MAP kinases (MAPKs), which control mitogenic signal transduction in all eukaryotic organisms, are inactivated by dual specificity MAPK phosphatases (MKPs). MKP-3, a prototypical MKP, achieves substrate specificity through its N-terminal domain binding to the MAPK ERK2, resulting in the activation of its C-terminal phosphatase domain. The solution structure and biochemical analysis of the ERK2 binding (EB) domain of MKP-3 show that regions that are essential for ERK2 binding partly overlap with its sites that interact with the C-terminal catalytic domain, and that these interactions are functionally coupled to the active site residues of MKP-3. Our findings suggest a novel mechanism by which the EB domain binding to ERK2 is transduced to cause a conformational change of the C-terminal catalytic domain, resulting in the enzymatic activation of MKP-3.

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CXC chemokines with a glutamate-leucine-arginine (ELR) tripeptide motif (ELR؉ CXC chemokines) play an important role in leukocyte trafficking into the tissues. For reasons that are not well elucidated, circulating leukocytes are recruited into the tissues mainly in small vessels such as capillaries and venules. Because ELR ؉ CXC chemokines are important mediators of endothelial-leukocyte interaction, we compared chemokine expression by microvascular and aortic endothelium to investigate whether differences in chemokine expression by various endothelial types could, at least partially, explain the microvascular localization of endothelial-leukocyte interaction. Both in vitro and in vivo models indicate that ELR ؉ CXC chemokine expression is higher in microvascular endothelium than in aortic endothelial cells. These differences can be explained on the basis of the preferential activation of endothelial chemokine production by low intensity shear stress. Low shear activated endothelial ELR ؉ CXC chemokine production via cell surface heparan sulfates, ␤ 3 -integrins, focal adhesion kinase, the mitogen-activated protein kinase p38␤, mitogen-and stress-associated protein kinase-1, and the transcription factor.

Studies of the Hepatitis C virus (HCV) life-cycle rely heavily upon Huh7.5 cells, but the reasons why these cells are exceptionally permissive for HCV replication are not clear. Based on recent clinical observations, we hypothesized that the Hedgehog (Hh) pathway, which has not been previously associated with HCV replication, may be involved in the Huh7.5 phenotype of increased permissiveness. We tested this hypothesis by comparing levels of a variety of Hh related cellular markers in Huh7.5 cells with the parental Huh7 cells, which are far less permissive. Here, we demonstrate that Huh7.5 cells, when compared to Huh7 cells, have substantially decreased expression of epithelial markers, increased levels of mesenchymal markers and markedly upregulated Hh pathway activity: Shh, >100 fold, Gli1, >30 fold, Ptc, 2 fold. In Huh7.5 cells, we found that cyclopamine, a Hh pathway antagonist, reduced HCV RNA levels by 50% compared to vehicle and inactive isomer controls. Moreover, in Huh7 cells, treatment with recombinant Shh ligand and SAG, both Hh pathway agonists, stimulated HCV replication by 2 fold and 4 fold, respectively. These effects were observed with both viral infections and a subgenomic replicon. Finally, we demonstrated that GDC-0449 decreased HCV RNA levels in a dose response manner. Conclusions We have identified a relationship between HCV and Hh signaling where upregulated pathway activity during infection promotes an environment conducive to replication. Given that Hh activity is very low in most hepatocytes, these findings may serve to further shift the model of HCV liver infection from modest widespread replication in hepatocytes to one where a subset of cells support high level replication. These findings also introduce Hh pathway inhibitors as potential anti-HCV therapeutics.

