Unraveling the effect of intra- and intercellular processes on acetaminophen-induced liver injury.

In high dosages, acetaminophen (APAP) can cause severe liver damage, but susceptibility to liver failure varies across individuals and is influenced by factors such as health status. Because APAP-induced liver injury and recovery is regulated by an intricate system of intra- and extracellular molecular signaling, we here aim to quantify the importance of specific modules in determining the outcome after an APAP insult and of potential targets for therapies that mitigate adversity. For this purpose, we integrated hepatocellular acetaminophen metabolism, DNA damage response induction and cell fate into a multiscale mechanistic liver lobule model which involves various cell types, such as hepatocytes, residential Kupffer cells and macrophages. Our model simulations show that zonal differences in metabolism and detoxification efficiency are essential determinants of necrotic damage. Moreover, the extent of senescence, which is regulated by intracellular processes and triggered by extracellular signaling, influences the potential to recover. In silico therapies at early and late time points after APAP insult indicated that prevention of necrotic damage is most beneficial for recovery, whereas interference with regulation of senescence promotes regeneration in a less pronounced way.

Adjuvant Use of PlasmaJet Device During Cytoreductive Surgery for Advanced-Stage Ovarian Cancer: Results of the PlaComOv-study, a Randomized Controlled Trial in The Netherlands.

OBJECTIVE: Standard surgical treatment of advanced-stage ovarian carcinoma with electrosurgery cannot always result in complete cytoreductive surgery (CRS), especially when many small metastases are found on the mesentery and intestinal surface. We investigated whether adjuvant use of a neutral argon plasma device can help increase the complete cytoreduction rate. PATIENTS AND METHODS: 327 patients with FIGO stage IIIB-IV epithelial ovarian cancer (EOC) who underwent primary or interval CRS were randomized to either surgery with neutral argon plasma (PlasmaJet) (intervention) or without PlasmaJet (control group). The primary outcome was the percentage of complete CRS. The secondary outcomes were duration of surgery, blood loss, number of bowel resections and colostomies, hospitalization, 30-day morbidity, and quality of life (QoL). RESULTS: Complete CRS was achieved in 119 patients (75.8%) in the intervention group and 115 patients (67.6%) in the control group (risk difference (RD) 8.2%, 95% confidence interval (CI) -0.021 to 0.181; P = 0.131). In a per-protocol analysis excluding patients with unresectable disease, complete CRS was obtained in 85.6% in the intervention group and 71.5% in the control group (RD 14.1%, 95% CI 0.042 to 0.235; P = 0.005). Patient-reported QoL at 6 months after surgery differed between groups in favor of PlasmaJet surgery (95% CI 0.455-8.350; P = 0.029). Other secondary outcomes did not differ significantly. CONCLUSIONS: Adjuvant use of PlasmaJet during CRS for advanced-stage ovarian cancer resulted in a significantly higher proportion of complete CRS in patients with resectable disease and higher QoL at 6 months after surgery. (Funded by ZonMw, Trial Register NL62035.078.17.) TRIAL REGISTRATION: Approved by the Medical Ethics Review Board of the Erasmus University Medical Center Rotterdam, the Netherlands, NL62035.078.17 on 20-11-2017. Recruitment started on 30-1-2018.

Incomplete surgical staging in clinical early-stage ovarian cancer: guidelines versus daily practice.

