Dataset Information

The regulation of ?-adrenergic receptor-mediated PKA activation by substrate stiffness via microtubule dynamics in human MSCs.

ABSTRACT: The mechanical microenvironment surrounding cells has a significant impact on cellular function. One prominent example is that the stiffness of the substrate directs stem cell differentiation. However, the underlying mechanisms of how mechanical cues affect stem cell functions are largely elusive. Here, we report that in human mesenchymal stem cells (HMSCs), substrate stiffness can regulate cellular responses to a ?-adrenergic receptor (?-AR) agonist, Isoproterenol (ISO). Fluorescence resonance energy transfer-based A-Kinase Activity Reporter revealed that HMSCs displayed low activity of ISO-induced protein kinase A (PKA) signal on soft substrate, whereas a significantly higher activity can be observed on hard substrate. Meanwhile, there is an increasing ISO-induced internalization of ?2-AR with increasing substrate stiffness. Further experiments revealed that the effects of substrate stiffness on both events were disrupted by interfering the polymerization of microtubules, but not actin filaments. Mechanistic investigation revealed that inhibiting ISO-induced PKA activation abolished ?2-AR internalization and vice versa, forming a feedback loop. Thus, our results suggest that the cellular sensing mechanism of its mechanical environment, such as substrate stiffness, affects its response to chemical stimulation of ?-AR signaling and PKA activation through the coordination of microtubules, which may contribute to how mechanical cues direct stem cell differentiation.

SUBMITTER: Kim TJ

PROVIDER: S-EPMC4144871 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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The regulation of β-adrenergic receptor-mediated PKA activation by substrate stiffness via microtubule dynamics in human MSCs.

Kim Tae-Jin TJ Sun Jie J Lu Shaoying S Zhang Jin J Wang Yingxiao Y

Biomaterials 20140624 29

The mechanical microenvironment surrounding cells has a significant impact on cellular function. One prominent example is that the stiffness of the substrate directs stem cell differentiation. However, the underlying mechanisms of how mechanical cues affect stem cell functions are largely elusive. Here, we report that in human mesenchymal stem cells (HMSCs), substrate stiffness can regulate cellular responses to a β-adrenergic receptor (β-AR) agonist, Isoproterenol (ISO). Fluorescence resonance ...[more]

PMID: 24973298

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Project description:In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) automatically triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These dramatic phenotypic switches confound interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we utilized poly (ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic cardiac ECM, respectively. As hypothesized, low hydrogel stiffness converted activated CFs into a quiescent state with reduced abundance of a-smooth muscle actin (a-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated b-galactosidase (SA-b-Gal) activity and increased expression of p16 and p21, which are cyclin-dependent kinase (CDK) inhibitors and markers of senescence. Using dynamically stiffening hydrogels with phototunable crosslinking capabilities, we demonstrate that substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low stiffness hydrogels, with a dramatic reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-kB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings further demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment to which they are exposed.

Dataset Information

The regulation of ?-adrenergic receptor-mediated PKA activation by substrate stiffness via microtubule dynamics in human MSCs.

Publications

The regulation of β-adrenergic receptor-mediated PKA activation by substrate stiffness via microtubule dynamics in human MSCs.

Similar Datasets

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