ABSTRACT
Shwachman-Diamond syndrome (SDS) is a rare autosomal recessive disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, and skeletal abnormalities, caused by loss-of-function mutations in the SBDS gene, a factor involved in ribosome biogenesis. By analyzing osteoblasts from SDS patients (SDS-OBs), we show that SDS-OBs displayed reduced SBDS gene expression and reduced/undetectable SBDS protein compared to osteoblasts from healthy subjects (H-OBs). SDS-OBs cultured in an osteogenic medium displayed a lower mineralization capacity compared to H-OBs. Whole transcriptome analysis showed significant differences in the gene expression of SDS-OBs vs. H-OBs, particularly in the ossification pathway. SDS-OBs expressed lower levels of the main genes responsible for osteoblastogenesis. Of all downregulated genes, Western blot analyses confirmed lower levels of alkaline phosphatase and collagen type I in SDS-OBs than in H-OBs. Interestingly, SDS-OBs showed higher protein levels of p53, an inhibitor of osteogenesis, compared to H-OBs. Silencing of Tp53 was associated with higher collagen type I and alkaline phosphatase protein levels and an increase in SDS-OB mineralization capacity. In conclusion, our results show that the reduced capacity of SDS-OBs to mineralize is mediated, at least in part, by the high levels of p53 and highlight an important role of SBDS in osteoblast functions.
Subject(s)
Calcification, Physiologic , Osteoblasts/metabolism , Shwachman-Diamond Syndrome/metabolism , Tumor Suppressor Protein p53/metabolism , Cells, Cultured , Female , Humans , Male , Osteoblasts/pathology , Proteins/genetics , Proteins/metabolism , Shwachman-Diamond Syndrome/genetics , Shwachman-Diamond Syndrome/pathology , Tumor Suppressor Protein p53/geneticsABSTRACT
The imbalance between osteogenesis and adipogenesis, which naturally accompanies bone marrow senescence, may contribute to the development of bone-associated diseases, like osteoporosis. In the present study, using primary human mesenchymal stromal cells (hMSCs) isolated from trabecular bone, we assessed the possible effect of GH on hMSC differentiation potential into adipocytes. GH (5â¯ng/ml) significantly inhibited the lipid accumulation in hMSCs cultured for 14â¯days in lipogenic medium. GH decreased the expression of the adipogenic genes, CCAAT/enhancer-binding protein alpha (C/EBPα) and adiponectin (ADN) as well as the expression of two lipogenesis-related enzymes, lipoprotein lipase (LPL) and acethylCoA carboxylase (ACACA). In parallel, GH induced an increase in the gene expression and protein levels of osterix (OSX) and osteoprotegerin (OPG). These effects were ascribed to enhanced Wnt signaling as GH significantly reduced Wnt inhibitors, Dickkopf 1 (DKK1) and the secreted frizzled protein 2 (SFRP2), and increased the expression of an activator of Wnt, Wnt3. Accordingly, the expression of ß-catenin and its nuclear levels were raised. Wnt involvement in GH anti-adipogenic effect was further confirmed by the silencing of ß-catenin. In silenced hMSC, both the inhibitory effect of GH on the expression of the adipogenic genes, ADN and C/EBPα and the lipogenesis enzymes LPL and ACACA, were prevented together with the stimulatory effect of GH on the osteogenic genes OSX and OPG. The present study supports the hypothesis that when GH secretion declines as in aging, the fat in the bone-marrow cavities increases and the osteogenic capacity of the MSC pool is reduced due to a decrease in Wnt signaling.