BACKGROUND: Incomplete surgical staging of patients with early-stage epithelial ovarian cancer (EOC) has been reported in up to 98% of cases, when based on the International Federation of Obstetrics and Gynecology (FIGO) staging procedure. The aim of the present retrospective study was to clarify the reasons for incomplete staging. METHODS: The PRISMA (Prevention Recovery Information System for Monitoring and Analysis) technique was used to evaluate cases with FIGO I-IIa EOC based on incomplete staging from five gynecologic oncologic center hospitals in the Netherlands in the period 2010-2014. RESULTS: Fifty cases with an incomplete surgical staging of EOC according to national guidelines were included. The most common reasons for incomplete staging were insufficient random biopsies of the peritoneum (n = 34, 68%), and less than ten lymph nodes being resected and/or found at pathology (n = 16, 32%). The most mentioned reason for not performing biopsies was, besides forgetting to do so, believing that after careful inspection and palpation, taking biopsies is irrelevant and/or already are being taken while performing a hysterectomy (peritoneum of cul-de-sac, bladder). The value of contralateral pelvic lymph node dissection in case of a unilateral ovarian malignancy was also doubted, influencing the number of lymph nodes resected. CONCLUSIONS: The most important reasons for incomplete staging in EOC are, besides omitting elements by accident, questioning the importance of obligatory elements of the staging procedure. A structured list of staging steps during surgery and more evidence-based consensus concerning these obligatory elements might increase the number of complete staging procedures in EOC.

Evaluation of effectiveness of the PlasmaJet surgical device in the treatment of advanced stage ovarian cancer (PlaComOv-study): study protocol of a randomized controlled trial in the Netherlands.

BACKGROUND: The most important goal for survival benefit of advanced stage ovarian cancer is to surgically remove all visible tumour, because complete cytoreductive surgery (CCS) has been shown to be associated with prolonged survival. In a remarkable number of women, CCS is very challenging. Especially in women with many small metastases on the peritoneum and intestinal surface, conventional CCS with electrosurgery is not able to be "complete" in removing safely all visible tumour. In this randomized controlled trail (RCT) we investigate whether the use of the PlasmaJet Surgical Device increases the rate of CCS, and whether this indeed leads to a longer progression free and overall survival. The main research question is: does the use of the PlasmaJet Surgical Device in surgery for advanced stage ovarian cancer result in an increased number of complete cytoreductive surgeries when compared with conventional surgical techniques. Secondary study objectives are: 30-day morbidity, duration of surgery, blood loss, length of hospitalisation, Quality of Life, disease-free survival, overall survival, percentage colostomy, cost-effectiveness. METHODS: The study design is a multicentre single-blinded superiority RCT in two university and nine non-university hospitals in The Netherlands. Three hundred and thirty women undergoing cytoreductive surgery for advanced stage ovarian carcinoma (FIGO Stage IIIB-IV) will be randomized into two arms: use of the PlasmaJet (intervention group) versus the use of standard surgical instruments combined with electrocoagulation (control group). The primary outcome is the rate of complete cytoreductive surgery in both groups. Secondary study objectives are: 30-day morbidity, duration of surgery, blood loss, length of hospitalisation, Quality of Life, disease-free survival, overall survival, percentage colostomy, cost-effectiveness. Quality of life will be evaluated using validated questionnaires at baseline, at 1 and 6 months after surgery and at 1, 2, 3 and 4 years after surgery. DISCUSSION: We hypothesize the additional value of the use of the PlasmaJet in CCS for advanced stage epithelial ovarian cancer. More knowledge about efficacy, side effects, recurrence rates, cost effectiveness and pathology findings after using the PlasmaJet Device is advocated. This RCT may aid in this void. TRIAL REGISTRATION: Dutch Trial Register NTR6624 . Registered 18 August 2017. Medical Ethical Committee approval number: NL62035.078.17 (Medical Ethical Committee Erasmus Medical Centre Rotterdam).

Outcome and Prognostic Impact of Surgical Staging in Serous Tubal Intraepithelial Carcinoma: A Cohort Study and Systematic Review.

