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C E Darling

Publications and source records attributed to C E Darling.

5 recordsLinked to original sources
PubMed2001

Activator protein 1 activation following hypoosmotic stress in HepG2 cells is actin cytoskeleton dependent.

BACKGROUND: Following hypoosmotic stress-induced cell volume change, the actin cytoskeleton reorganizes itself. The role of this reorganization in the activation of the phosphatidylinositol 3-OH-kinase/protein kinase B/activator protein 1 (PI-3-K/PKB/AP-1) proliferative signaling cascade is unknown. Focal adhesion kinase (FAK) participates in the cytoskeleton-based activation of PI-3-K. We hypothesized that hypoosmotic stress-induced activation of PKB and AP-1 in HepG2 cells is dependent on an intact actin cytoskeleton and subsequent FAK phosphorylation. METHODS: HepG2 cells were incubated for 1 h with or without 20 microM cytochalasin D, an actin disrupter, and were then exposed for up to 30 min to hypoosmotic medium (200 mOsm/L) to induce swelling. Tumor necrosis factor alpha (1.4 nM) and medium alone served as positive and negative controls, respectively. Western blots measured cytoplasmic phosphorylated or total FAK and PKB. EMSAs measured nuclear AP-1. All experiments were performed in triplicate. RESULTS: Exposure to hypoosmotic stress resulted in activation of the following signaling messengers in a sequential fashion: (1) phosphorylation of FAK occurred by 2 min, (2) phosphorylation of PKB occurred by 10 min, (3) nuclear translocation of AP-1 occurred by 30 min. All three signaling events were abolished when these cells were pretreated with cytochalasin D. CONCLUSION: Actin reorganization following hypoosmotic stress is essential for the FAK-mediated activation of the PI-3-K/PKB/AP-1 proliferative cascade. These data delineate a possible mechanism by which the cell swelling-induced cytoskeletal changes can initiate proliferative signal transduction in human liver cancer.

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PubMed2000

Hypoosmotic stress activates p38, ERK 1 and 2, and SAPK/JNK in rat hepatocytes.

BACKGROUND: Following hepatocyte injury, changes in the perihepatocyte milieu modulate cell volume and influence growth. Hypoosmotic stress activates nuclear factor-kappa B (NF-kappaB), a transcription factor believed to prime cell cycle progression in hepatocytes. In this study, we investigate the role of mitogen-activated protein kinases (MAPKs) in the activation of NF-kappaB. MATERIALS AND METHODS: Quiescent primary hepatocytes were exposed to hypoosmotic serum-free William's E (WE) medium (200 mOsm/liter), with or without a 1-h pretreatment with either PD 98059 (15 microM) or SB 202190 (3 microM). Parallel experiments were conducted using hepatocyte growth factor (HGF) at 0.1 mg/ml and normoosmotic WE medium as positive and negative controls, respectively (n = 3). Relative densitometries of Western blots measured phosphorylated cytoplasmic p38, ERK 1 and 2, and SAPK/JNK. Electromobility shift assays examined nuclear NF-kappaB activation. RESULTS: (i) Hypoosmolar WE medium phosphorylated p38, ERK 1 and 2, and SAPK/JNK by 5 min. (ii) Hypoosmolar WE medium activated NF-kappaB at 60 min. (iii) HGF phosphorylated all three MAPKs and activated NF-kappaB with profiles similar to those of hypoosmotic stress. (iv) Both PD 98059 and SB 202190 abrogated the activation of NF-kappaB in HGF-stimulated cells but not in hypoosmotically stressed cells. CONCLUSION: (i) Both hypoosmotic cell swelling and HGF phosphorylate p38, ERK 1 and 2, and SAPK/JNK, and (ii) HGF, but not hypoosmotic stress, activates NF-kappaB via p38 and ERK 1 and 2 phosphorylation. These data suggest that cell swelling activates NF-kappaB through a pathway separate from that of growth factors.

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PubMed1998

Mesenteric and celiac duplex scanning: a validation study.

