The Role of Hypoxia-Inducible Factor 1 Alpha in Acute-on-Chronic Liver Failure.

Acute-on-chronic liver failure (ACLF) is associated with increased mortality. Specific therapy options are limited. Hypoxia-inducible factor 1 alpha (HIF-1α) has been linked to the pathogenesis of chronic liver disease (CLD), but the role of HIF-1α in ACLF is poorly understood. In the current study, different etiologies of CLD and precipitating events triggering ACLF were used in four rodent models. HIF-1α expression and the intracellular pathway of HIF-1α induction were investigated using real-time quantitative PCR. The results were verified by Western blotting and immunohistochemistry for extrahepatic HIF-1α expression using transcriptome analysis. Exploratory immunohistochemical staining was performed to assess HIF-1α in human liver tissue. Intrahepatic HIF-1α expression was significantly increased in all animals with ACLF, regardless of the underlying etiology of CLD or the precipitating event. The induction of HIF-1α was accompanied by the increased mRNA expression of NFkB1 and STAT3 and resulted in a marked elevation of mRNA levels of its downstream genes. Extrahepatic HIF-1α expression was not elevated. In human liver tissue samples, HIF-1α expression was elevated in CLD and ACLF. Increased intrahepatic HIF-1α expression seems to play an important role in the pathogenesis of ACLF, and future studies are pending to investigate the role of therapeutic HIF inhibitors in ACLF.

A host-vector toolbox for improved secretory protein overproduction in Bacillus subtilis.

Target proteins in biotechnological applications are highly diverse. Therefore, versatile flexible expression systems for their functional overproduction are required. In order to find the right heterologous gene expression strategy, suitable host-vector systems, which combine different genetic circuits, are useful. In this study, we designed a novel Bacillus subtilis expression toolbox, which allows the overproduction and secretion of potentially toxic enzymes. This toolbox comprises a set of 60 expression vectors, which combine two promoter variants, four strong secretion signals, a translation-enhancing downstream box, and three plasmid backbones. This B. subtilis toolbox is based on a tailor-made, clean deletion mutant strain, which is protease and sporulation deficient and exhibits reduced autolysis and secondary metabolism. The appropriateness of this alternative expression platform was tested for the overproduction of two difficult-to-produce eukaryotic model proteins. These included the sulfhydryl oxidase Sox from Saccharomyces cerevisiae, which forms reactive hydrogen peroxide and undesired cross-linking of functional proteins, and the human interleukin-1ß, a pro-inflammatory cytokine. For the best performing Sox and interleukin, overproducing and secreting variants of these new B. subtilis toolbox fermentation strategies were developed and tested. This study demonstrates the suitability of the prokaryotic B. subtilis host-vector system for the extracellular production of two eukaryotic proteins with biotechnological relevance. KEY POINTS: • Construction of a versatile Bacillus subtilis gene expression toolbox. • Verification of the toolbox by the secretory overproduction of two difficult-to-express proteins. • Fermentation strategy for an acetoin-controlled overproduction of heterologous proteins.

High Expression of Casein Kinase 2 Alpha Is Responsible for Enhanced Phosphorylation of DNA Mismatch Repair Protein MLH1 and Increased Tumor Mutation Rates in Colorectal Cancer.

DNA mismatch repair (MMR) deficiency plays an essential role in the development of colorectal cancer (CRC). We recently demonstrated in vitro that the serine/threonine casein kinase 2 alpha (CK2α) causes phosphorylation of the MMR protein MLH1 at position serine 477, which significantly inhibits the MMR. In the present study, CK2α-dependent MLH1 phosphorylation was analyzed in vivo. Using a cohort of 165 patients, we identified 88 CRCs showing significantly increased nuclear/cytoplasmic CK2α expression, 28 tumors with high nuclear CK2α expression and 49 cases showing a general low CK2α expression. Patients with high nuclear/cytoplasmic CK2α expression demonstrated significantly reduced 5-year survival outcome. By immunoprecipitation and Western blot analysis, we showed that high nuclear/cytoplasmic CK2α expression significantly correlates with increased MLH1 phosphorylation and enriched somatic tumor mutation rates. The CK2α mRNA levels tended to be enhanced in high nuclear/cytoplasmic and high nuclear CK2α-expressing tumors. Furthermore, we identified various SNPs in the promotor region of CK2α, which might cause differential CK2α expression. In summary, we demonstrated that high nuclear/cytoplasmic CK2α expression in CRCs correlates with enhanced MLH1 phosphorylation in vivo and seems to be causative for increased mutation rates, presumably induced by reduced MMR. These observations could provide important new therapeutic targets.