The optimal management of breast cancer susceptibility gene (BRCA)1/2 carriers with isolated serous tubal intraepithelial carcinoma (STIC) found at risk-reducing salpingo-oophorectomy (RRSO) is unclear. The prevalence of occult carcinoma and STIC in a consecutive series of BRCA1/2 carriers undergoing RRSO is reported. The outcome of staging procedures in BRCA1/2 carriers with isolated STIC at RRSO as well as the relationship between staging, chemotherapy treatment and risk of recurrence was assessed via a systematic review of the literature. Our series included 235 BRCA1/2 carriers who underwent RRSO. Federation of Gynaecology and Obstetrics stage IA carcinoma or STIC was found at RRSO in three (1.3%) and two (0.9%) patients, respectively. A systematic review of the literature included 82 BRCA1/2 carriers with isolated STIC found at RRSO. In 13/82 (16%) cases with STIC, staging was reported. In none of these cases staging revealed more advanced disease. Recurrent disease was found in four of 36 patients with reported follow-up. The estimated risk of recurrence in patients with isolated STIC at RRSO was about 11% (95% confidence interval 3-26%) after a median follow-up of 42 months (range 7-138). No recurrences were reported in those patients with STIC at RRSO who underwent staging or received chemotherapy. We found 1.3% occult carcinoma and 0.9% STIC at RRSO in our cohort of BRCA1/2 carriers. A systematic review of the literature suggests that additional treatment after RRSO, i.e. staging and/or chemotherapy, is associated with a lower risk of recurrence. However, data on staging and follow-up are limited.

Accelerated HIV testing for PMTCT in maternity and labour wards is vital to capture mothers at a critical point in the programme at district level in Malawi.

Round the clock (24 hours×7 days) HIV testing is vital to maintain a high prevention of mother to child transmission (PMTCT) coverage for women delivering in district health facilities. PMTCT coverage increases when most of the pregnant women will have their HIV status tested. Therefore routine offering of HIV testing should be integrated and seen as a part of comprehensive antenatal care. For women who miss antenatal care and deliver in a health facility without having had their HIV status tested, the labour and maternity ward could still serve as other entry points.

Effects of electric stimulation-assisted cycling training in people with chronic stroke.

OBJECTIVE: To evaluate whether leg cycling training in subjects with chronic stroke can improve cycling performance, aerobic capacity, muscle strength, and functional performance and to determine if electric stimulation (ES) to the contralateral (paretic) leg during cycling has additional effects over cycling without ES. DESIGN: A randomized controlled trial, with a partial double-blind design. SETTING: A rehabilitation center. PARTICIPANTS: Twelve stroke patients (range, 18-70 y), more than 5 months poststroke, with lower-extremity hemiparesis. INTERVENTION: Subjects were randomly assigned to groups that performed cycling exercise, one with ES evoking muscle contractions and a control group with ES not evoking muscle contractions. Subjects, blinded for group assignment, trained twice a week for 6 weeks. MAIN OUTCOME MEASURES: Changes in aerobic capacity and maximal power output, functional performance, and lower-limb muscle strength. RESULTS: Aerobic capacity and maximal power output significantly increased by 13.8%+/-19.1% and 38.1%+/-19.8%, but muscle strength was not significantly enhanced after training. Functional performance improved (ie, scores on the Berg Balance Scale increased by 6.9%+/-5.8% (P=.000) and the six-minute walk test improved by 14.5%+/-14.1% (P=.035). There was no significant effect on the Rivermead Mobility Index (P=.165). Training-induced changes were not significantly different between the 2 groups. Changes in cycling performance and aerobic capacity were not significantly related to changes in functional performance. CONCLUSIONS: This study showed that a short cycling training program on a semirecumbent cycle ergometer can markedly improve cycling performance, aerobic capacity, and functional performance of people with chronic stroke. The use of ES had no additional effects in this specific group of subjects with chronic stroke.

[Diagnostic image (310). A man with a knife in his head]. / Diagnose in beeld (310). Een man met een mes in zijn hoofd.

A 30-year-old man presented with a knife in his head; it had perforated the left temporal area and its point was located in the mouth. After surgical removal of the knife, the patient left the hospital without functional deficits.

Speciation: more likely through a genetic or through a learned habitat preference?

