It has been shown that bone marrow mesenchymal stromal cells (MSCs) from patients with myelodysplastic syndromes (MDSs) display defective proliferative potential. We have probed the impaired replicative capacity of culture-expanded MSCs in MDS patients (n=30) compared with healthy subjects (n=32) by studying senescence characteristics and gene expression associated with WNT/transforming growth factor-β1 (TGFB1) signaling pathways. We have also explored the consequences of the impaired patient MSC proliferative potential by investigating their differentiation potential and the capacity to support normal CD34(+) cell growth under coculture conditions. Patient MSCs displayed decreased gene expression of the senescence-associated cyclin-dependent kinase inhibitors CDKN1A, CDKN2A, and CDKN2B, along with PARG1, whereas the mean telomere length was upregulated in patient MSCs. MDS-derived MSCs exhibited impaired capacity to support normal CD34(+) myeloid and erythroid colony formation. No significant changes were observed between patients and controls in gene expression related to TGFB1 pathway. Patient MSCs displayed upregulated non-canonical WNT expression, combined with downregulated canonical WNT expression and upregulated canonical WNT inhibitors. MDS-derived MSCs displayed defective osteogenic and adipogenic lineage priming under non-differentiating culture conditions. Pharmacological activation of canonical WNT signaling in patient MDSs led to an increase in cell proliferation and upregulation in the expression of early osteogenesis-related genes. This study indicates abnormal WNT signaling in MSCs of MDS patients and supports the concept of a primary MSC defect that might have a contributory effect in MDS natural history.
Starch binding domain-containing protein 1 (Stbd1) is a carbohydrate-binding protein that has been proposed to be a selective autophagy receptor for glycogen. Here, we show that mouse Stbd1 is a transmembrane endoplasmic reticulum (ER)-resident protein with the capacity to induce the formation of organized ER structures in HeLa cells. In addition to bulk ER, Stbd1 was found to localize to mitochondria-associated membranes (MAMs), which represent regions of close apposition between the ER and mitochondria. We demonstrate that N-myristoylation and binding of Stbd1 to glycogen act as major determinants of its subcellular targeting. Moreover, overexpression of non-myristoylated Stbd1 enhanced the association between ER and mitochondria, and further induced prominent mitochondrial fragmentation and clustering. Conversely, shRNA-mediated Stbd1 silencing resulted in an increase in the spacing between ER and mitochondria, and an altered morphology of the mitochondrial network, suggesting elevated fusion and interconnectivity of mitochondria. Our data unravel the molecular mechanism underlying Stbd1 subcellular targeting, support and expand its proposed function as a selective autophagy receptor for glycogen and uncover a new role for the protein in the physical association between ER and mitochondria.
Imbalances in endoplasmic reticulum (ER) homeostasis provoke a condition known as ER stress and activate the unfolded protein response (UPR) pathway, an evolutionary conserved cell survival mechanism. Here, we show that mouse myoblasts respond to UPR activation by stimulating glycogenesis and the formation of α-amylase-degradable, glycogen-containing, ER structures. We demonstrate that, the glycogen-binding protein Stbd1 is markedly upregulated through the PERK signalling branch of the UPR pathway and is required for the build-up of glycogen structures in response to ER stress activation. In the absence of ER stress, Stbd1 overexpression is sufficient to induce glycogen clustering but does not stimulate glycogenesis. Glycogen structures induced by ER stress are degraded under conditions of glucose restriction through a process which does not depend on autophagosome-lysosome fusion. Furthermore, we provide evidence that failure to induce glycogen clustering during ER stress is associated with enhanced activation of the apoptotic pathway. Our results reveal a so far unknown response of mouse myoblasts to ER stress and uncover a novel specific function of Stbd1 in this process, which may have physiological implications during myogenic differentiation.
The regions at which the ER and mitochondria come into close proximity, known as ER-mitochondria contact sites provide essential platforms for the exchange of molecules between the two organelles and the coordination of various fundamental cellular processes. In addition to the well-established role of ER-mitochondria interfaces in calcium and lipid crosstalk, emerging evidence supports that a proper communication between ER and mitochondria is critical for the regulation of mitochondrial morphology and the initiation of autophagy. Within this context, our recent data indicate that glycogen is targeted to ER-mitochondria contacts through the Stbd1 protein, a proposed autophagy receptor for glycogen. Glycogen-bound Stbd1 influences ER-mitochondria tethering and the morphology of the mitochondrial network. We here suggest possible roles of glycogen recruitment to ER-mitochondria contact sites. Stbd1-mediated targeting of glycogen to ER-mitochondria junctions could represent a mechanism through which glycogen is sequestered into autophagosomes for lysosomal degradation, a process described as glycogen autophagy or glycophagy. Additionally, we discuss a possible mechanism which links the observed effects of Stbd1 on mitochondrial morphology with the previously reported impact of nutrient availability on mitochondrial dynamics. In this model we propose that glycogen-bound Stbd1 signals nutrient status to ER-mitochondria junctions resulting in adaptations in the morphology of the mitochondrial network.
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