that silencing SMOX or SAT1 within MSCs will lead to the opposite effects of silencing SMS, allowing increased proliferation and osteogenic differentiation of MSCs. This hypothesis is grounded in the idea that spermine is necessary for MSC differentiation, and with the knockdown of the proposed genes, higher levels of spermine will permit increased osteocyte production.