Consistent alteration of chain length-specific ceramides in human and mouse fibrotic kidneys.

BACKGROUND: Several studies revealed alterations of single sphingolipid species, such as chain length-specific ceramides, in plasma and serum of patients with kidney diseases. Here, we investigated whether such alterations occur in kidney tissue from patients and mice suffering from renal fibrosis, the common endpoint of chronic kidney diseases. METHODS: Human fibrotic kidney samples were collected from nephrectomy specimens with hydronephrosis and/or pyelonephritis. Healthy parts from tumor nephrectomies served as nonfibrotic controls. Mouse fibrotic kidney samples were collected from male C57BL/6J mice treated with an adenine-rich diet for 14 days or were subjected to 7 days of unilateral ureteral obstruction (UUO). Kidneys of untreated mice and contralateral kidneys (UUO) served as respective controls. Sphingolipid levels were detected by LC-MS/MS. Fibrotic markers were analyzed by TaqMan® analysis and immunohistology. RESULTS: Very long-chain ceramides Cer d18:1/24:0 and Cer d18:1/24:1 were significantly downregulated in both fibrotic human kidney cortex and fibrotic murine kidney compared to respective control samples. These effects correlate with upregulation of COL1α1, COL3α1 and αSMA expression in fibrotic human kidney cortex and fibrotic mouse kidney. CONCLUSION: We have shown that very long-chain ceramides Cer d18:1/24:0 and Cer d18:1/24:1 are consistently downregulated in fibrotic kidney samples from human and mouse. Our findings support the use of in vivo murine models as appropriate translational means to understand the involvement of ceramides in human kidney diseases. In addition, our study raises interesting questions about the possible manipulation of ceramide metabolism to prevent progression of fibrosis and the use of ceramides as potential biomarkers of chronic kidney disease.

Validation of highly selective sphingosine kinase 2 inhibitors SLM6031434 and HWG-35D as effective anti-fibrotic treatment options in a mouse model of tubulointerstitial fibrosis.

Renal fibrosis is characterized by chronic inflammation and excessive accumulation of extracellular matrix and progressively leads to functional insufficiency and even total loss of kidney function. In this study we investigated the anti-fibrotic potential of two highly selective and potent SK2 inhibitors, SLM6031434 and HWG-35D, in unilateral ureter obstruction (UUO), a model for progressive renal fibrosis, in mice. In both cases, treatment with SLM6031434 or HWG-35D resulted in an attenuated fibrotic response to UUO in comparison to vehicle-treated mice as demonstrated by reduced collagen accumulation and a decreased expression of collagen-1 (Col1), fibronectin-1 (FN-1), connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA). Similar to our previous study in Sphk2-/- mice, we found an increased protein expression of Smad7, a negative regulator of the pro-fibrotic TGFß/Smad signalling cascade, accompanied by a strong accumulation of sphingosine in SK2 inhibitor-treated kidneys. Treatment of primary renal fibroblasts with SLM6031434 or HWG-35D dose-dependently increased Smad7 expression and ameliorated the expression of Col1, FN-1 and CTGF. In summary, these data prove the anti-fibrotic potential of SK2 inhibition in a mouse model of renal fibrosis, thereby validating SK2 as pharmacological target for the treatment of fibrosis in chronic kidney disease.

Design, Synthesis, and Structure-Activity Relationship Studies of Dual Inhibitors of Soluble Epoxide Hydrolase and 5-Lipoxygenase.

