Steffan N. Ho

The NFAT-Related Protein NFATL1 (TonEBP/NFAT5) Is Induced Upon T Cell Activation in a Calcineurin-Dependent Manner

NFAT DNA binding complexes regulate programs of cellular activation and differentiation by translating receptor-dependent signaling events into specific transcriptional responses. NFAT proteins, originally defined as calcium/calcineurin-dependent regulators of cytokine gene transcription in T lymphocytes, are expressed in many different cell types and represent critical signaling intermediates that mediate an increasingly wide spectrum of biologic responses. Recent studies have identified a novel protein containing a region of similarity to the NFAT DNA binding domain. Here we demonstrate that this protein, designated NFATL1 (also known as tonicity enhancer binding protein and NFAT5) is expressed at high levels in the thymus but is undetectable in mature lymphocytes. However, NFATL1 can be induced in both primary quiescent T lymphocytes and differentiated Th1 and Th2 cell populations upon mitogen- or Ag receptor-dependent activation. The induction of NFATL1 protein, as well as NFATL1-dependent transcription, is inhibited by cyclosporin A and FK506, and expression of constitutively active calcineurin induces NFATL1-dependent transcription. Overexpression of NFATc1 and inhibition of NFATc activity through the use of a dominant negative NFATc1 protein have no affect on NFATL1-dependent transcription, indicating that NFATc proteins do not play a role in the calcineurin-dependent induction of NFATL1. Interestingly, induction of NFATL1 by a hyperosmotic stimulus is not blocked by the inhibition of calcineurin. Moreover, osmotic stress response genes such as aldose reductase are not induced upon T cell activation. Thus inducible expression of NFATL1 represents a mechanism by which receptor-dependent signals as well as osmotic stress signals are translated into transcriptional responses that regulate cell function.

The Osmoprotective Function of the NFAT5 Transcription Factor in T Cell Development and Activation

The NFAT5/TonEBP transcription factor, a recently identified rel/NF-κB family member, activates transcription of osmocompensatory genes in response to extracellular hyperosmotic stress. However, the function of NFAT5 under isosmotic conditions present in vivo remains unknown. Here we demonstrate that NFAT5 is necessary for optimal T cell development in vivo and allows for optimal cell growth ex vivo under conditions associated with osmotic stress. Transgenic mice expressing an inhibitory form of NFAT5 in developing and mature T cells exhibited a 30% reduction in thymic cellularity evenly distributed among thymic subsets, consistent with the uniform expression and nuclear localization of NFAT5 in each subset. This was associated with a 25% reduction in peripheral CD4+ T cells and a 50% reduction in CD8+ T cells. While transgenic T cells exhibited no impairment in cell growth or cytokine production under normal culture conditions, impaired cell growth was observed under both hyperosmotic conditions and isosmotic conditions associated with osmotic stress. Transgenic thymocytes also demonstrated increased sensitivity to osmotic stress. Consistent with this, the system A amino acid transporter gene ATA2 exhibited NFAT5 dependence under hypertonic conditions but not in response to amino acid deprivation. Expression of the TNF-α gene, a putative NFAT5 target, was not altered in transgenic T cells. These results not only demonstrate an osmoprotective function for NFAT5 in primary cells but also show that NFAT5 is necessary for optimal thymic development in vivo, suggesting that developing thymocytes within the thymic microenvironment are subject to an osmotic stress that is effectively countered by NFAT5-dependent responses.

Intracellular water homeostasis and the mammalian cellular osmotic stress response

The cellular response to osmotic stress ensures that the concentration of water inside the cell is maintained within a range that is compatible with biologic function. Single cell organisms are particularly dependent on mechanisms that permit adaptation to osmotic stress because each individual cell is directly exposed to the external environment. Mammals, however, limit osmotic stress by establishing an internal aqueous environment in which intravascular water and electrolytes are subject to sensitive and dynamic, organism‐based homeostatic regulation. Recent studies of NFAT5/TonEBP, an essential mammalian osmoregulatory transcription factor, demonstrate the unexpected yet critical significance of cell‐based osmotic regulation in vivo. These results highlight the fundamental importance of maintaining intracellular water homeostasis in the face of varying cellular metabolic activity and distinct tissue microenvironments.

TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress: organic osmolyte-dependent and -independent pathways

TonEBP (tonicity-responsive enhancer binding protein) is a transcription factor that promotes cellular accumulation of organic osmolytes in the hypertonic renal medulla by stimulating expression of its target genes. Genetically modified animals with deficient TonEBP activity in the kidney suffer from severe medullary atrophy in association with cell death, demonstrating that TonEBP is essential for the survival of the renal medullary cells. Using both TonEBP knockout cells and RNA interference of TonEBP, we found that TonEBP promoted cellular adaptation to hypertonic stress. Microarray analyses revealed that the genetic response to hypertonicity was dominated by TonEBP in that expression of totally different sets of genes was increased by hypertonicity in those cells with TonEBP vs. those without TonEBP activity. Of over 100 potentially new TonEBP-regulated genes, we selected seven for further analyses and found that their expressions were all dependent on TonEBP. RNA interference experiments showed that some of these genes, asporin, insulin-like growth factor-binding protein-5 and -7, and an extracellular lysophospholipase D, plus heat shock protein 70, a known TonEBP target gene, contributed to the adaptation to hypertonicity without promoting organic osmolyte accumulation. We conclude that TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress in addition to organic osmolyte accumulation.

Optimization and direct comparison of the dimerizer and reverse tet transcriptional control systems.

Exogenously controlled gene expression systems are essential for both the in vivo analysis of gene function and the regulated delivery of therapeutic gene products. However, differences in experimental methods used to characterize the various systems prohibit informative comparisons. The purpose of this study was to identify an optimal system for regulated gene expression studies through a rigorous direct comparison of the dimerizer and the reverse tet transactivator (rtTA) transcriptional switch systems.

CHAPTER 289 – Transcriptional Control through Regulated Nuclear Transport

A defining characteristic of eukaryotic cells is the compartmentalization of the genome into the nucleus, a membrane-bound subcellular organelle that is essentially impermeable to the passive diffusion of macromolecules. This spatial segregation of genomic DNA within the cell provides a means to control the expression of genome information that takes place during cell growth and differentiation through the regulated localization of transcriptional control proteins to the nucleus. The regulation of nuclear transport has thus been widely utilized in a variety of biological contexts to regulate transcription in a signal-dependent manner. The regulated nuclear transport of DNA-binding transcription factors as well as non-DNA-binding transcriptional regulatory proteins represents an important means of regulating transcription in response to receptor-mediated signaling events. Both nuclear import and export can be regulated by either direct modification of transport signals, masking or unmasking of transport signals through intramolecular or intermolecular protein interactions, or by interaction and co-transport with a carrier protein that contains a transport signal. The coordinated regulation of both nuclear import and export by various combinations of these mechanisms provides a rapid, sensitive, and highly responsive means to dynamically translate intracellular signaling events into transcriptional responses in the nucleus.