AMP-activated protein kinase inhibits TGF-ß-induced fibrogenic responses of hepatic stellate cells by targeting transcriptional coactivator p300.

Liver fibrosis is a common consequence of various chronic liver injuries, including virus infection and ethanol. Activated hepatic stellate cells (HSCs) contribute to liver fibrosis through the accumulation of extracellular matrix proteins, including type I alpha collagen (COL1A). The activation of adenosine monophosphate-activated protein kinase (AMPK) modulates HSCs activation, but its underlying mechanism remains unclear. Here, we report that AMPK inhibits transforming growth factor (TGF)-ß-induced fibrogenic property of HSCs by regulating transcriptional coactivator p300. We treated human (LX-2) and rat (CFSC-2G) HSC lines with TGF-ß to induce fibrogenic activation of HSCs. Pharmacological activation of AMPK by treatment with 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), metformin, or adiponectin lowered TGF-ß-induced expression of COL1A and myofibroblast marker alpha-smooth muscle actin (α-SMA). Transient transduction of constitutively active AMPKα (caAMPKα) was sufficient to attenuate COL1A and α-SMA expression, whereas an AMPK inhibitor considerably abrogated the inhibitory effect of AICAR on fibrogenic gene expression. Although AMPK significantly suppressed Smad-dependent transcription, it did not affect TGF-ß-stimulated phosphorylation, nuclear localization, or DNA-binding activity of Smad2/3. AICAR rather attenuated TGF-ß-induced Smad3 interaction with transcriptional coactivator p300 accompanying with reduction of Smad3 acetylation. Moreover, AICAR induced not only physical interaction between AMPK and p300 but also proteasomal degradation of p300 protein. Our data provide substantial evidence that AMPK could be a novel therapeutic target for treatment of liver fibrosis, by demonstrating the underlying mechanism of AMPK-induced antifibrotic function in HSCs.

Laparoendoscopic single-site bladder diverticulectomy: technique and initial experience.

PURPOSE: To present our initial experience with the laparoendoscopic single-site surgery (LESS) approach to bladder diverticulectomy. PATIENTS AND METHODS: Four patients underwent LESS bladder diverticulectomy for the management of diverticula that were associated with persistent symptoms. The median diameter of the diverticula was 8.5 cm (range 4-9 cm). Immediately before, a double-pigtail ureteral catheter was inserted in the ureter on the side of the diverticulum. An 18F urethral catheter was placed via guidewire, and its balloon was inflated into the diverticulum under fluoroscopic control. A TriPort inserted in the umbilicus and a combination of prebent and conventional laparoscopic instruments were used. The balloon inside the diverticulum was inflated and the diverticulum were incised. The diverticulum was dissected circumferentially and removed. Suturing of the bladder lesion followed. The specimen was extracted through the umbilical incision. Perioperative parameters were recorded. Postoperative follow-up included cystography, uroflowmetry, and ultrasonography measurement of postvoid residual urine. RESULTS: Average patient age was 51 years (range 42-66 y) and the average body mass index was 26 kg/m(2) (range 23.7-28.7 kg/m(2)). Average operative time was 130 minutes (range 101-154 min). Blood loss was minimal, with 150 mL the higher loss. Complications were not observed during the follow-up period. The catheter was removed on postoperative day (POD) 8 (n=3) and on POD 9 (n=1). Histologic examination did not reveal malignancy. Postoperative pain and analgesic medication requirement were minimal. CONCLUSIONS: LESS bladder diverticulectomy proved to be feasible with comparable postoperative outcome to that of the laparoscopic procedure. Further clinical evaluation is deemed necessary.

Comparative assessment of laparoscopic single-site surgery instruments to conventional laparoscopic in laboratory setting.

INTRODUCTION: Laparoendoscopic single-site surgery (LESS) represents the latest innovation in laparoscopic surgery. We compare in dry and animal laboratory the efficacy of recently introduced pre-bent instruments with conventional laparoscopic and flexible instruments in terms of time requirement, maneuverability, and ease of handling. MATERIALS AND METHODS: Participants of varying laparoscopic experience were included in the study and divided in groups according to their experience. The participants performed predetermined tasks in dry laboratory using all sets of instruments. An experienced laparoscopic surgeon performed 24 nephrectomies in 12 pigs using all sets of instruments. Single port was used for all instrument sets except for the conventional instruments, which were inserted through three ports. The time required for the performance of dry laboratory tasks and the porcine nephrectomies was recorded. Errors in the performance of dry laboratory tasks of each instrument type were also recorded. RESULTS: Pre-bent instruments had a significant advantage over flexible instruments in terms of time requirement to accomplish tasks and procedures as well as maneuverability. Flexible instruments were more time consuming in comparison to the conventional laparoscopic instruments during the performance of the tasks. There were no significant differences in the time required for the accomplishment of dry laboratory tasks or steps of nephrectomy using conventional instruments through appropriate number of ports in comparison to pre-bent instruments through single port. DISCUSSION: Pre-bent instruments were less time consuming and with better maneuverability in comparison to flexible instruments in experimental single-port access surgery. Further clinical investigations would elucidate the efficacy of pre-bent instruments.