Inhibition of multiple enzymes of the arachidonic acid cascade leads to synergistic anti-inflammatory effects. Merging of 5-lipoxygenase (5-LOX) and soluble epoxide hydrolase (sEH) pharmacophores led to the discovery of a dual 5-LOX/sEH inhibitor, which was subsequently optimized in terms of potency toward both targets and metabolic stability. The optimized lead structure displayed cellular activity in human polymorphonuclear leukocytes, oral bioavailability, and target engagement in vivo and demonstrated profound anti-inflammatory and anti-fibrotic efficiency in a kidney injury model caused by unilateral ureteral obstruction in mice. These results pave the way for investigating the therapeutic potential of dual 5-LOX/sEH inhibitors in other inflammation- and fibrosis-related disease models.

Sorafenib Treatment and Modulation of the Sphingolipid Pathway Affect Proliferation and Viability of Hepatocellular Carcinoma In Vitro.

Hepatocellular carcinoma (HCC) shows a remarkable heterogeneity and is recognized as a chemoresistant tumor with dismal prognosis. In previous studies, we observed significant alterations in the serum sphingolipids of patients with HCC. This study aimed to investigate the in vitro effects of sorafenib, which is the most widely used systemic HCC medication, on the sphingolipid pathway as well as the effects of inhibiting the sphingolipid pathway in HCC. Huh7.5 and HepG2 cells were stimulated with sorafenib, and inhibitors of the sphingolipid pathway and cell proliferation, viability, and concentrations of bioactive metabolites were assessed. We observed a significant downregulation of cell proliferation and viability and a simultaneous upregulation of dihydroceramides upon sorafenib stimulation. Interestingly, fumonisin B1 (FB1) and the general sphingosine kinase inhibitor SKI II were able to inhibit cell proliferation more prominently in HepG2 and Huh7.5 cells, whereas there were no consistent effects on the formation of dihydroceramides, thus implying an involvement of distinct metabolic pathways. In conclusion, our study demonstrates a significant downregulation of HCC proliferation upon sorafenib, FB1, and SKI II treatment, whereas it seems they exert antiproliferative effects independently from sphingolipids. Certainly, further data would be required to elucidate the potential of FB1 and SKI II as putative novel therapeutic targets in HCC.

Renal Mesangial Cells Isolated from Sphingosine Kinase 2 Transgenic Mice Show Reduced Proliferation and are More Sensitive to Stress-Induced Apoptosis.

BACKGROUND/AIMS: Sphingosine 1-phosphate (S1P) is considered as a key molecule regulating various cell functions including cell growth and death. It is produced by two sphingosine kinases (SK) denoted as SK-1 and SK-2. Whereas SK-1 has been extensively studied and has been appointed a role in promoting cell growth, the function of SK-2 is controversial, and both pro-proliferative and pro-apoptotic functions have been suggested. In this study we investigated whether renal mesangial cells isolated from transgenic mice overexpressing the human Sphk2 gene (hSK2-tg) showed an altered cell response towards growth-inducing and apoptotic stimuli. METHODS: hSK2-tg mice were generated by using a Quick KnockinR strategy. Renal mesangial cells were isolated by a differential sieving method and further cultivated in vitro. Lipids were quantified by mass spectrometry. Protein expression was determined by Western blot analysis, cell proliferation was determined by 3H-thymidine incorporation, and apoptosis was determined by a DNA fragmentation ELISA. RESULTS: We show here that kidneys and mesangial cells from hSK2-tg mice express the hSK2 as well as the endogenous mouse mSK2. hSK2 and mSK2 predominantly resided in the cytosol of quiescent transgenic cells. However, S1P accumulated strongly in the nucleus and only minimally in the cytosol of transgenic cells. Functionally, hSK2-tg cells proliferated less than control cells under normal growth conditions and were also more sensitive towards stress-induced apoptosis. On the molecular level, this was reflected by reduced ERK and Akt/PKB activation, and upon staurosporine treatment, by a sensitized mitochondrial pathway as manifested by reduced anti-apoptotic Bcl-XL expression and increased cleavage of caspase-9, downstream caspase-3 and PARP-1. CONCLUSION: Altogether, these data demonstrate that SK-2 exerts an antiproliferative and apoptosis-sensitizing effect in renal mesangial cells which suggests that selective inhibitors of SK-2 may promote proliferation and reduce apoptosis and this may have impact on the outcome of proliferation-associated diseases such as mesangioproliferative glomerulonephritis.

Sphingosine Kinase-2 Deficiency Ameliorates Kidney Fibrosis by Up-Regulating Smad7 in a Mouse Model of Unilateral Ureteral Obstruction.

