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ATF4 licenses C/EBP? activity in human mesenchymal stem cells primed for adipogenesis.

ABSTRACT: A well-established cascade of transcription factor (TF) activity orchestrates adipogenesis in response to chemical cues, yet how cell-intrinsic determinants of differentiation such as cell shape and/or seeding density inform this transcriptional program remain enigmatic. Here, we uncover a novel mechanism licensing transcription in human mesenchymal stem cells (hMSCs) adipogenically primed by confluence. Prior to adipogenesis, confluency promotes heterodimer recruitment of the bZip TFs C/EBP? and ATF4 to a non-canonical C/EBP DNA sequence. ATF4 depletion decreases both cell-density-dependent transcription and adipocyte differentiation. Global profiling in hMSCs and a novel cell-free assay reveals that ATF4 requires C/EBP? for genomic binding at a motif distinct from that bound by the C/EBP? homodimer. Our observations demonstrate that C/EBP? bridges the transcriptional programs in naïve, confluent cells and early differentiating pre-adipocytes. Moreover, they suggest that homo- and heterodimer formation poise C/EBP? to execute diverse and stage-specific transcriptional programs by exploiting an expanded motif repertoire.

SUBMITTER: Cohen DM

PROVIDER: S-EPMC4501333 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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ATF4 licenses C/EBPβ activity in human mesenchymal stem cells primed for adipogenesis.

Cohen Daniel M DM Won Kyoung-Jae KJ Nguyen Nha N Lazar Mitchell A MA Chen Christopher S CS Steger David J DJ

eLife 20150625

A well-established cascade of transcription factor (TF) activity orchestrates adipogenesis in response to chemical cues, yet how cell-intrinsic determinants of differentiation such as cell shape and/or seeding density inform this transcriptional program remain enigmatic. Here, we uncover a novel mechanism licensing transcription in human mesenchymal stem cells (hMSCs) adipogenically primed by confluence. Prior to adipogenesis, confluency promotes heterodimer recruitment of the bZip TFs C/EBPβ an ...[more]

PMID: 26111340

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Project description:BackgroundHuman bone marrow-derived mesenchymal stem cells (hBMSCs) can differentiate into adipocytes upon stimulation and are considered an appropriate cell source for adipose tissue engineering. In addition to biochemical cues, the stiffness of a substrate that cells attach to has also been shown to affect hBMSC differentiation potential. Of note, most current studies are conducted on monolayer cultures which do not directly inform adipose tissue engineering, where 3-dimensional (3D) scaffolds are often used to create proper tissue architecture. In this study, we aim to examine the adipogenic differentiation of hBMSCs within soft or stiff scaffolds and investigate the molecular mechanism mediating the response of hBMSCs to substrate stiffness in 3D culture, specifically the involvement of the integral membrane protein, caveolin-1 (CAV1), known to regulate signaling in MSCs via compartmentalizing and concentrating signaling molecules.MethodsBy adjusting the photo-illumination time, photocrosslinkable gelatin scaffolds with the same polymer concentration but different stiffnesses were created. hBMSCs were seeded within soft and stiff scaffolds, and their response to adipogenic induction under different substrate mechanical conditions was characterized. The functional involvement of CAV1 was assessed by suppressing its expression level using CAV1-specific siRNA.ResultsThe soft and stiff scaffolds used in this study had a compressive modulus of ~0.5 kPa and ~23.5 kPa, respectively. hBMSCs showed high viability in both scaffold types, but only spread out in the soft scaffolds. hBMSCs cultured in soft scaffolds displayed significantly higher adipogenesis, as revealed by histology, qRT-PCR, and immunostaining. Interestingly, a lower CAV1 level was observed in hBMSCs in the soft scaffolds, concomitantly accompanied by increased levels of Yes-associated protein (YAP) and decreased YAP phosphorylation, when compared to cells seeded in the stiff scaffolds. Interestingly, reducing CAV1 expression with siRNA was shown to further enhance hBMSC adipogenesis, which may function through activation of the YAP signaling pathway.ConclusionsSoft biomaterials support superior adipogenesis of encapsulated hBMSCs in 3D culture, which is partially mediated by the CAV1-YAP axis. Suppressing CAV1 expression levels represents a robust method in the promotion of hBMSC adipogenesis.

Project description:Bone marrow-derived human mesenchymal stem cells (hMSCs) have shown promise in in vitro neuronal differentiation and in cellular therapy for neurodegenerative disorders, including Parkinson' disease. However, the effects of intracerebral transplantation are not well defined, and studies do not agreed on the optimal neuronal differentiation method. Here, we investigated three growth factor-based neuronal differentiation procedures (using FGF-2/EGF/PDGF/SHH/FGF-8/GDNF), and found all to be capable of eliciting an immature neural phenotype, in terms of cell morphology and gene/protein expression. The neuronal-priming (FGF-2/EGF) method induced neurosphere-like formation and the highest NES and NR4A2 expression by hMSCs. Transplantation of undifferentiated and neuronal-primed hMSCs into the striatum and substantia nigra of 6-OHDA-lesioned hemiparkinsonian rats revealed transient graft survival of 7 days, despite the reported immunosuppressive properties of MSCs and cyclosporine-immunosuppression of rats. Neither differentiation of hMSCs nor induction of host neurogenesis was observed at injection sites, and hMSCs continued producing mesodermal fibronectin. Strategies for improving engraftment and differentiation post-transplantation, such as prior in vitro neuronal-priming, nigral and striatal grafting, and co-transplantation of olfactory ensheathing cells that promote neural regeneration, were unable to provide advantages. Innate inflammatory responses (Iba-1-positive microglia/macrophage and GFAP-positive astrocyte activation and accumulation) were detected around grafts within 7 days. Our findings indicate that growth factor-based methods allow hMSC differentiation toward immature neuronal-like cells, and contrary to previous reports, only transient survival and engraftment of hMSCs occurs following transplantation in immunosuppressed hemiparkinsonian rats. In addition, suppression of host innate inflammatory responses may be a key factor for improving hMSC survival and engraftment.

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ATF4 licenses C/EBP? activity in human mesenchymal stem cells primed for adipogenesis.

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ATF4 licenses C/EBPβ activity in human mesenchymal stem cells primed for adipogenesis.

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