A problem in understanding sympatric speciation is establishing how reproductive isolation can arise when there is disruptive selection on an ecological trait. One of the solutions that has been proposed is that a habitat preference evolves, and that mates are chosen within the preferred habitat. We present a model where the habitat preference can evolve either by means of a genetic mechanism or by means of learning. Employing an adaptive-dynamical analysis, we show that evolution proceeds either to a single population of specialists with a genetic preference for their optimal habitat, or to a population of generalists without a habitat preference. The generalist population subsequently experiences disruptive selection. Learning promotes speciation because it increases the intensity of disruptive selection. An individual-based version of the model shows that, when loci are completely unlinked and learning confers little cost, the presence of disruptive selection most probably leads to speciation via the simultaneous evolution of a learned habitat preference. For high costs of learning, speciation is most likely to occur via the evolution of a genetic habitat preference. However, the latter only happens when the effect of mutations is large, or when there is linkage between genes coding for the different traits.

Speciation through the learning of habitat features.

Learning of environmental features can influence both mating behaviour and the location where young are produced. This may lead to speciation in three steps: (i) colonization of a new habitat, (ii) genetic divergence of the two groups by adaptation to the habitats, and (iii) a decrease of genetic mixing between the lineages (similar to reinforcement). In a previous paper we showed that steps (i) and (ii) occur readily for a wide range of fixed mating and habitat preferences. Here, we study whether this can ultimately lead to speciation through selective changes in these preferences. We show that this indeed occurs, and, furthermore, it is very general: for a large class of models there is selection toward producing young more frequently in the natal habitat. Once habitat preference is strong, there is selection toward stronger assortative mating. Even when steps (i) and (ii) initially fail, genetic divergence may succeed at a later evolutionary stage, after which a decrease of genetic mixing completes speciation. Our results show that speciation by the learning of habitat features is an extremely effective mechanism.

Metabolic cost of lengthening, isometric and shortening contractions in maximally stimulated rat skeletal muscle.

AIM: The present study investigated the energy cost of lengthening, isometric and shortening contractions in rat muscle (n = 19). METHODS: With electrical stimulation the rat medial gastrocnemius muscle was maximally stimulated to perform 10 lengthening, isometric and shortening contractions (velocity 25 mm s(-1)) under experimental conditions (e.g. temperature, movement velocity) that resemble conditions in human movement. RESULTS: Mean +/- SD force-time-integral of the first contraction was significantly different between the three protocols, 2.4 +/- 0.2, 1.7 +/- 0.2 and 1.0 +/- 0.2 N s, respectively (P < 0.05). High-energy phosphate consumption was not significantly different between the three modes of exercise but a trend could be observed from lengthening (7.7 +/- 2.7 micromol approximately P muscle(-1)) to isometric (8.9 +/- 2.2 micromol approximately P muscle(-1)) to shortening contractions (10.4 +/- 1.6 micromol approximately P muscle(-1)). The ratio of high-energy phosphate consumption to force-time-integral was significantly lower for lengthening [0.3 +/- 0.1 micromol approximately P (N s)(-1)] and isometric [0.6 +/- 0.2 micromol approximately P (N s)(-1)] contractions compared with shortening [1.2 +/- 0.2 micromol approximately P (N s)(-1)] contractions (P < 0.05). CONCLUSION: The present results of maximally stimulated muscles are comparable with data in the literature for voluntary human exercise showing that the energy cost of force production during lengthening exercise is approximately 30% of that in shortening exercise. The present study suggests that this finding in humans probably does reflect intrinsic muscle properties rather than effects of differential recruitment and/or coactivation.

Metabolically assessed muscle fibre recruitment in brief isometric contractions at different intensities.

