Knockdown of the Shwachman-Diamond syndrome gene, SBDS, induces galectin-1 expression and impairs cell growth.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. The depletion of SBDS protein by RNA interference has been shown to cause inhibition of cell proliferation in several cell lines. However, the precise mechanism by which the loss of SBDS leads to inhibition of cell growth remains unknown. To evaluate the impaired growth of SBDS-knockdown cells, we analyzed Epstein-Barr virus-transformed lymphoblast cells (LCLs) derived from two patients with SDS (c. 183_184TA > CT and c. 258 + 2 T > C). After 3 days of culture, the growth of LCL-SDS cell lines was considerably less than that of control donor cells. By annealing control primer-based GeneFishing PCR screening, we found that galectin-1 (Gal-1) mRNA expression was elevated in LCL-SDS cells. Western blot analysis showed that the level of Gal-1 protein expression was also increased in LCL-SDS cells as well as in SBDS-knockdown 32Dcl3 murine myeloid cells. We confirmed that recombinant Gal-1 inhibited the proliferation of both LCL-control and LCL-SDS cells and induced apoptosis (as determined by annexin V-positive staining). These results suggest that the overexpression of Gal-1 contributes to abnormal cell growth in SBDS-deficient cells.

Mislocalization or low expression of mutated Shwachman-Bodian-Diamond syndrome protein.

Shwachman-Diamond syndrome (SDS) is an autosomal-recessive disorder characterized by exocrine pancreatic insufficiency and bone marrow failure. Mutations in the SBDS gene are identified in most patients with SDS. Recent studies have shown that SBDS is involved in ribosome biogenesis and is localized to the nucleolus. The significance of cellular localization in SBDS is unknown, particularly as SBDS does not exhibit canonical nuclear localization signals. In this study, we have constructed wild-type deletion mutants of the critical domains and disease-associated mutants of the SBDS gene. These constructs were expressed in HeLa cells to explore the subcellular distribution of normal and mutant proteins. Wild-type SBDS was detected in the nucleus. However, constructs lacking N-terminal Domain I and two disease-associated mutants (C31W and N34I) failed to localize SBDS to the nucleus. Moreover, the amount of mutated SBDS protein was decreased. When N-terminal Domain I was overexpressed in HeLa cells, the localization of endogenous SBDS protein was changed from nuclei to cytosolic fraction. These data indicate that the N-terminal Domain I is responsible for nuclear localization. Furthermore, low expression of SBDS, as exhibited in some of the disease-associated mutants, may be associated with the pathogenesis of SDS.