Specific tyrosine phosphorylation of focal adhesion kinase mediated by Fer tyrosine kinase in suspended hepatocytes.

Cell adhesion to the extracellular matrix (ECM) can activate signaling via focal adhesion kinase (FAK) leading to dynamic regulation of cellular morphology. Mechanistic basis for the lack of effective intracellular signaling by non-attached epithelial cells is poorly understood. To examine whether signaling in suspended cells is regulated by Fer cytoplasmic tyrosine kinase, we investigated the effect of ectopic Fer expression on signaling in suspended or adherent hepatocytes. We found that ectopic Fer expression in Huh7 hepatocytes in suspension or on non-permissive poly-lysine caused significant phosphorylation of FAK Tyr577, Tyr861, or Tyr925, but not Tyr397 or Tyr576. Fer-mediated FAK phosphorylation in suspended cells was independent of c-Src activity or growth factor stimulation, but dependent of cortactin expression. Consistent with these results, complex formation between FAK, Fer, and cortactin was observed in suspended cells. The Fer-mediated effect correlated with multiple membrane protrusions, even on poly-lysine. Together, these observations suggest that Fer may allow a bypass of anchorage-dependency for intracellular signal transduction in hepatocytes.

O-GlcNAc modulation at Akt1 Ser473 correlates with apoptosis of murine pancreatic beta cells.

O-GlcNAc transferase (OGT)-mediated modification of protein Ser/Thr residues with O-GlcNAc influences protein activity, similar to the effects of phosphorylation. The anti-apoptotic Akt1 is both activated by phosphorylation and modified with O-GlcNAc. However, the nature and significance of the Akt1 O-GlcNAc modification is unknown. The relationship of O-GlcNAc modification and phosphorylation at Akt1 Ser473 was examined with respect to apoptosis of murine beta-pancreatic cells. Glucosamine treatment induced apoptosis, which correlated with enhanced O-GlcNAc modification of Akt1 and concomitant reduction in Ser473 phosphorylation. Pharmacological inhibition of OGT or O-GlcNAcase revealed an inverse correlation between O-GlcNAc modification and Ser473 phosphorylation of Akt1. MALDI-TOF/TOF mass spectrometry analysis of Akt1 immunoprecipitates from glucosamine-treated cells, but not untreated controls, showed a peptide containing S473/T479 that was presumably modified with O-GlcNAc. Furthermore, in vitro O-GlcNAc-modification analysis of wildtype and mutant Akt1 revealed that S473 was targeted by recombinant OGT. A S473A Akt1 mutant demonstrated reduced basal and glucosamine-induced Akt1 O-GlcNAc modification compared with wildtype Akt1. Furthermore, wildtype Akt1, but not the S473A mutant, appeared to be associated with OGT following glucosamine treatment. Together, these observations suggest that Akt1 Ser473 may undergo both phosphorylation and O-GlcNAc modification, and the balance between these may regulate murine beta-pancreatic cell fate.

Tetraspanin TM4SF5 mediates loss of contact inhibition through epithelial-mesenchymal transition in human hepatocarcinoma.

The growth of normal cells is arrested when they come in contact with each other, a process known as contact inhibition. Contact inhibition is lost during tumorigenesis, resulting in uncontrolled cell growth. Here, we investigated the role of the tetraspanin transmembrane 4 superfamily member 5 (TM4SF5) in contact inhibition and tumorigenesis. We found that TM4SF5 was overexpressed in human hepatocarcinoma tissue. TM4SF5 expression in clinical samples and in human hepatocellular carcinoma cell lines correlated with enhanced p27Kip1 expression and cytosolic stabilization as well as morphological elongation mediated by RhoA inactivation. These TM4SF5-mediated effects resulted in epithelial-mesenchymal transition (EMT) via loss of E-cadherin expression. The consequence of this was aberrant cell growth, as assessed by S-phase transition in confluent conditions, anchorage-independent growth, and tumor formation in nude mice. The TM4SF5-mediated effects were abolished by suppressing the expression of either TM4SF5 or cytosolic p27Kip1, as well as by reconstituting the expression of E-cadherin. Our observations have revealed a role for TM4SF5 in causing uncontrolled growth of human hepatocarcinoma cells through EMT.

