Charakterisierung der transkriptionellen Aktivierung des cadBA-Operons durch den Transmembranregulator CadC aus Escherichia coli

Das Cad-System von Escherichia coli gehört zu den pH-induzierbaren Aminosäure-Decarboxylase- Systemen. Der Aktivator des Cad-Systems ist der membrangebundene Transkriptionsregulator CadC. CadC ist gleichzeitig Sensor für die Umweltreize pH und Lysin, und Effektorprotein, das die Expression des cadBA-Operons induziert. Im Rahmen dieser Arbeit wurden der molekulare Mechanismus der transkriptionellen Aktivierung des cadBA-Operons durch CadC und verschiedene Modelle für die Aktivierung eines membranintegrierten Transkriptionsaktivators untersucht. Im Rahmen dieser Arbeit konnten durch Footprint-Analysen innerhalb der regulatorischen Region des cadBA-Operons die zwei CadC-Bindestellen Cad1 (erstreckt sich von bp -150 bis -112, relativ zum Transkriptionsstart des cadBA-Operons) und Cad2 (bp -89 bis -59) identifiziert werden. DNA-Bindeexperimente in vitro zeigten, dass CadC mit einer höheren Affinität an Cad1 als an Cad2 bindet. Die Affinität von CadC zu Cad1 und Cad2 wurde durch unterschiedliche pH-Werte oder durch Lysin und Cadaverin nicht signifikant beeinflusst. Die Analyse der Bindestellen Cad1 und Cad2 in vivo ergab, dass das Vorhandensein beider Bindestellen für die Induktion der cadBA-Expression durch Lysin und einen niedrigen externen pH-Wert essentiell ist. Desweiteren wurde die Repression des cadBA-Operons unter nicht-induzierenden Bedingungen durch den globalen Repressor H-NS untersucht. Deletionsanalysen der regulatorischen Region des cadBA-Operons indizierten zwei H-NS-Bindestellen stromaufwärts der CadC-Bindestellen. Rechner-gestützte Sequenzanalysen legten die Existenz von zwei weiteren H-NS-Bindestellen nahe, die mit den CadC-Bindestellen und der -35/-10-Region von PCad überlappen. In hns- Deletionsstämmen war die cadBA-Expression sowohl unter induzierenden als auch unter nicht-induzierenden Bedingungen signifikant erhöht. Für die Aktivierung des cadBA-Operons war CadC essentiell. Biochemische und molekularbiologische Untersuchungen zum Oligomerisationszustand von CadC indizierten, dass CadC Tetramere ausbildet. Die periplasmatische Domäne war für die Oligomerisierung von CadC essentiell. Die Tetramere traten sowohl unter induzierenden als auch unter nicht-induzierenden Bedingungen auf. Daher scheint eine Aktivierung von CadC durch eine Oligomerisierung von CadC-Monomeren, die durch Umgebungsbedingungen wie den pH-Wert und die Lysin-Konzentration moduliert wird, unwahrscheinlich. Basierend auf den oben angeführten Daten wurde ein Modell für die transkriptionelle Aktivierung des cadBA-Operons entwickelt. Demzufolge bildet H-NS unter nicht-induzierenden Bedingungen innerhalb der regulatorischen Region des cadBA-Operons einen Repressionskomplex. Unter induzierenden Bedingungen bindet CadC als Tetramer zunächst an die Bindestelle Cad1, wodurch die anschließende Bindung an Cad2 erleichtert und stabilisiert wird. Durch die Bindung von CadC wird der H-NS vermittelte Repressionskomplex aufgelöst, wodurch eine Interaktion der RNA-Polymerase mit der -35/-10-Region von PCad und die cadBA-Transkription ermöglicht werden. Verschiedene membranintegrierte Transkriptionsfaktoren in eukaryontischen Zellen werden durch eine Regulierte Proteolyse (RP) aktiviert. Biochemische und molekularbiologische Untersuchungen zum molekularen Mechanismus des membran-integrierten Transkriptionsaktivators CadC ergaben bisher keine Hinweise darauf, dass CadC unter induzierenden Bedingungen durch einen Mechanismus ähnlich den der Regulierten Proteolyse aktiviert wird. Um die Funktion der Transmembrandomäne und der periplasmatischen Domäne für die Aktivierung von CadC genauer zu analysieren, wurden verschiedene C-terminal verkürzte CadC-Derivate hinsichtlich ihrer Funktionalität untersucht. Dabei zeigte sich, dass eine Membranassoziation oder -integration von CadC für die Induktion der cadBA-Expression notwendig war. Desweiteren war die periplasmatische Domäne für die CadC-Aktivierung essentiell. In Zusammenarbeit mit dem Department für Physik der LMU München wurde ein in silico Modell für die Regulation der cadBA-Expression erstellt. Zur Überprüfung des Modells wurde die Expression des Cad-Systems während einer simulierten Magen-Passage in vivo analysiert. Die experimentellen Daten stimmten mit dem Modell sehr gut überein. Das Modell ist also in der Lage, die in vivo-Daten zu abzubilden. Ein weiterer Aspekt dieser Arbeit war die Untersuchung der genauen physiologischen Funktion des Cad-Systems. Es konnte nachgewiesen werden, dass das Cad-System eine wichtige Funktion für die Säureresistenz von E. coli bei extremen Säurestress bei pH-Werten <3 hat. Dieser Effekt ist vor allem auf die pH-Wert steigernde Wirkung von ausgeschleustem Cadaverin zurückzuführen. Bei mildem Säurestress bis pH 5 erfüllt das Cad-System vermutlich keine wichtige Funktion

