Sinusoidal endothelial cells (SEC) constitute a permeable barrier between hepatocytes and blood. SEC are exposed to high concentrations of bile salts from the enterohepatic circulation. Whether SEC are responsive to bile salts is unknown. TGR5, a G-protein-coupled bile acid receptor, which triggers cAMP formation, has been discovered recently in macrophages. In this study, rat TGR5 was cloned and antibodies directed against the C-terminus of rat TGR5 were developed, which detected TGR5 as a glycoprotein in transfected HepG2-cells. B ile salts are required for cholesterol excretion and lipid absorption. 1 However, high concentrations of lipophilic bile salts have toxic effects. Bile salts alter membrane fluidity 2 and can act pro-or anti-apoptotic. [3][4][5][6] Many liver diseases are aggravated by the cholestatic potential of lipophilic bile salts. Therefore, several mechanisms exist to maintain bile salt homeostasis. These include the coordinated expression and action of bile salt transporters at the sinusoidal and canalicular membrane of liver parenchymal cells, 7 alternative pathways for bile salt synthesis and metabolism, 8,9 and the involvement of extrahepatic tissues (such as the gut and the kidneys) in bile salt excretion. [10][11][12] These mechanisms are closely regulated by nuclear receptors sensitive for bile salts, which control the expression of transporter proteins and enzymes. They comprise the farnesoid X receptor, 13-15 the pregnane X receptor, 16,17 and the vitamin D receptor. 18 Recently, a G-protein-coupled plasma membrane receptor responsive to bile salts has been discovered by highthroughput screening. This receptor, named TGR5, 19 M-BAR, or BG37, 20 stimulates adenylate cyclase on activation and increases the production of cyclic adenosine monophosphate (cAMP). Thereby, bile salts not only may be involved in the regulation of transcription but also may influence rapid, cAMP-dependent mechanisms in TGR5 expressing cells. So far, TGR5 expression has been demonstrated in enteroendocrine cells, 21 where bile salts stimulate the secretion of glucagon-like peptide-1 via TGR5 and in alveolar macrophages, 19 which secrete smaller amounts of cytokines in response to endotoxin, when bile salts are present. Recently, bile salts were shown to influence energy consumption in brown adipose tissue

The rat tissue inhibitor of metalloproteinase 1 (TIMP-1) gene is expressed in rat hepatocytes, and this expression is up-regulated by interleukin 6 (IL-6). We report here the cloning of the 5' flanking region of the rat TIMP-1 gene and identification of an IL-6/oncostatin M (OSM) response element at -64 to -36 which functions in hepatic cells. Within this element we have identified two functional binding sites for transcription factors AP-1 (activatory protein-1) and STAT (signal transducer and activator of transcription). IL-6/OSM stimulation induces binding of a protein, identified as STAT3, to the IL-6/OSM response element, while binding of the AP-1 protein was constitutive. Binding sites for both AP-1 and STAT3 are necessary for full responsiveness of the TIMP-1 promoter to IL-6/OSM, as shown by deletion and mutation analysis. Furthermore, the entire IL-6/OSM response element conferred responsiveness onto a heterologous promoter, whereas this has not been observed when AP-1 and STAT elements were separately tested.

Numerous cytokines, growth, and differentiation factors elicit their intracellular responses via Janus tyrosine kinases (Jaks) and transcription factors of the STAT (signal transducer and activator of transcription) family. Additionally, environmental stress (UV light, heat, aniso-osmolarity, and radicals) has recently been shown to activate intracellular signaling cascades such as the stress-activated protein kinases and nuclear factor-B. In this study, we demonstrate that in different cell lines a particular stress, namely hyperosmolarity, results in tyrosine phosphorylation of the Janus kinases Jak1, Jak2, and Tyk2 and in the activation of STAT1 and/or STAT3. Both transcription factors are phosphorylated at a specific tyrosine residue and translocation to the nucleus was demonstrated by the use of a STAT3/ green fluorescent protein fusion protein. A prominent role for Jak1 in the activation of STATs by hypertonicity was demonstrated by the use of Jak-deficient cell lines. Stress-activated STAT1 and STAT3 transactivate a reporter gene containing the acute-phase response element of the rat ␣ 2 -macroglobulin promoter. Experiments using a diffusible solute suggest that not the increase in intracellular osmolarity but the resultant cell shrinkage is the trigger for Jak/STAT activation.

Environmental stress (e.g. aniso-osmolarity and UV light), hypoxia/reoxygenation, and reactive oxygen species activate intracellular signaling cascades such as the "stress-responsive" mitogen-activated protein kinases and nuclear factor B. We have recently shown that the Janus tyrosine kinase/signal transducer and activator of transcription (Jak/STAT) pathway is ligand-independently activated by hyperosmotic shock. In the present study, we show that besides STAT1 also the tyrosine phosphatase SHP2 became tyrosine-phosphorylated upon hyperosmolarity. SB 202190 and SB 203580 (specific inhibitors of p38) inhibited both STAT activation and tyrosine phosphorylation of SHP2 induced by hyperosmotic stress. Overexpression of wild-type p38 mitogen-activated protein kinase and its upstream activator mitogen-activated protein kinase kinase 6 (MKK6) resulted in an enhanced STAT1 tyrosine phosphorylation upon osmotic shock. Accordingly, overexpression of dominant negative mutants of p38 and MKK6 largely decreased hyperosmotic STAT1 activation and tyrosine phosphorylation of SHP2. Furthermore, we provide evidence that a genistein-sensitive tyrosine kinase different from Jak1 is involved in stress-activation of STAT1 and tyrosine phosphorylation of SHP2. These results strongly suggest that hyperosmotic shock activates STAT1 and SHP2 via p38 and its upstream activator MKK6. Mitogen-activated protein (MAP)1 kinases are important mediators of signal transduction from the cell surface to the nucleus. Regulation by MAP kinases has been implicated in many cellular processes such as proliferation, differentiation, and apoptosis. In mammals, MAP kinases are divided into at least three subfamilies: the "classical" extracellular signal-regulated kinases (extracellular signal-regulated kinases 1 and 2), the stress-activated protein kinases/c-Jun N-terminal kinases (JNK), and the cytokine-suppressive anti-inflammatory drugbinding protein/p38. Whereas extracellular signal-regulated kinase-type MAP kinases are preferentially activated by a variety of cell growth and differentiation stimuli and by hypoosmolarity, JNK and p38 are primarily activated by various environmental stresses (for reviews, see Refs. 1-3). p38 has substantial similarity to the S. cerevisiae HOG1 kinase, a yeast MAP kinase required for cellular osmoregulation (4). Like HOG1, p38 is activated in response to changes in environmental osmolarity. It further appears to be involved in the signal transduction of lipopolysaccharide and inflammatory mediators such as tumor necrosis factor ␣ and IL-1 (4 -6). The major upstream activators of p38 are the recently discovered dual specific MAP kinase kinases (MKKs) MKK3 and MKK6, while the related MKK4 activates both p38 and JNK (7,8).Another key signaling system involved in the signal transduction of numerous interleukins and the interferons as well as a number of growth and differentiation factors is the Janus kinase (Jak)/signal transducer and activator of transcription (STAT) pathway. The binding of mediators to their respective r...