Transcriptome-Wide Analysis Reveals a Role for Extracellular Matrix and Integrin Receptor Genes in Otic Neurosensory Differentiation from Human iPSCs.
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ABSTRACT:
SUBMITTER: Johnson Chacko L
PROVIDER: S-EPMC8509699 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Integrins provide the primary link between mesenchymal stem cells (MSCs) and their surrounding extracellular matrix (ECM), with different integrin pairs having specificity for different ECM molecules or peptide sequences contained within them. It is widely acknowledged that the type of ECM present can influence MSC differentiation; however, it is yet to be determined how specific integrin-ECM interactions may alter this or how they change during differentiation. We determined that human bone marrow-derived mesenchymal stem cells (hMSCs) express a broad range of integrins in their undifferentiated state and show a dramatic, but transient, increase in the level of ?5 integrin on day 7 of osteogenesis and an increase in ?6 integrin expression throughout adipogenesis. We used a nonfouling polystyrene-block-poly(ethylene oxide)-copolymer (PS-PEO) surface to present short peptides with defined integrin-binding capabilities (RGD, IKVAV, YIGSR, and RETTAWA) to hMSCs and investigate the effects of such specific integrin-ECM contacts on differentiation. hMSCs cultured on these peptides displayed different morphologies and had varying abilities to differentiate along the osteogenic and adipogenic lineages. The peptide sequences most conducive to differentiation (IKVAV for osteogenesis and RETTAWA and IKVAV for adipogenesis) were not necessarily those that were bound by those integrin subunits seen to increase during differentiation. Additionally, we also determined that presentation of RGD, which is bound by multiple integrins, was required to support long-term viability of hMSCs. Overall we confirm that integrin-ECM contacts change throughout hMSC differentiation and show that surfaces presenting defined peptide sequences can be used to target specific integrins and ultimately influence hMSC differentiation. This platform also provides information for the development of biomaterials capable of directing hMSC differentiation for use in tissue engineering therapies.
| S-EPMC3425135 | biostudies-literature
Project description:BACKGROUND:Human mesenchymal stem cell (hMSC) differentiation into osteoblasts has important clinical significance in treating bone injury, and the stiffness of the extracellular matrix (ECM) has been shown to be an important regulatory factor for hMSC differentiation. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through integrin α5/β1, FAK, and Wnt signaling, subsequently regulating the osteogenic phenotype of hMSCs. METHODS:hMSCs were cultured on tunable polyacrylamide hydrogels coated with fibronectin with stiffness corresponding to a Young's modulus of 13-16 kPa and 62-68 kPa. After hMSCs were cultured on gels for 1 week, gene expression of alpha-1 type I collagen, BGLAP, and RUNX2 were evaluated by real-time PCR. After hMSCs were cultured on gels for 24 h, signaling molecules relating to integrin α5 (FAK, ERK, p-ERK, Akt, p-Akt, GSK-3β, p-GSK-3β, and β-catenin) were evaluated by western blot analysis. RESULTS:Osteogenic differentiation was increased on 62-68 kPa ECM, as evidenced by alpha-1 type I collagen, BGLAP, and RUNX2 gene expression, calcium deposition, and ALP staining. In the process of differentiation, gene and protein expression of integrin α5/β1 increased, together with protein expression of the downstream signaling molecules FAK, p-ERK, p-Akt, GSK-3β, p-GSK-3β, and β-catenin, indicating that these molecules can affect the osteogenic differentiation of hMSCs. An antibody blocking integrin α5 suppressed the stiffness-induced expression of all osteoblast markers examined. In particular, alpha-1 type I collagen, RUNX2, and BGLAP were significantly downregulated, indicating that integrin α5 regulates hMSC osteogenic differentiation. Downstream expression of FAK, ERK, p-ERK, and β-catenin protein was unchanged, whereas Akt, p-Akt, GSK-3β, and p-GSK-3β were upregulated. Moreover, expression of Akt and p-Akt was upregulated with anti-integrin α5 antibody, but no difference was observed for FAK, ERK, and p-ERK between the with or without anti-integrin α5 antibody groups. At the same time, expression of GSK-3β and p-GSK-3β was upregulated and β-catenin levels showed no difference between the groups with or without anti-integrin α5 antibody. Since Akt, p-Akt, GSK-3β, and p-GSK-3β displayed the same changes between the groups with or without anti-integrin α5 antibody, we then detected the links among them. Expression of p-Akt and p-GSK-3β was reduced effectively in the presence of the Akt inhibitor Triciribine. However, Akt, GSK-3β, and β-catenin were unchanged. These results suggested that expression of p-GSK-3β was regulated by p-Akt on 62-68 kPa ECM. CONCLUSIONS:Taken together, our results provide evidence that matrix stiffness (62-68 kPa) affects the osteogenic outcome of hMSCs through mechanotransduction events that are mediated by integrin α5.