Dual effect of lithium on NFAT5 activity in kidney cells

Lithium salts are used widely for treatment of bipolar and other mental disorders. Lithium therapy is accompanied frequently by renal side effects, such as nephrogenic diabetes insipidus or chronic kidney disease (CKD),but the molecular mechanisms underlying these effects are still poorly understood. In the present study we examined the effect of lithium on the activity of the osmosensitive transcriptional activator nuclear factor of activated T cells 5 (NFAT5, also known as TonEBP),which plays a key role in renal cellular osmoprotection and urinary concentrating ability. Interestingly, we found different effects of lithium on NFAT5 activity, depending on medium osmolality and incubation time. When cells were exposed to lithium for a relative short period (24 h),NFAT5 activity was significantly increased, especially under isosmotic conditions, resulting in an enhanced expression of the NFAT5 target gene heat shock protein 70 (HSP70). Further analysis revealed that the increase of NFAT5 activity depended primarily on an enhanced activity of the c-terminal transactivation domain (TAD),while NFAT5 protein abundance was largely unaffected. Enhanced activity of the TAD is probably mediated by lithium-induced inhibitory phosphorylation of glycogen synthase kinase 3 beta (GSK-3 beta),which is in accordance with previous studies. When cells were exposed to lithium for a longer period (96 h),cellular NFAT5 activity and subsequently expression of HSP70 significantly decreased under hyperosmotic conditions, due to diminished NFAT5 protein abundance, also resulting from GSK-3 beta inhibition. Taken together, our results provide evidence that lithium has opposing effects on NFAT5 activity, depending on environmental osmolality and exposure duration. The potential impacts of these observations on the diverse effects of lithium on kidney function are discussed

NFAT5 Contributes to Osmolality-Induced MCP-1 Expression in Mesothelial Cells

Increased expression of the C-C chemokine monocyte chemoattractant protein-1 (MCP-1) in mesothelial cells in response to high glucose concentrations and/or high osmolality plays a crucial role in the development of peritoneal fibrosis during continuous ambulatory peritoneal dialysis (CAPD). Recent studies suggest that in kidney cells osmolality-induced MCP-1 upregulation is mediated by the osmosensitive transcription factor, nuclear factor of activated T cells 5 (NFAT5). The present study addressed the question of whether activation of NFAT5 by hyperosmolality, as present in PD fluids, contributes to MCP-1 expression in the mesothelial cell line Met5A. Hyperosmolality, induced by addition of glucose, NaCl, or mannitol to the growth medium, increased NFAT5 activity and stimulated MCP-1 expression in Met5A cells. siRNA-mediated knockdown of NFAT5 attenuated osmolality-induced MCP-1 upregulation substantially. Hyperosmolality also induced activation of nuclear factor-κB (NF-κB). Accordingly, pharmacological inhibition of NF-κB significantly decreased osmolality-induced MCP-1 expression. Taken together, these results indicate that high osmolalities activate the transcription factor NFAT5 in mesothelial cells. NFAT5 in turn upregulates MCP-1, likely in combination with NF-κB, and thus may participate in the development of peritoneal fibrosis during CAPD

NFAT5-mediated expression of S100A4 contributes to proliferation and migration of renal carcinoma cells

The osmosensitive transcription factor nuclear factor of activated T-cells (NEAT) 5, also known as tonicity enhancer binding protein (TonEBP),has been associated with the development of a variety of tumor entities, among them breast cancer, colon carcinoma, and melanoma. The aim of the present study was to determine whether NFAT5 is also involved in the development of renal cell carcinoma (RCC). The most common type of RCC, the clear cell RCC, originates from the proximal convoluted tubule. We tested our hypothesis in the clear cell RCC cell line CaKi-1 and the non-cancerous proximal tubule cell line HK-2, as control. Basal expression of NFAT5 and NFAT5 activity in CaKi-1 cells was several times higher than in HK-2 cells. Osmotic stress induced an increased NFAT5 activity in both CaKi-1 and HK-2 cells, again with significantly higher activities in CaKi-1 cells. Analysis of NFAT5-regulating signaling pathways in CaKi-1 cells revealed that inhibition of the MAP kinases p38, c-Jun-terminal kinase (JNK) and extracellular regulated kinase (ERK) and of the focal adhesion kinase (FAK) partially blunted NFAT5 activity. FAK and ERK were both constitutively active, even under isotonic conditions, which may contribute to the high basal expression and activity of NFAT5 in CaKi-1 cells. In contrast, the MAP kinases p38 and JNK were inactive under isotonic conditions and became activated under osmotic stress conditions, indicating that p38 and JNK mediate upregulation of NFAT5 activity under these conditions. siRNA-mediated knockdown of NFAT5 in CaKi-1 cells reduced the expression of S100A4, a member of the S100 family of proteins, which promotes metastasis. Knockdown of NFAT5 was accompanied by a significant decrease in proliferation and migration activity. Taken together, our results indicate that NFAT5 induces S100A4 expression in CaKi-1 cells, thereby playing an important role in RCC proliferation and migration

