Lkb1 deletion in periosteal mesenchymal progenitors induces osteogenic tumors through mTORC1 activation.
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ABSTRACT:
SUBMITTER: Han Y
PROVIDER: S-EPMC6486357 | biostudies-literature | 2019 May
REPOSITORIES: biostudies-literature
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Project description:The translation of stem cell-based regenerative solutions from the laboratory to the clinic is often hindered by the culture conditions used to expand cell populations. Although fetal bovine serum (FBS) is widely used, regulatory bodies and safety concerns encourage alternative, xeno-free culturing practices. In an attempt to apply this approach to a bone-forming combination product of human periosteal progenitors (human periosteum derived cells) on a clinically used calcium phosphate carrier, FBS was substituted for human allogeneic serum (hAS) during cell expansion. It was found that cell proliferation was increased in hAS along with an apparent commitment to the osteogenic lineage, indicated by enhanced Runx2 expression, as well as alkaline phosphatase activity and matrix mineralization. Following analysis of signaling pathways, it was found that interferon-mediated signaling was downregulated, whereas JAK-STAT signaling was upregulated. STAT3 phosphorylation was enhanced in hAS-cultured human periosteum derived cells, inhibition of which ablated the proliferative effect of hAS. Furthermore, following in vivo implantation of hAS-cultured cells on NuOss scaffolds, enhanced bone formation was observed compared with FBS (71% increase, p < .001). Interestingly, the de novo-formed bone appeared to have a higher ratio of immature regions to mature regions, indicating that after 8 weeks implantation, tissue-formation processes were continuing. Integration of the implant with the environment appeared to be altered, with a decrease in calcium phosphate grain size and surface area, indicative of accelerated resorption. This study highlights the advantages of using humanized culture conditions for the expansion of human periosteal progenitors intended for bone regeneration.
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Project description:Congenital pseudarthrosis of the tibia (CPT) is a refractory condition characterized by the decreased osteogenic ability in tibial pseudarthrosis repair. Periosteal mesenchymal stem cells (PMSCs) are multipotent cells involved in bone formation regulation. However, the mechanisms underlying its role in CPT remain unclear. In this study, we observed downregulation of circ_0000888 and pleiotrophin (PTN), as well as upregulation of miR-338-3p in CPT derived PMSCs (CPT-dPMSCs). Our results demonstrated that circ_0000888 and PTN likely enhanced the viability, proliferation, and osteogenic ability of PMSCs, while miR-338-3p had the opposite effect. Further analysis confirmed the regulatory relationship circ_0000888 suppressed the activity of miR-338-3p and upregulated the expression of its downstream target PTN by binding to miR-338-3p, consequently promoting the viability and osteogenic differentiation ability of CPT-dPMSCs. Our findings unveil an unexpected link between circ_0000888/miR-338-3p/PTN in promoting osteogenic ability and indicate the potential pathogenic mechanisms of CPT.
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Project description:IntroductionInactivation of serine/threonine kinase 11 (STK11 or LKB1) is common in lung cancer, and understanding the pathways and phenotypes altered as a consequence will aid the development of targeted therapeutic strategies. Gene and protein expressions in a murine model of v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (Kras)-mutant lung cancer have been studied to gain insight into the biology of these tumors. However, the molecular consequences of LKB1 loss in human lung cancer have not been fully characterized.MethodsWe studied gene expression profiles associated with LKB1 loss in resected lung adenocarcinomas, non-small-cell lung cancer cell lines, and murine tumors. The biological significance of dysregulated genes was interpreted using gene set enrichment and transcription factor analyses and also by integration with somatic mutations and proteomic data.ResultsLoss of LKB1 is associated with consistent gene expression changes in resected human lung cancers and cell lines that differ substantially from the mouse model. Our analysis implicates novel biological features associated with LKB1 loss, including altered mitochondrial metabolism, activation of the nuclear respiratory factor 2 (NRF2) transcription factor by kelch-like ECH-associated protein 1 (KEAP1) mutations, and attenuation of the phosphatidylinositiol 3-kinase and v-akt murine thymoma viral oncogene homolog (PI3K/AKT) pathway. Furthermore, we derived a 16-gene classifier that accurately predicts LKB1 mutations and loss by nonmutational mechanisms. In vitro, transduction of LKB1 into LKB1-mutant cell lines results in attenuation of this signature.ConclusionLoss of LKB1 defines a subset of lung adenocarcinomas associated with characteristic molecular phenotypes and distinctive gene expression features. Studying these effects may improve our understanding of the biology of these tumors and lead to the identification of targeted treatment strategies.
