Depletion of the Shwachman-Diamond syndrome gene product, SBDS, leads to growth inhibition and increased expression of OPG and VEGF-A
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 Studies of the yeast ortholog of SBDS suggest a role in ribosomal subunit function [[13] and J.B. Moore IV et al., unpublished data], and human SBDS has been found to associate with 60S ribosomal subunits [14].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.
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Cells and molecular reagents
HeLa and NIH3T3 cells were purchased from ATCC (Manassas, VA). Cells were grown in 5% CO2 at 37 °C in DMEM medium with 10% fetal calf serum and 1% penicillin and streptomycin. Reagents for Real-Time PCR were purchased from Bio-Rad (Hercules, CA). Vectors, siRNA plasmids and TaqMan-based Real-Time PCR oligos for human SBDS, murine Sbds, human and murine GAPDH genes were designed using the software provided at www.genscript.com.
Design of siRNA plasmid vectors against SBDS
Six potential SBDS (Genbank sequence #AY169963) siRNA sequences were
Depletion of SBDS inhibits growth and alters expression of genes in multiple pathways
To study the effect of depleting SBDS in mammalian cells, we created human HeLa and murine NIH3T3 cells stably expressing siRNA against SBDS mRNA. Each of the siRNA plasmids was confirmed to knock down human or mouse SBDS expression, by real-time PCR, except for siRNA #1, which did not reliably silence SBDS expression in stably transfected NIH3T3 cells, and we focused on clones stably expressing siRNA #4 and #7 (mixture of siRNAs #1–6) shown in Table 1. The growth of HeLa and NIH3T3 stable
Discussion
Constitutive and inducible knockdown of SBDS resulted in significant growth abnormalities, which mirrored those seen in yeast cells depleted of the Sdo1 ortholog ([13]and J.B. Moore IV et al., unpublished data) and in most other mammalian knockdown models [19], [20]. In inducible knockdown HeLa cells, we demonstrated modestly increased levels of apoptosis by flow cytometry, suggesting a partial contribution of this process to growth inhibition. In addition, we also found an increased propensity
Acknowledgments
We acknowledge funding support from the Shwachman-Diamond Syndrome Foundation and thank the Microarray Core Facility at Mount Sinai School of Medicine for the GeneChip experiments.
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Cited by (19)
M phase-specific interaction between SBDS and RNF2 at the mitotic spindles regulates mitotic progression
2023, Biochemical and Biophysical Research CommunicationsSBDS interacts with RNF2 and is degraded through RNF2-dependent ubiquitination
2022, Biochemical and Biophysical Research CommunicationsDiagnosis, Treatment, and Molecular Pathology of Shwachman-Diamond Syndrome
2018, Hematology/Oncology Clinics of North AmericaCitation Excerpt :SBDS may also be involved in marrow stromal function.76,77 Increased expression of vascular endothelial growth factor-A and osteoprotegerin are seen in SBDS knockdown cell lines, which are known to influence monocyte and macrophage migration, osteoclast differentiation, and angiogenesis.58 Targeted deletion of SBDS in murine osteoprogenitors resulted in significant marrow abnormalities including lymphopenia, leukopenia, and myelodysplasia in the setting of bony changes.57
Gene clustering analysis in human osteoporosis disease and modifications of the jawbone
2013, Archives of Oral BiologyCitation Excerpt :Interleukin 10, primarily produced by monocytes and bone morphogenetic proteins (BMPs) belonging to the group of multifunctional peptides (TGFβ superfamily) that control proliferation and differentiation in many cell types, yielded inconclusive results regarding an association with osteoporosis44,45 as attested to by the absence of these two genes from all the more recent reviews and meta-analyses regarding genome-wide association studies.1,12,31 Another two genes, although belonging to upper clusters, appeared not to be associated to osteoporosis after research performed among genome-wide-significant loci of GWAS meta-analyses1,12,31: IGF1, encoding a protein similar to insulin in function and structure, which, interacting with some variants of ESR2 (Oestrogen Receptor-β), influences the risk of fracture in postmenopausal women,46 and the growth factor encoded by VEGFA, that shows several effects on osteoclast differentiation, angiogenesis, and monocyte/macrophage migration.47 The last class B gene was TNFSF11, a member of the tumour necrosis factor (TNF) cytokine family, which is a ligand for osteoprotegerin and functions as a key factor for osteoclast differentiation and activation.48
Clinical and Molecular Pathophysiology of Shwachman-Diamond Syndrome. An Update.
2013, Hematology/Oncology Clinics of North AmericaCitation Excerpt :Around 90% of patients with SDS harbor mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene located on chromosome 7q11. SBDS encodes a novel protein involved in ribosomal maturation and implicated in additional functions, such as cell proliferation and mitosis,2 as well as in the stromal microenvironment.3,4 Although the majority (90%)5 of patients clinically diagnosed with SDS harbor mutations in the SBDS gene, phenotype varies widely between patients and even within the same individual over time, posing challenges for diagnosis and treatment.
Non-diamond Blackfan anemia disorders of ribosome function: Shwachman diamond syndrome and 5q- syndrome
2011, Seminars in HematologyCitation Excerpt :Furthermore, SBDS depletion in HeLa cells has been shown to result in significant upregulation of vascular endothelial growth factor-A (VEGF-A) and osteoprotegerin. Together these findings suggest an association between SBDS depletion and dysregulated angiogenesis and osteoclast activity.29 In addition to a role for SBDS in normal stromal function, a cell-intrinsic role in hematopoietic cells has been demonstrated in both human and mouse models.