Focal adhesion kinase regulates the activity of the osmosensitive transcription factor TonEBP/NFAT5 under hypertonic conditions

Ton EBP/NFAT5 is a major regulator of the urinary concentrating process and is essential for the osmoadaptation of renal medullary cells. Focal adhesion kinase (FAK) is a mechanosensitive non-receptor protein tyrosine kinase expressed abundantly in the renal medulla. Since osmotic stress causes cell shrinkage, the present study investigated the contribution of FAK on TonEBP/NFAT5 activation. Osmotic stress induced time-dependent activation of FAK as evidenced by phosphorylation at Tyr-397, and furosemide reduces FAK Tyr-397 phosphorylation in the rat renal medulla. Both pharmacological inhibition of FAK and siRNA-mediated knockdown of FAK drastically reduced TonEBP/NFAT5 transcriptional activity and target gene expression in HEK293 cells. This effect was not mediated by impaired nuclear translocation or by reduced transactivating activity of TonEBP/NFAT5. However, TonEBP/NFAT5 abundance under hypertonic conditions was diminished by 50% by FAK inhibition or siRNA knockdown of FAK. FAK inhibition only marginally reduced transcription of the TonEBP/NFAT5 gene. Rather, TonEBP/NFAT5 mRNA stability was diminished significantly by FAK inhibition, which correlated with reduced reporter activity of the TonEBP/NFAT5 mRNA 3' untranslated region (3'-UTR). In conclusion, FAK is a major regulator of TonEBP/NFAT5 activity by increasing its abundance via stabilization of the mRNA. This in turn, depends on the presence of the TonEBP/NFAT5 3'-UTR

Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone.

Lower urinary tract infections are among the most common human bacterial infections, but extension to the kidneys is rare. This has been attributed to mechanical forces, such as urine flow, that prevent the ascent of bladder microbes. Here, we show that the regional hypersalinity, required for the kidney's urine-concentrating function, instructs epithelial cells to produce chemokines that localize monocyte-derived mononuclear phagocytes (MNPs) to the medulla. This hypersaline environment also increases the intrinsic bactericidal and neutrophil chemotactic activities of MNPs to generate a zone of defense. Because MNP positioning and function are dynamically regulated by the renal salt gradient, we find that patients with urinary concentrating defects are susceptible to kidney infection. Our work reveals a critical accessory role for the homeostatic function of a vital organ in optimizing tissue defense

Generation of a conditional knockout allele for the NFAT5 gene in mice

The osmosensitive transcription factor nuclear factor of activated T-cells 5 (NFAT5),also known as tonicity enhancer element binding protein (TonEBP) plays a crucial role in protection of renal medullary cells against hyperosmotic stress, urinary concentration, the adaptive immune response, and other physiological systems. Since it is also important for development, conventional homozygous-null mutations result in perinatal death, which hinders the analysis of NFAT5 function in specific tissues in vivo. Here we describe the generation of mice with a conditional-null allele, in which loxP sites are inserted around exon 4. Mice harboring the floxed allele (NFAT5(flx)) were mated to a strain expressing a tamoxifen-inducible derivative of the Cre-recombinase (Cre(+)) under the control of the ubigitinC promoter. The resultant homozygous conditional knockout mice (Cre+ NFAT5(flx/flx)) are viable, fertile, and show normal expression of NFAT5 and NFAT5 target genes, indicating that the conditional alleles retain their wild-type function. Induction of Cre-mediated recombination by administration of tamoxifen in 8-week-old mice resulted in a decrease in NFAT5 expression of about 70-90% in all tested tissues (renal cortex, renal outer medulla, renal inner medulla, heart, lung, spleen, skeletal muscle). Accordingly, the expression of the NFAT5 target genes aldose reductase and heat shock protein 70 in the renal medulla was also significantly decreased. Mice harboring this conditional knockout allele should be useful in future studies for gaining a better understanding of tissue and cell-type specific functions of NFAT5 in adult animals under physiological and pathophysiological conditions