PURPOSE: To validate the accuracy of previously established duplex ultrasound criteria for > or =50% superior mesenteric artery (SMA) and celiac artery (CA) stenosis by comparison with arteriography. METHODS: Duplex criteria established retrospectively in our laboratory in 1991 identified an end-diastolic velocity (EDV) > or =45 cm/sec, or no flow signal, as highly sensitive (100%) and specific (92%) indicators for SMA stenosis > or =50% or occlusion. EDV was more accurate (95%) than peak systolic velocity (PSV), which had a maximal accuracy of 86% at a PSV > or =300 cm/sec, with low sensitivity (62%), but high specificity (100%). For CA, accurate velocity thresholds were not identified, but we subsequently noted that retrograde common hepatic artery flow direction from SMA collateral was highly predictive of severe CA stenosis or occlusion. Since publication of those findings, 243 mesenteric duplex scans were performed for clinical evaluation of suspected chronic mesenteric ischemia. Angiographic confirmation was available for a subset of 46. SMA and CA diameters were measured on lateral aortograms by observers blinded to the duplex results, and the original duplex diagnostic criteria were tested for accuracy. In addition, receiver operator characteristic curve analysis was performed on the velocity data to identify the most accurate velocity thresholds in the new data. RESULTS: Duplex was technically adequate in 98% of SMA, 96% of CA, and 89% of hepatic arteries, and arteriograms were adequate in 100% of SMA and 98% of CA. For the SMA, EDV > or =45 cm/sec again provided the best sensitivity (90%), specificity (91%), positive predictive value (90%), negative predictive value (91%), and overall accuracy (91%). As in the retrospective study, PSV > or =300 cm/sec provided low overall accuracy (81%), low sensitivity (60%), but high specificity (100%). Lowering the PSV threshold improved sensitivity but reduced accuracy. For CA, retrograde common hepatic artery flow direction was 100% predictive of severe CA stenosis or occlusion. Velocity data in CA provided accuracy not found in the original study. EDV > or =55 cm/sec or no flow signal had best overall accuracy (95%) with high sensitivity (93%) and specificity (100%). PSV > or =200 cm/sec or no signal also had excellent accuracy (93%), sensitivity (93%), and specificity (94%). In addition, three of four anatomic anomalies were correctly identified by duplex. These included one right hepatic and one common hepatic artery originating from the SMA, and one common celiacomesenteric trunk. CONCLUSION: This validation analysis confirms that duplex velocity criteria are accurate in the identification of mesenteric occlusive disease. Retrograde common hepatic artery flow direction correctly predicts severe CA stenosis or occlusion. Duplex ultrasound may also identify mesenteric anatomic variants that can influence study interpretation.

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PubMedDate not supplied

Hypoosmotic stress stimulates growth in HepG2 cells via protein kinase B-dependent activation of activator protein-1.

Although hypoosmotic stress-induced cell swelling activates phosphatidylinositol-3-kinase, its impact on the downstream signal protein kinase B and cell growth is unknown. Activator protein-1 is in part phosphatidylinositol-3-kinase dependent, and is important in proliferation. We hypothesized that cell swelling modulates proliferation in HepG2 cells via the protein kinase B-dependent activation of activator protein-1. HepG2 cells pretreated with or without LY294002 were exposed for up to 30 minutes to hypoosmotic medium (160 mOsm/L). Tumor necrosis factor-alpha (1.4 nmol/L) or normoosmolar medium (270 mOsm/L) served as positive and negative controls, respectively. Western immunoblots measured cytoplasmic phosphorylated and total protein kinase B. Electromobility shift assays measured nuclear activator protein-1. Methylene blue assays measured cell proliferation at 24, 48, and 72 hours after stimulation. Hypoosmotic stress phosphorylated protein kinase B by 10 minutes. Subsequently, hypoosmotic exposure stimulated activator protein-1 by 30 minutes. Pulse exposure to hypoosmotic stress potentiated HepG2 proliferation by 72 hours as compared to both negative controls and LY-inhibited cells (n = 4 per group, P = 0.009 and P = 0.004, respectively; P <0.001 analysis of variance. All three activation events were abolished with LY294002 pretreatment. In HepG2 cells, hypoosmotic stress-induced swelling stimulates proliferation via protein kinase B-mediated activation of activator protein-1. These data delineate a possible mechanism linking changes in cell volume to growth in human liver cancer.

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