Depletion of the Shwachman-Diamond syndrome gene product, SBDS, leads to growth inhibition and increased expression of OPG and VEGF-A

Abstract

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 RNA interference. The growth of HeLa cells constitutively depleted of SBDS was markedly hindered when compared to cells stably transfected with siRNA against an irrelevant control gene. Similarly, growth of HeLa cells induced to express siRNA against SBDS was specifically inhibited. Inducible SBDS knockdown was associated with modestly increased levels of apoptosis, suggesting a partial contribution of this process to growth inhibition. 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.

Introduction

Shwachman-Diamond syndrome (SDS, MIM 260400), first described in 1964, is an autosomal recessive disorder characterized by neutropenia, exocrine pancreatic insufficiency, growth abnormalities, and metaphyseal dysostosis [1], [2]. Neutropenia, found in 88–100% of patients, is the hematologic hallmark of SDS [reviewed in [3]], and in some patients there are also defects in neutrophil chemotaxis [4] and immune cell action [5]. Pancytopenia occurs in 10–65% of cases and may precede progressive bone marrow dysfunction characterized by severe aplastic anemia, myelodysplastic syndrome (MDS) or acute leukemia, usually of myeloid origin (AML) [6]. In addition to these hematologic abnormalities, pancreatic acinar dysfunction (and resulting fat maldigestion) is an invariable feature of SDS that often remits with age [7]. Many, but not all, patients exhibit skeletal abnormalities, which typically include delayed secondary ossification, abnormal development of growth plates and metaphyses, and generalized osteopenia [8] and low-turnover osteoporosis. Angiogenesis, a process involved in the development of a variety of hematological malignancies including MDS [9] and AML [10], may be accelerated in bone marrow cells from SDS patients [11].

Most cases of SDS are caused by mutations in the SBDS gene [12]. While SBDS is highly conserved in species as divergent as archaebacteria, little is known regarding the precise function of its gene product. Studies of the yeast ortholog of SBDS suggest a role in ribosomal subunit function [[13] and J.B. Moore IV et al., unpublished data], and human SBDS has been found to associate with 60S ribosomal subunits [14]. These data indicate that SDS may belong to an emerging class of inherited bone marrow failure syndromes linked to defects in ribosome synthesis [15]. However, a recent report has suggested an unexpected role for SBDS in stabilizing the mitotic spindle [16].

Loss of the mouse Sbds ortholog results in early embryonic lethality, indicating an important role in development [17]. To clarify the function of SBDS, we examined mammalian cells in which expression of SBDS was reduced by RNA interference.