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Project description:The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue shapes and regulates cell behaviors such as cell adhesion, differentiation and proliferation. The mechanism by which the ECM influences the cell cycle in vivo is poorly understood. Here we demonstrate that the β integrin PAT-3 regulates the localization and expression of CKI-1, a C. elegans homologue of the cyclin dependent kinase inhibitor p27(KIP1). In nematodes expressing wild type PAT-3, CKI-1::GFP localizes primarily to nucleoli in hypodermal cells, whereas in animals expressing mutant pat-3 with a defective splice junction, CKI-1::GFP appears clumped and disorganized in nucleoplasm. RNAi analysis links cell adhesion genes to the regulation of CKI-1. RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization. Additional RNAi experiments revealed that the SCF E3 ubiquitin-ligase complex genes, skpt-1/SKP2, cul-1/CUL1 and lin-23/F-box, are required for the proper localization and expression of CKI-1, suggesting that integrin signaling and SCF E3 ligase work together to regulate the cellular distribution of CKI-1. These data also suggest that integrin plays a major role in maintaining proper CKI-1/p27(KIP1) levels in the cell. Perturbed integrin signaling may lead to the inhibition of SCF ligase activity, mislocalization and elevation of CKI-1/p27(KIP1). These results suggest that adhesion signaling is crucial for cell cycle regulation in vivo.
| S-EPMC3412830 | biostudies-literature
Project description:Epidermal growth factor-like repeats and discoidin I-like domain 3 (Edil3) is an extracellular matrix protein containing an Arg-Gly-Asp (RGD) motif that binds integrin. Recently, Edil3 has been implicated in various biological processes, including angiogenesis and cellular differentiation. It can inhibit inflammatory bone destruction. The objective of this study was to explore the role of Edil3 in osteoblast differentiation and its underlying molecular mechanisms. In wild-type mice, high expression levels of Edil3 mRNA were observed in isolated calvaria and tibia/femur bones. Immunohistochemical analysis showed that Edil3 protein was localized along periosteum and calcified regions surrounding bone tissues. When murine calvaria-derived MC3T3-E1 cells were cultured in osteogenic medium containing 50 ?g/ml ascorbic acid and 5 mM ?-glycerophosphate, Edil3 mRNA and protein expression levels were increased. Treatment with Edil3 protein in growth media increased expression levels of alkaline phosphatase and osteocalcin gene and phosphorylation level of extracellular signal-regulated kinase (ERK). Edil3 treatment with osteogenic medium induced mineralization. Treatment with a neutralizing antibody against ?5?1 and MEK inhibitor U0126 inhibited Edil3-enhanced osteogenic marker gene expression and mineral deposition. Edil3 increased protein expression levels of transcription factor runt-related transcription factor2 (Runx2). Edil3-induced Runx2 protein expression was suppressed by pretreatment with U0126. Taken together, these results suggest that Edil3 may stimulate osteoblast differentiation and matrix mineralization by increasing expression of Runx2 through ?5?1 integrin /ERK pathway.
| S-EPMC5705136 | biostudies-literature
Project description:It is well established that extracellular matrix (ECM) stiffness plays a significant role in regulating the phenotypes and behaviors of many cell types. However, the mechanism underlying the sensing of mechanical cues and subsequent elasticity-triggered pathways remains largely unknown. We observed that stiff ECM significantly enhanced the expression level of several members of the Wnt/?-catenin pathway in both bone marrow mesenchymal stem cells and primary chondrocytes. The activation of ?-catenin by stiff ECM is not dependent on Wnt signals but is elevated by the activation of integrin/ focal adhesion kinase (FAK) pathway. The accumulated ?-catenin then bound to the wnt1 promoter region to up-regulate the gene transcription, thus constituting a positive feedback of the Wnt/?-catenin pathway. With the amplifying effect of positive feedback, this integrin-activated ?-catenin/Wnt pathway plays significant roles in mediating the enhancement of Wnt signal on stiff ECM and contributes to the regulation of mesenchymal stem cell differentiation and primary chondrocyte phenotype maintenance. The present integrin-regulated Wnt1 expression and signaling contributes to the understanding of the molecular mechanisms underlying the regulation of cell behaviors by ECM elasticity.
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Project description:The development of an alternative source for donor lungs would change the paradigm of lung transplantation. Recent studies have demonstrated the potential feasibility of using decellularized lungs as scaffolds for lung tissue regeneration and subsequent implantation. However, finding a reliable cell source and the ability to scale up for recellularization of the lung scaffold still remain significant challenges. To explore the possibility of regeneration of human lung tissue from stem cells in vitro, populations of lung progenitor cells were generated from human iPSCs. To explore the feasibility of producing engineered lungs from stem cells, we repopulated decellularized human lung and rat lungs with iPSC-derived epithelial progenitor cells. The iPSCs-derived epithelial progenitor cells lined the decellularized human lung and expressed most of the epithelial markers when were cultured in a lung bioreactor system. In decellularized rat lungs, these human-derived cells attach and proliferate in a manner similar to what was observed in the decellularized human lung. Our results suggest that repopulation of lung matrix with iPSC-derived lung epithelial cells may be a viable strategy for human lung regeneration and represents an important early step toward translation of this technology.