This study investigated the recruitment of type I, IIA and IIAX fibres after seven isometric contractions at 40, 70 and 100% maximal voluntary knee extension torque (MVC, 1 s on/1 s off). Biopsies of the vastus lateralis muscle were collected from seven subjects at rest and immediately post-exercise. Fibre fragments were dissected from the freeze-dried samples and characterized as type I, IIA and IIAX using mATPase staining. Phosphocreatine (PCr) and creatine (Cr) content were measured in the remaining part of characterized fibres. A decline in the ratio of PCr to Cr (PCr/Cr) was used as an indication of activation. The mean peak torques were, respectively, 39 (2), 72 (2) and 87 (6)% MVC. Cumulative distributions of type I and IIA fibres were significantly shifted to lower PCr/Cr ratios at all intensities (Kolmogorov-Smirnov test, P<0.05). The cumulative distribution of type IIAX fibres showed a significant leftward shift only at 87% MVC ( P<0.05). A hierarchical order of fibre activation with increasing intensity of exercise was found, with some indication of rate coding for type I and IIA fibres. Evidence for activation of type IIAX fibres was only found at 87% MVC.

Voluntary activation level and muscle fiber recruitment of human quadriceps during lengthening contractions.

Voluntary activation levels during lengthening, isometric, and shortening contractions (angular velocity 60 degrees/s) were investigated by using electrical stimulation of the femoral nerve (triplet, 300 Hz) superimposed on maximal efforts. Recruitment of fiber populations was investigated by using the phosphocreatine-to-creatine ratio (PCr/Cr) of single characterized muscle fibers obtained from needle biopsies at rest and immediately after a series of 10 lengthening, isometric, and shortening contractions (1 s on/1 s off). Maximal voluntary torque was significantly higher during lengthening (270 +/- 55 N.m) compared with shortening contractions (199 +/- 47 N.m, P < 0.05) but was not different from isometric contractions (252 +/- 47 N.m). Isometric torque was higher than torque during shortening (P < 0.05). Voluntary activation level during maximal attempted lengthening contractions (79 +/- 8%) was significantly lower compared with isometric (93 +/- 5%) and shortening contractions (92 +/- 3%, P < 0.05). Mean PCr/Cr values of all fibers from all subjects at rest were 2.5 +/- 0.6, 2.0 +/- 0.7, and 2.0 +/- 0.7, respectively, for type I, IIa, and IIax fibers. After 10 contractions, the mean PCr/Cr values for grouped fiber populations (regardless of fiber type) were all significantly different from rest (1.3 +/- 0.2, 0.7 +/- 0.3, and 0.8 +/- 0.6 for lengthening, isometric, and shortening contractions, respectively; P < 0.05). The cumulative distributions of individual fiber populations after either contraction mode were significantly different from rest (P < 0.05). Curves after lengthening contractions were less shifted compared with curves from isometric and shortening contractions (P < 0.05), with a smaller shift for the type IIax compared with type I fibers in the lengthening contractions. The results indicate a reduced voluntary drive during lengthening contractions. PCr/Cr values of single fibers indicated a hierarchical order of recruitment of all fiber populations during maximal attempted lengthening contractions.

Changes in PCr/Cr ratio in single characterized muscle fibre fragments after only a few maximal voluntary contractions in humans.

AIM: This methodological study investigated the number of brief maximal voluntary isometric contractions (MVC) needed to show evidence of fibre activation, as indicated by changes in the phosphocreatine to creatine (PCr/Cr) ratio. METHODS: Subjects performed series of four, seven and/or 10 MVC (1 s on, 1 s off) of the m. quadriceps (60 degrees -flexion angle). Biopsy samples of the m. vastus lateralis were taken at rest and immediately post-exercise. Single muscle fibres were dissected from the freeze-dried samples and classified as types I, IIA or IIAX, using mATPase stainings. Fragments of characterized fibres were analysed for PCr and Cr content. Analyses of variance were performed to investigate changes in PCr/Cr per fibre group over time, followed by Bonferroni post-hoc test (P < 0.01). The fifth percentile of resting values of each fibre group was determined. RESULTS: Mean PCr/Cr ratio after four, seven and 10 MVCs were significantly lower for all fibre groups (P < 0.01). The mean decreases were 44, 64 and 76%, respectively. However, only after seven and 10 contractions PCr/Cr ratios of all, but three type I and two type IIAX fibres, individual fibres were below the fifth percentile. CONCLUSION: In very short duration exercise, involving seven brief maximal voluntary contractions, changes in the PCr/Cr ratio indicated activation of different characterized muscle fibre fragments. The results suggest that this approach may be useful for investigating the pattern of fibre type activation in exercise of very short duration.