Cell adhesion-dependent cofilin serine 3 phosphorylation by the integrin-linked kinase.c-Src complex.

Integrin-linked kinase (ILK) is involved in signal transduction by integrin-mediated cell adhesion that leads to dynamic actin reorganization. Actin (de)polymerization is regulated by cofilin, the Ser(3) phosphorylation (pS(3)cofilin) of which inhibits its actin-severing activity. To determine how ILK regulates pS(3)cofilin, we examined the effects of ILK on pS(3)cofilin using normal RIE1 cells. Compared with suspended cells, fibronectin-adherent cells showed enhanced pS(3)cofilin, depending on ILK expression and c-Src activity. The ILK-mediated pS(3)cofilin in RIE1 cells did not involve Rho-associated kinase, LIM kinase, or testicular protein kinases, which are known to be upstream of cofilin. The kinase domain of ILK, including proline-rich regions, appeared to interact physically with the Src homology 3 domain of c-Src. In vitro kinase assay revealed that ILK immunoprecipitates phosphorylated the recombinant glutathione S-transferase-cofilin, which was abolished by c-Src inhibition. Interestingly, epidermal growth factor treatment abolished the ILK effects, indicating that the linkage from ILK to cofilin is biologically responsive to extracellular cues. Altogether, this study provides evidence for a new signaling connection from ILK to cofilin for dynamic actin polymerization during cell adhesion, depending on the activity of ILK-associated c-Src.

PKCdelta and cofilin activation affects peripheral actin reorganization and cell-cell contact in cells expressing integrin alpha5 but not its tailless mutant.

Integrin-mediated cell adhesion transduces signaling activities for actin reorganization, which is crucially involved in cellular function and architectural integrity. In this study, we explored the possibility of whether cell-cell contacts might be regulated via integrin-alpha5beta1-mediated actin reorganization. Ectopic expression of integrin alpha5 in integrin-alpha5-null intestinal epithelial cells resulted in facilitated retraction, cell-cell contact loss, and wound healing depending on Src and PI3K (phosphoinositide 3-kinase) activities by a reagent that affects actin organization. However, cytoplasmic tailless integrin alpha5 (hereafter referred to as alpha5/1) expression caused no such effects but rather sustained peripheral actin fibers, regardless of Src and PI3K signaling activities. Furthermore, integrin alpha5 engagement with fibronectin phosphorylated Ser643 of PKCdelta, upstream of FAK and Src and at a transmodulatory loop with PI3K/Akt. Pharmacological PKCdelta inactivation, dominant-negative PKCdelta adenovirus or inactive cofilin phosphatase (SSH1L mutant) retrovirus infection of alpha5-expressing cells sustained peripheral actin organization and blocked the actin reorganizing-mediated loss of cell-cell contacts. Meanwhile, wild-type PKCdelta expression sensitized alpha5/1-expressing cells to the actin disruptor to induce cell scattering. Altogether, these observations indicate that integrin alpha5, but not alpha5/1, mediates PKCdelta phosphorylation and cofilin dephosphorylation, which in turn modulate peripheral actin organization presumably leading to an efficient regulation of cell-cell contact and migration.

EGFR phosphorylation-dependent formation of cell-cell contacts by Ras/Erks cascade inhibition.

Cell-cell contacts play important roles in the homeostasis of normal epithelium and in the steps of metastasis of tumor cells, although signaling mechanisms to regulate cell-cell contacts are unclear. In this study, we observed that phenotype of no cell-cell contacts in rat intestinal epithelial cell subline (RIE1-Sca) correlated with increased Erk1/2 signaling activity, compared to that of parental RIE1 cells growing in colonies. Furthermore, cell-cell contacts between RIE1-Sca cells were reformed by treatment with a specific MEK inhibitor (U0126), with translocation of ZO1 and beta-catenin to cell-cell contacts, without changes of their expression levels. U0126 treatment also increased EGFR phosphorylation in a ligand-independent manner. Pretreatment with EGFR kinase inhibitor abolished U0126 treatment-mediated EGFR phosphorylation, and expression of dominant negative H-Ras N17 allowed EGFR phosphorylation and cell-cell contacts even without U0126 treatment. Furthermore, the expression of a nonphosphorylatable EGFR Y5F mutant abolished U0126-mediated cell-cell contacts. U0126 treatment also caused less efficient wound healing by keeping monolayer integrity intact, compared to control untreated cells. This U0126-mediated reduction in wound healing was further altered either by pretreatment of EGFR kinase inhibitor or expression of H-Ras N17 or EGFR Y5F. Taken together, this study supports a unique mechanism of cell-cell contact formation through MEK/Erks inhibition-mediated EGFR phosphorylation.