Hyperosmotic stress regulates the distribution and stability of myocardin-related transcription factor, a key modulator of the cytoskeleton

Abstract

Hyperosmotic stress initiates several adaptive responses, including the remodeling of the cytoskeleton. Besides maintaining structural integrity, the cytoskeleton has emerged as an important regulator of gene transcription. Myocardin-related transcription factor (MRTF), an actin-regulated coactivator of serum response factor, is a major link between the actin skeleton and transcriptional control. We therefore investigated whether MRTF is regulated by hyperosmotic stress. Here we show that hypertonicity induces robust, rapid, and transient translocation of MRTF from the cytosol to the nucleus in kidney tubular cells. We found that the hyperosmolarity-triggered MRTF translocation is mediated by the RhoA/Rho kinase (ROK) pathway. Moreover, the Rho guanine nucleotide exchange factor GEF-H1 is activated by hyperosmotic stress, and it is a key contributor to the ensuing RhoA activation and MRTF translocation, since siRNA-mediated GEF-H1 downregulation suppresses these responses. While the osmotically induced RhoA activation promotes nuclear MRTF accumulation, the concomitant activation of p38 MAP kinase mitigates this effect. Moderate hyperosmotic stress (600 mosM) drives MRTF-dependent transcription through the cis-element CArG box. Silencing or pharmacological inhibition of MRTF prevents the osmotic stimulation of CArG-dependent transcription and renders the cells susceptible to osmotic shock-induced structural damage. Interestingly, strong hyperosmolarity promotes proteasomal degradation of MRTF, concomitant with apoptosis. Thus, MRTF is an osmosensitive and osmoprotective transcription factor, whose intracellular distribution is regulated by the GEF-H1/RhoA/ROK and p38 pathways. However, strong osmotic stress destabilizes MRTF, concomitant with apoptosis, implying that hyperosmotically induced cell death takes precedence over epithelial-myofibroblast transition, a potential consequence of MRTF-mediated phenotypic reprogramming.

Peptide affinity analysis of proteins that bind to an unstructured NH2-terminal region of the osmoprotective transcription factor NFAT5

“…Therefore, we are intrigued that we now find protein-to-protein association of NFAT5, with many proteins involved in effects of hypertonicity, but not known to be transcriptional targets of NFAT5. Thus, hypertonicity interrupts the cell cycle (9), and NFAT5 associates with numerous proteins involved in cell proliferation (see RESULTS); development is aberrant in NFAT5 Ϫ/Ϫ fetuses (37,40), and NFAT5 associates with proteins involved in development (see RE-SULTS); hypertonicity affects activity of numerous enzymes (19), and we find that NFAT5 associates with several enzymes (see RESULTS); hypertonicity affects the cytoskeleton (8,39,45,48), and NFAT5 associates with cytoskeletal proteins and their regulators (see results); hypertonicity affects mitochondria (42,54,56), and NFAT5 associates with mitochondrial ribosomal proteins, transporters and structural proteins. We do not know whether and, if so, how protein-to-protein association of NFAT5 with these other proteins affects them, but the association raises interesting questions.…”

Myocardin-related Transcription Factor Regulates Nox4 Protein Expression

“…To address the above question, initially we confirmed the need for the MRTF/ SRF axis for the regulation of the Nox4 promoter using yet another (pharmacological) approach. CCG-1423 is a potent inhibitor of MRTF or the MRTF/SRF interaction (47). Pretreatment with this drug strongly reduced the activity of the Nox4 promoter induced by the double hit scheme compared with vehicle control (Fig.…”

Central role of the exchange factor GEF-H1 in TNF-α–induced sequential activation of Rac, ADAM17/TACE, and RhoA in tubular epithelial cells

“…, 2012; Nie et al. , 2012; Ly et al. , 2013), and its overexpression leads to cell transformation (Mizuarai et al.…”

“…, 2010), exposure to the immunosuppressant drugs cyclosporine and sirolimus (Martin-Martin et al. , 2012), and hyperosmotic shock (Ly et al. , 2013), also require GEF-H1 for RhoA activation.…”