The homeodomain protein Cux1 is highly expressed in the nephrogenic zone of the developing kidney where it functions to regulate cell proliferation. Here we show that Cux1 directly interacts with the co-repressor Grg4 (Groucho 4), a known effector of Notch signaling. Promoter reporter based luciferase assays revealed enhanced repression of p27kip1 promoter activity by Cux1 in the presence of Grg4. Chromatin immunoprecipitation (ChIP) assays demonstrated the direct interaction of Cux1 with p27kip1 in newborn kidney tissue in vivo. ChIP assays also identified interactions of Cux1, Grg4, HDAC1, and HDAC3 with p27kip1 at two separate sites in the p27kip1 promoter. DNAse1 footprinting experiments revealed that Cux1 binds to the p27kip1 promoter on the sequence containing two Sp1 sites and a CCAAT box ~500 bp from the transcriptional start site, and to an AT rich sequence ~1.5 KB from the transcriptional start site. Taken together, these results identify Grg4 as an interacting partner for Cux1 and suggest a mechanism of p27kip1 repression by Cux1 during kidney development.

Inactivation of mitogen-activated protein kinases (MAPKs) by MAPK phosphatases (MKPs) is accomplished via substrate-induced activation of the latter enzymes; however, the structural basis for the underlying mechanism remains elusive. Here, we report the three-dimensional solution structure of the C-terminal phosphatase domain of the prototypical MKP PAC-1, determined when bound to phosphate. Structural and biochemical analyses reveal unique active site geometry of the enzyme important for binding to phosphorylated threonine and tyrosine of MAPK ERK2. Our study further demonstrates that the dynamic interaction between the N-terminal kinase binding domain and the C-terminal phosphatase domain of an MKP is directly coupled to MAPK-induced conformational change of the phosphatase active site, which is essential for eliciting its full enzymatic activity.

Hepatitis C virus (HCV) infection exacerbates alcoholic liver injury by mechanisms that include enhanced oxidative stress. The forkhead box transcription factor FOXO3 is an important component of the antioxidant stress response that can be altered by HCV. To test whether FOXO3 is protective for alcoholic liver injury, we fed alcohol to FOXO3 À/À mice. After 3 weeks, one third of these mice developed severe hepatic steatosis, neutrophilic infiltration, and >10-fold alanine aminotransferase (ALT) elevations. In cell culture, either alcohol or HCV infection alone increased FOXO3 transcriptional activity and expression of target genes, but the combination of HCV and alcohol together caused loss of nuclear FOXO3 and decreased its transcriptional activity. This was accompanied by increased phosphorylation of FOXO3. Mice expressing HCV structural proteins on a background of reduced expression of superoxide dismutase 2 (SOD2; Sod2 þ/À ) also had increased liver sensitivity to alcohol, with elevated ALT, steatosis, and lobular inflammation. Elevated ALT was associated with an alcohol-induced decrease in SOD2 and redistribution of FOXO3 to the cytosol. These results demonstrate that FOXO3 functions as a protective factor preventing alcoholic liver injury. The combination of HCV and alcohol, but not either condition alone, inactivates FOXO3, causing a decrease in expression of its target genes and an increase in liver injury. Modulation of the FOXO3 pathway is a potential therapeutic approach for HCV-alcoholeinduced liver injury. (Am J Pathol 2013 http://dx

Purpose Acute burn resuscitation in initial 24 h remains a challenge to plastic surgeons. Though various formulae for fluid infusion are available but consensus is still lacking, resulting in under resuscitation or over resuscitation. Parkland formula is widely used but recently its adequacy is questioned in studies. This study was conducted to see how closely the actual volume of fluid given in our center matches with that of calculated volume by Parkland formula. Methods All patients admitted with more than 20% flame burn injury and within 8 h of incident were included in this study. Crystalloid solution for infusion was calculated as per Parkland formula; however, it was titrated according to the urine output. Data on fluid infusion were collected from patient's inpatient records and analyzed. Results The study included a total of 90 patients, about 86.7% ( n = 78) of the patients received fluid less than the calculated Parkland formula. Rate of fluid administered over 24 h in our study was 3.149 mL/kg/h. Mean hourly urine output was found to be 0.993 mL/kg/h. The mean difference between fluid administered and fluid calculated by Parkland formula was 3431.825 mL which was significant ( p < 0.001). Conclusion The study showed a significant difference in the fluid infused based on urine output and the fluid calculated by Parkland formula. This probably is because fluid infused based on end point of resuscitation was more physiological than fluid calculated based on formulae.