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Control of matrix stiffness promotes endodermal lineage specification by regulating SMAD2/3 via lncRNA LINC00458.

ABSTRACT: During endoderm formation, cell identity and tissue morphogenesis are tightly controlled by cell-intrinsic and cell-extrinsic factors such as biochemical and physical inputs. While the effects of biochemical factors are well studied, the physical cues that regulate cell division and differentiation are poorly understood. RNA sequencing analysis demonstrated increases of endoderm-specific gene expression in hPSCs cultured on soft substrate (Young's modulus, 3 ± 0.45 kPa) in comparison with hard substrate (Young's modulus, 165 ± 6.39 kPa). Further analyses revealed that multiple long noncoding RNAs (lncRNAs) were up-regulated on soft substrate; among them, LINC00458 was identified as a stiffness-dependent lncRNA specifically required for hPSC differentiation toward an early endodermal lineage. Gain- and loss-of-function experiments confirmed that LINC00458 is functionally required for hPSC endodermal lineage specification induced by soft substrates. Our study provides evidence that mechanical cues regulate the expression of LINC00458 and induce differentiation of hPSC into hepatic lineage progenitors.

SUBMITTER: Chen YF

PROVIDER: S-EPMC7002135 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundVascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular aging. Sox9 (SRY-box transcription factor 9) has been implicated in vascular smooth muscle cell (VSMC) osteo/chondrogenic conversion; however, its relationship with aging and calcification has not been studied.MethodsImmunohistochemistry was performed on human aortic samples from young and aged patients. Young and senescent primary human VSMCs were induced to produce ECM, and Sox9 expression was manipulated using adenoviral overexpression and depletion. ECM properties were characterized using atomic force microscopy and proteomics, and VSMC phenotype on hydrogels and the ECM were examined using confocal microscopy.ResultsIn vivo, Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16 (cyclin-dependent kinase inhibitor 2A). In vitro Sox9 showed mechanosensitive responses with increased expression and nuclear translocation in senescent cells and on stiff matrices. Sox9 was found to regulate ECM stiffness and organization by orchestrating changes in collagen (Col) expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs, whereby senescent cells plated on ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. LH3 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3) was identified as an Sox9 target and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles and Sox9 promotes extracellular vesicle secretion, leading to increased LH3 deposition within the ECM.ConclusionsThese findings highlight the crucial role of ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle, whereby cellular senescence and increased ECM stiffening promote Sox9 expression, which, in turn, drives further ECM modifications to further accelerate stiffening and senescence.

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Control of matrix stiffness promotes endodermal lineage specification by regulating SMAD2/3 via lncRNA LINC00458.

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