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Project description:Alix (ALG-2-interacting protein X), a cytoplasmic adaptor protein involved in endosomal sorting and actin cytoskeleton assembly, is required for the maintenance of fibroblast morphology. As Alix has sequence similarity to adhesin in Entamoeba histolytica, and we observed that Alix is secreted, we determined whether extracellular Alix affects fibroblast morphology. Here, we demonstrate that secreted Alix is deposited on the substratum of non-immortalized WI38 fibroblasts. Antibody binding to extracellular Alix retards WI38 cell adhesion and spreading on fibronectin and vitronectin. Alix knockdown in WI38 cells reduces spreading and fibronectin assembly, and the effect is partially complemented by coating recombinant Alix on the cell substratum. Immortalized NIH/3T3 fibroblasts deposit less Alix on the substratum and have defects in alpha5beta1-integrin functions. Coating recombinant Alix on the culture substratum for NIH/3T3 cells promotes alpha5beta1-integrin-mediated cell adhesions and fibronectin assembly, and these effects require the aa 605-709 region of Alix. These findings demonstrate that a sub-population of Alix localizes extracellularly and regulates integrin-mediated cell adhesions and fibronectin matrix assembly.
| S-EPMC2516883 | biostudies-other
Project description:Sphingolipids are bioactive lipid components of cell membranes with important signal transduction functions in health and disease. Ceramide is the central building block for sphingolipid biosynthesis and is processed to form structurally and functionally distinct sphingolipids. Ceramide can be phosphorylated by ceramide kinase (CERK) to generate ceramide-1-phosphate, a cytoprotective signaling molecule that has been widely studied in multiple tissues and organs, including the developing otocyst. However, little is known about ceramide kinase regulation during inner ear development. Using chicken otocysts, we show that genes for CERK and other enzymes of ceramide metabolism are expressed during the early stages of inner ear development and that CERK is developmentally regulated at the otic vesicle stage. To explore its role in inner ear morphogenesis, we blocked CERK activity in organotypic cultures of otic vesicles with a specific inhibitor. Inhibition of CERK activity impaired proliferation and promoted apoptosis of epithelial otic progenitors. CERK inhibition also compromised neurogenesis of the acoustic-vestibular ganglion. Insulin-like growth factor-1 (IGF-1) is a key factor for proliferation, survival and differentiation in the chicken otocyst. CERK inhibition decreased IGF-1-induced AKT phosphorylation and blocked IGF-1-induced cell survival. Overall, our data suggest that CERK is activated as a central element in the network of anti-apoptotic pro-survival pathways elicited by IGF-1 during early inner ear development.
| S-EPMC8220815 | biostudies-literature
Project description:BackgroundThe unique lactation strategy of the tammar wallaby (Macropus eugeni) has been invaluable in evaluating the role of lactogenic hormones and the extracellular matrix (ECM) in the local control of mammary gland function. However molecular pathways through which hormones and ECM exert their effect on wallaby mammary gland function remain unclear. This study undertakes transcriptome analysis of wallaby mammary epithelial cells (WallMEC) following treatment with mammary ECM from two distinct stages of lactation.MethodsWallMEC from MID lactation mammary glands were cultured on ECM from MID or LATE lactation and treated for 5 days with 1 μg/ml cortisol, 1 μg/ml insulin, 0.2 µg/ml prolactin, 650 pg/ml triodothyronine and 1 pg/ml estradiol to induce lactation. WallMEC RNA from triplicate ECM treatments was used to perform RNAseq.ResultsECM from MID and LATE lactation differentially regulated key genes in sugar and lipid metabolism. Seven pathways including galactose metabolism, lysosome, cell adhesion molecules (CAM), p53 signaling, the complement and coagulation and Nod-like receptor signaling pathways were only significantly responsive to ECM in the presence of hormones. The raw RNA-seq data for this project are available on the NCBI Gene Expression Omnibus (GEO) browser (accession number GSE81210).ConclusionsA potential role of ECM in regulation of the caloric content of milk, among other functions including apoptosis, cell proliferation and differentiation has been identified.General significanceThis study has used a non-eutherian lactation model to demonstrate the synergy between ECM and hormones in the local regulation of mammary function.
| S-EPMC5613237 | biostudies-literature
Project description:Extracellular matrix (ECM), which provides critical scaffolds for all adhesive cells, regulates proliferation, differentiation, and apoptosis. Different cell types employ customized ECMs, which are thought to play important roles in the generation of so-called niches that contribute to cell-specific functions. The molecular entities of these customized ECMs, however, have not been elucidated. Here, we describe a strategy for transcriptome-wide identification of ECM proteins based on computational screening of >60,000 full-length mouse cDNAs for secreted proteins, followed by in vitro functional assays. These assays screened the candidate proteins for ECM-assembling activities, interactions with other ECM molecules, modifications with glycosaminoglycans, and cell-adhesive activities, and were then complemented with immunohistochemical analysis. We identified 16 ECM proteins, of which seven were localized in basement membrane (BM) zones. The identification of these previously unknown BM proteins allowed us to construct a body map of BM proteins, which represents the comprehensive immunohistochemistry-based expression profiles of the tissue-specific customization of BMs.
| S-EPMC2529034 | biostudies-literature