Mechanotransduction at the nucleus level: Understanding glucocorticoid receptor activation and the role of nuclear lamina in response to hemodynamic forces

Arman Nayebosadri, Purdue University

Abstract

The endothelium that lines the blood vessels has been of considerable interest with respect to understanding the onset and development of vascular diseases such as atherosclerosis. Endothelial cells are capable of initiating a multitude of physiological or pathological responses, both at the molecular and cellular level in response to the pattern of blood flow such as the tangential shear stress force and the circumferential vessel deformation. Ultimately, gene transcription becomes activated or repressed, and this activity is regulated by transcription factors. One type of transcription factor, the glucocorticoid receptor (GR), is widely recognized for its anti-inflammatory effects via upregulation of immunosuppressive genes and downregulation of inflammatory genes, while serving as a therapeutic target for numerous diseases. Recently, it was shown that steady, physiological levels of shear stress on vascular endothelial cells activate GR in the absence of its ligand and initiate its subsequent nuclear translocation. As a continuation of this work, we have conducted live fluorescent time-lapse imaging of GR sub-cellular movement in response to fluid shear stress, and for the first time, quantitative analysis of GR nuclear translocation was performed using a unique image analysis algorithm based on an expectation- maximization/maximization of posterior marginals algorithm. Moreover, we have determined that GR translocation in endothelial cells is independent of an intact cytoskeleton. We have also looked into the role of the nuclear lamina in GR intracellular movement by silencing lamin A/C. The nuclear lamina not only gives the nucleus its structure, but also serves as a scaffold for numerous nuclear proteins as well as the cell's genetic machinery. Mutation or absence of the lamin A/C gene, LMNA, has been shown to lead to an array of diseases called laminopathies. Interestingly, there was no dependence on nuclear lamina for GR import through the nucleus, but downstream transcription of glucocorticoid responsive genes was influenced significantly as demonstrated by reporter assays and real time-polymerase chain reaction of genes containing glucocorticoid response elements. Further experiments suggested that this might be due to aberrant function of histones proteins that are normally sequestered at the nuclear lamina and regulate the opening as well as closing of chromatin for gene transcription or repression, respectively. Glucocorticoid-responsive genes also showed significant changes in mRNA expression between lamin-intact and lamin-deficient endothelial cells. Finally, for the first time we observed the effects of another hemodynamic force, cyclic stretch, on GR activity in normal and lamin-silenced endothelial cells under both physiological and pathological conditions. Interestingly, GR only showed signs of nuclear translocation in response to pathological degrees of stretch in cells with intact lamina; it failed to translocate in response to any stretch stimulus in lamin-deficient cells. Inhibition of mitogen-activated protein kinases in cells with lamin A/C suggested potential roles of JNK and ERK on GR activation, whereas only p38 inhibition in lamin-deficient cells appeared to marginally enhance nuclear presence of GR. In search of potential explanations as to why GR was held within the cytoplasm under both mechanical strain and pharmacological drug inhibition, we found mRNA levels of four mitogen-activated protein kinase phosphatases to be significantly altered in lamin-deficient cells, thus affecting upstream kinases and potentially affecting GR sensitivity to cyclic stretch. Furthermore, we believe that the substrate used for mechanical stimuli studies may be significant in understanding intracellular protein activity in the case of dysfunctional lamin A/C. These findings will allow us to learn more about the response of the endothelial glucocorticoid receptor to hemodynamic forces such as fluid shear stress and cyclic stretch. Moreover, the introduction of a novel image analysis algorithm to quantify the intracellular movement of GR as well as any other fluorescently-labeled protein within a cell gives new and improved insights into protein dynamics. Our groundbreaking work on the relationship between GR and nuclear lamina has also been intriguing with respect to their function following lamin A/C silencing and application of mechanical stimuli, and our observations necessitate further studies that will emphasize live-cell imaging of new proteins of interest, mechanical stimuli and lamin A/C-induced GR phosphorylation, as well as other potential paths to better link their mutual dependence. Ultimately, we anticipate that the intracellular activity of GR may become recognized as a critical mediator for cardiovascular disease pathogenesis, and its altered function as a result of dysfunctional lamin A/C may provide new insight in understanding aberrant protein behavior in laminopathies.

Degree

Ph.D.

Advisors

Ji, Purdue University.

Subject Area

Biomedical engineering

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