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Project description:Bone morphogenetic protein 9 (BMP-9) is a member of the transforming growth factor (TGF)-beta/BMP superfamily, and we have demonstrated that it is one of the most potent BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). Here, we sought to investigate if canonical Wnt/beta-catenin signalling plays an important role in BMP-9-induced osteogenic differentiation of MSCs. Wnt3A and BMP-9 enhanced each other's ability to induce alkaline phosphatase (ALP) in MSCs and mouse embryonic fibroblasts (MEFs). Wnt antagonist FrzB was shown to inhibit BMP-9-induced ALP activity more effectively than Dkk1, whereas a secreted form of LPR-5 or low-density lipoprotein receptor-related protein (LRP)-6 exerted no inhibitory effect on BMP-9-induced ALP activity. beta-Catenin knockdown in MSCs and MEFs diminished BMP-9-induced ALP activity, and led to a decrease in BMP-9-induced osteocalcin reporter activity and BMP-9-induced expression of late osteogenic markers. Furthermore, beta-catenin knockdown or FrzB overexpression inhibited BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, resulting in immature osteogenesis and the formation of chondrogenic matrix. Chromatin immunoprecipitation (ChIP) analysis indicated that BMP-9 induced recruitment of both Runx2 and beta-catenin to the osteocalcin promoter. Thus, we have demonstrated that canonical Wnt signalling, possibly through interactions between beta-catenin and Runx2, plays an important role in BMP-9-induced osteogenic differentiation of MSCs.
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Project description:Mesenchymal stem cells (MSCs) are promising cell sources for regenerative medicine. Growing evidence has indicated that mechanical stimuli are crucial for their lineage-specific differentiation. However, the effect of mechanical loading on redox balance and the intracellular antioxidant system in MSCs was unknown. In this study, human bone marrow-derived MSCs (BM-MSCs) were subjected to cyclic stretch at the magnitude of 2.5%, 5%, and 10%. Cell proliferation, intracellular reactive oxygen species (ROS), expression of antioxidant enzymes, and osteogenic differentiation were evaluated. RNA was extracted and subjected to DNA microarray analysis. Sirtinol and compound C were used to investigate the underlying mechanisms involved silent information regulator type 1 (SIRT1) and AMP-activated protein kinase (AMPK). Our results showed that mechanical stretch at appropriate magnitudes increased cell proliferation, up-regulated extracellular matrix organization, and down-regulated matrix disassembly. After 3 days of stretch, intracellular ROS in BM-MSCs were decreased but the levels of antioxidant enzymes, especially superoxide dismutase 1 (SOD1), were up-regulated. Osteogenesis was improved by 5% stretch rather than 10% stretch, as evidenced by increased matrix mineralization and osteogenic marker gene expression. The expression of SIRT1 and phosphorylation of AMPK were enhanced by mechanical stretch; however, inhibition of SIRT1 or AMPK abrogated the stretch-induced antioxidant effect on BM-MSCs and inhibited the stretch-mediated osteogenic differentiation. Our findings reveal that mechanical stretch induced antioxidant responses, attenuated intracellular ROS, and improved osteogenesis of BM-MSCs. The stretch-induced antioxidant effect was through activation of the AMPK-SIRT1 signaling pathway. Our findings demonstrated that appropriate mechanical stimulation can improve MSC antioxidant functions and benefit bone regeneration.
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Project description:25-Hydroxyvitamin D3 [25(OH)D3] has recently been found to be an active hormone. Its biological actions are demonstrated in various cell types. 25(OH)D3 deficiency results in failure in bone formation and skeletal deformation. Here, we investigated the effect of 25(OH)D3 on osteogenic differentiation of human mesenchymal stem cells (hMSCs). We also studied the effect of 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3], a metabolite of 25(OH)D3. One of the vitamin D responsive genes, 25(OH)D3-24-hydroxylase (cytochrome P450 family 24 subfamily A member 1) mRNA expression is up-regulated by 25(OH)D3 at 250-500 nM and by 1α,25-(OH)2D3 at 1-10 nM. 25(OH)D3 and 1α,25-(OH)2D3 at a time-dependent manner alter cell morphology towards osteoblast-associated characteristics. The osteogenic markers, alkaline phosphatase, secreted phosphoprotein 1 (osteopontin), and bone gamma-carboxyglutamate protein (osteocalcin) are increased by 25(OH)D3 and 1α,25-(OH)2D3 in a dose-dependent manner. Finally, mineralisation is significantly increased by 25(OH)D3 but not by 1α,25-(OH)2D3. Moreover, we found that hMSCs express very low level of 25(OH)D3-1α-hydroxylase (cytochrome P450 family 27 subfamily B member 1), and there is no detectable 1α,25-(OH)2D3 product. Taken together, our findings provide evidence that 25(OH)D3 at 250-500 nM can induce osteogenic differentiation and that 25(OH)D3 has great potential for cell-based bone tissue engineering.
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