Objective: Classical homocystinuria due to cystathionine beta-synthase (CBS) deficiency, is a rare autosomal recessively inherited disease characterized by the multiple involvement on different organs. While the most striking cause of morbidity and mortality is thromboembolism, patients develop a marked osteoporosis at early age along with many other skeletal abnormalities. As CBS normally converts homocysteine to cystathionine, the result of CBS deficiency is an accumulation of homocysteine. However, it seems to be conceivable that other mechanisms than high levels of homocysteine cause the defect in bone homeostasis, as betaine, the gold standard therapy for thromboembolism, is unable to completely prevent osteoporosis. Restoration of Hydrogen sulfide (H2S) levels in CBS knockout mice has been recently found to restore the BMMSC impairment and osteopenic phenotype. However CBS-H2S regulation of physiologic bone homeostasis is far from being fully elucidated. In particular, whether CBS expression is linked to the process of osteogenic differentiation of human MSCs is still unclear.
Methods: In vitro osteogenic differentiation of h-MSCs; ex-vivo comparison of h-MSCs and mature h-OBs and in vivo expression in human bone tissue biopsies were performed. h-MSC were separated into mineralizing vs non-mineraling based on Alizarin Red staining quantification at the end of culture. CBS and alkaline phosphatase (ALP) expression were evaluated by RT-PCR, immunohistochemistry and Western blot analyses.
Results: CBS was found to be expressed in human bone tissue as well as in ex-vivo primary cultures of bone cells. CBS expression was selectively up-regulated in h-MSCs undergoing mineralization (P<0.0001) while ALP was up-regulated in both mineralizing and non mineralizing h-MSCs (P<0.01). As a consequence, CBS expression significantly correlated (P<0.0001) with in vitro mineralization and outperformed ALP as a marker of mineralization. Consistently, CBS expression was significantly higher in mature h-OBs vs h-MSCs obtained from same patients (P<0.001). Finally, stimulation of h-MSCs with exogenous H2S (50200 μM) significantly stimulated (P<0.05), while pharmacological inhibition of CBS partially abrogated, in vitro mineralization.
Conclusions: CBS expression is unexpectedly correlated to the osteogenic differentiation of h-MSCs and identifies a new marker of mineralization behaviour. Moreover, functional data support the hypothesis that H2S may be the link between defective CBS and low bone mass in homocystinuria.
The study is funded by the Italian Ministry of Health (Grant Ricerca Finalizzata # PE-2011-02348395). Dr Gambari is partially supported by European Social Fund, Emilia-Romagna, Spinner Consortium.
Disclosure: The authors declared no competing interests.
27 - 30 Jun 2015