Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure and leukemia predisposition, pancreatic exocrine dysfunction, and skeletal abnormalities, manifesting as skeletal dysplasia and osteoporosis. Mutations in SBDS have been shown to cause SDS, but the function of the SBDS gene product is unclear. Accelerated angiogenesis has recently been described in bone marrow cells from SDS patients. To clarify the unknown function of SBDS, we performed experiments analyzing the cellular effects of depleting SBDS by constitutive or inducible RNA interference. The growth of HeLa and NIH3T3 cells constitutively depleted of SBDS was markedly hindered when compared to cells stably transfected with siRNA against an irrelevant control gene. However, the growth abnormality was related to a significantly increased propensity to apoptosis in NIH3T3 more than in HeLa cells. We believe these differences are related to inactivation of p53 in HPV-18 positive HeLa cells, suggesting that depletion of SBDS may partially sensitize cells to p53-mediated apoptosis. As in constitutive knockdown, HeLa cells induced by doxycycline to express siRNA against SBDS exhibited growth inhibition, which was associated with modestly increased levels of apoptosis, suggesting a partial contribution of this process in inducible knockdown cells. By microarray analysis of knockdown cells, we found marked differences in expression of genes in multiple pathways, and we chose to examine a selected subset more closely using quantitative PCR arrays. In constitutive and inducible SBDS-depleted HeLa cell clones, we found 3- to 6-fold elevated mRNA levels of osteoprotegerin (OPG or TNFRSF11B) and vascular endothelial growth factor-A (VEGF-A). We confirmed significant overexpression of both secreted proteins by ELISA from supernatants of SBDS-depleted HeLa cells. Osteoprotegerin and VEGF-A are known to have diverse effects on osteoclast differentiation, angiogenesis, and monocyte/macrophage migration, all processes that may be aberrant in SDS, and we propose that overexpression of these factors may contribute to its pathology.
Disclosures: No relevant conflicts of interest to declare.