Person:

Lotinun, Sutada

Mesenchymal stem cells (MSCs) are multi-potent cells that can differentiate into osteoblasts, adipocytes, chondrocytes and myocytes. This potential declines with aging. We investigated whether the sirtuin SIRT1 had a function in MSCs by creating MSC specific SIRT1 knock-out (MSCKO) mice. Aged MSCKO mice (2.2 years old) showed defects in tissues derived from MSCs; i.e. a reduction in subcutaneous fat, cortical bone thickness and trabecular volume. Young mice showed related but less pronounced effects. MSCs isolated from MSCKO mice showed reduced differentiation towards osteoblasts and chondrocytes in vitro, but no difference in proliferation or apoptosis. Expression of β-catenin targets important for differentiation was reduced in MSCKO cells. Moreover, while β-catenin itself (T41A mutant resistant to cytosolic turnover) accumulated in the nuclei of wild-type MSCs, it was unable to do so in MSCKO cells. However, mutating K49R or K345R in β-catenin to mimic deacetylation restored nuclear localization and differentiation potential in MSCKO cells. We conclude that SIRT1 deacetylates β-catenin to promote its accumulation in the nucleus leading to transcription of genes for MSC differentiation.

The effects of retinoids, the structural derivatives of vitamin A (retinol), on post-natal peak bone density acquisition and skeletal remodeling are complex and compartment specific. Emerging data indicates that retinoids, such as all trans retinoic acid (ATRA) and its precursor all trans retinaldehyde (Rald), exhibit distinct and divergent transcriptional effects in metabolism. Despite these observations, the role of enzymes that control retinoid metabolism in bone remains undefined. In this study, we examined the skeletal phenotype of mice deficient in retinaldehyde dehydrogenase 1 (Aldh1a1), the enzyme responsible for converting Rald to ATRA in adult animals. Bone densitometry and micro-computed tomography (µCT) demonstrated that Aldh1a1-deficient (Aldh1a1−/−) female mice had higher trabecular and cortical bone mass compared to age and sex-matched control C57Bl/6 wild type (WT) mice at multiple time points. Histomorphometry confirmed increased cortical bone thickness and demonstrated significantly higher bone marrow adiposity in Aldh1a1−/− mice. In serum assays, Aldh1a1−/− mice also had higher serum IGF-1 levels. In vitro, primary Aldh1a1−/− mesenchymal stem cells (MSCs) expressed significantly higher levels of bone morphogenetic protein 2 (BMP2) and demonstrated enhanced osteoblastogenesis and adipogenesis versus WT MSCs. BMP2 was also expressed at higher levels in the femurs and tibias of Aldh1a1−/− mice with accompanying induction of BMP2-regulated responses, including expression of Runx2 and alkaline phosphatase, and Smad phosphorylation. In vitro, Rald, which accumulates in Aldh1a1−/− mice, potently induced BMP2 in WT MSCs in a retinoic acid receptor (RAR)-dependent manner, suggesting that Rald is involved in the BMP2 increases seen in Aldh1a1 deficiency in vivo. Collectively, these data implicate Aldh1a1 as a novel determinant of cortical bone density and marrow adiposity in the skeleton in vivo through modulation of BMP signaling.

Osteoblasts are responsible for the formation and mineralization of the skeleton. To identify novel regulators of osteoblast differentiation, we conducted an unbiased forward genetic screen using a lentiviral-based shRNA library. This functional genomics analysis led to the identification of the microtubule-associated protein DCAMKL1 (Doublecortin-like and CAM kinase–like 1) as a novel regulator of osteogenesis. Mice with a targeted disruption of Dcamkl1 displayed elevated bone mass secondary to increased bone formation by osteoblasts. Molecular experiments demonstrated that DCAMKL1 represses osteoblast activation by antagonizing Runx2, the master transcription factor in osteoblasts. Key elements of the cleidocranial dysplasia phenotype observed in Runx2+/− mice are reversed by the introduction of a Dcamkl1-null allele. Our results establish a genetic linkage between these two proteins in vivo and demonstrate that DCAMKL1 is a physiologically relevant regulator of anabolic bone formation.

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stemand progenitor cell (HSPC) localization.HSPCegressed fromBMto spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions. (Blood. 2014;124(19):2937-2947).