Kidney fibrosis is a hallmark of chronic kidney disease and leads to extracellular matrix accumulation, organ scarring, and loss of kidney function. In this study, we investigated the role of sphingosine kinase-2 (SPHK2) on the progression of tubular fibrosis by using a mouse unilateral ureteral obstruction (UUO) model. We found that SPHK2 protein and activity are up-regulated in fibrotic renal tissue. Functionally, Sphk2-deficient (Sphk2-/-) mice showed an attenuated fibrotic response to UUO compared with wild-type mice, as demonstrated by reduced collagen abundance and decreased expression of fibronectin-1, collagen I, α-smooth muscle actin, connective tissue growth factor (CTGF), and plasminogen activator inhibitor (PAI-1). More important, these changes were associated with increased expression of the antifibrotic protein Smad7 and higher levels of sphingosine in Sphk2-/- UUO kidneys. Mechanistically, sphingosine ameliorates transforming growth factor-ß-induced collagen accumulation, CTGF, and PAI-1 expression, but enhances Smad7 protein expression in primary kidney fibroblasts. In a complementary approach, in human Sphk2-overexpressing mice, UUO resulted in exacerbated signs of fibrosis with increased collagen accumulation, higher expression levels of fibronectin-1, collagen I, α-smooth muscle actin, CTGF, and PAI-1, but decreased Smad7 expression. SPHK2 plays an important role in kidney fibrogenesis by modulating transforming growth factor-ß signaling. Thus, SPHK2 might be an attractive new target for the treatment of fibrosis in chronic kidney disease.

Role of phospholipases D1 and 2 in astroglial proliferation: effects of specific inhibitors and genetic deletion.

Phospholipase D (PLD) activity has been linked to proliferation in many cell types including tumor cells. In the present study, we investigated the effects of genetic deletion of PLD1 and PLD2 and of specific PLD1 and PLD2 inhibitors on PLD activity and cell proliferation in primary mouse astrocytes. Basal and stimulated PLD activity was negligible in PLD1/2 double knockouts. PLD activity was significantly reduced in PLD1-deficient cells when fetal calf serum (FCS), insulin-like growth factor 1 (IGF-1) or phorbol ester was used as a stimulant. The specificity of PLD inhibitors VU0359595 and VU0285655-1 at 500nM was confirmed in phorbol ester-stimulated cells. Significant reductions of cell proliferation were observed in PLD-deficient cell lines under basal and stimulated conditions. At 500nM, the PLD1 inhibitor VU0359595 reduced proliferation in PLD2-deficient cells, but also in PLD1-deficient cells stimulated by IGF-1 or phorbol ester. Vice versa, at 500nM, the PLD2 inhibitor VU0285655-1 reduced proliferation in PLD1-deficient cells, but also in PLD2-deficient cells exposed to IGF-1. At 5µM, both inhibitors showed non-specific effects because they inhibited cell proliferation even in PLD1/2 double knockouts. Summarizing, inhibition of PLD occurs in parallel with reduced cell proliferation in astrocytes which are deficient in PLD1 or PLD2. Synthetic PLD inhibitors show high specificity for PLD in low (nanomolar) concentrations, but have additional, non-specific effects on cell proliferation when used at high (micromolar) concentrations.

Impaired brain development and reduced cognitive function in phospholipase D-deficient mice.

The phospholipases D (PLD1 and 2) are signaling enzymes that catalyze the hydrolysis of phosphatidylcholine to phosphatidic acid, a lipid second messenger involved in cell proliferation, and choline, a precursor of acetylcholine (ACh). In the present study, we investigated development and cognitive function in mice that were deficient for PLD1, or PLD2, or both. We found that PLD-deficient mice had reduced brain growth at 14-27 days post partum when compared to wild-type mice. In adult PLD-deficient mice, cognitive function was impaired in social and object recognition tasks. Using brain microdialysis, we found that wild-type mice responded with a 4-fold increase of hippocampal ACh release upon behavioral stimulation in the open field, while PLD-deficient mice released significantly less ACh. These results may be relevant for cognitive dysfunctions observed in fetal alcohol syndrome and in Alzheimer' disease.