Effect of antagonist muscle fatigue on knee extension torque.

The effect of hamstring fatigue on knee extension torque was examined at different knee angles for seven male subjects. Before and after a dynamic flexion fatigue protocol (180 degrees s(-1), until dynamic torque had declined by 50%), maximal voluntary contraction extension torque was measured at four knee flexion angles (90 degrees, 70 degrees, 50 degrees and 30 degrees ). Maximal torque generating capacity and voluntary activation of the quadriceps muscle were determined using electrical stimulation. Average rectified EMG of the biceps femoris was determined. Mean dynamic flexion torque declined by 48+/-11%. Extensor maximal voluntary contraction torque, maximal torque generating capacity, voluntary activation and average rectified EMG at the four knee angles were unaffected by the hamstring fatigue protocol. Only at 50 degrees knee angle was voluntary activation significantly lower (15.7%) after fatigue ( P<0.05). In addition, average rectified EMG before fatigue was not significantly influenced by knee angle. It was concluded that a fatigued hamstring muscle did not increase the maximal voluntary contraction extension torque and knee angle did not change coactivation. Three possible mechanisms may explain the results: a potential difference in recruited fibre populations in antagonist activity compared with the fibres which were fatigued in the protocol, a smaller loss in isometric torque generating capacity of the hamstring muscle than was expected from the dynamic measurements and/or a reduction in voluntary activation.

No acute effects of short-term creatine supplementation on muscle properties and sprint performance.

In a double-blind, placebo, controlled study, we investigated the acute effects of short-term oral creatine supplementation (20 g.day-1 for 6 days) on muscle activation, fatigue and recovery of the m. quadriceps femoris during electrical stimulation, and on maximal performance during sprint cycling. The quadriceps muscles of 23 well-trained rowers were stimulated at different frequencies (10, 20, 50, 100, 150 and 200 Hz). Furthermore, 40 repetitive, electrically stimulated (duration 220 ms, stimulation frequency 150 Hz) concentric contractions were imposed at a constant angular velocity of 180 degrees.s-1 over a range of 50 degrees (from 90 to 140 degrees knee angle), each extension/flexion cycle lasting 1200 ms. To determine recovery, torque was measured at 20, 50, 80, 120, 180 and 300 s after the last contraction. In addition, two maximal 30-s sprints were performed on a cycle ergometer with 4 min rest in between. Following short-term creatine supplementation, body mass [mean (SEM)] increased (P < 0.05) from 85.7 (2.7) kg to 87.3 (2.9) kg. Creatine supplementation had no effect on maximal voluntary isometric torque and muscle activation, or on fatigue and recovery of dynamic exercise. There was also no significant effect on peak power, time to peak power and work to peak power, or total work during both sprints on the cycle ergometer. It was concluded that short-term oral creatine supplementation resulted in increased body mass, but did not enhance muscle performance or maximal output during sprint cycling.

C3 toxin activates the stress signaling pathways, JNK and p38, but antagonizes the activation of AP-1 in rat-1 cells.

Lysophosphatidic acid (LPA) stimulates the c-Fos serum response element (SRE) by activating two distinct signal pathways regulated by the small GTPases, Ras and RhoA. Ras activates the ERK cascade leading to phosphorylation of the transcription factors Elk-1 and Sap1a at the Ets/TCF site. RhoA regulates an undefined pathway required for the activation of the SRF/CArG site. Here we have examined the role of the Ras and RhoA pathways in activation of the SRE and c-Fos expression in Rat-1 cells. Pertussis toxin and PD98059 strongly inhibited LPA-stimulated c-Fos expression and activation of a SRE:Luc reporter. C3 toxin completely inhibited RhoA function, partially inhibited SRE:Luc activity, but had no effect on LPA-stimulated c-Fos expression. Thus, in a physiological context the Ras-Raf-MEK-ERK pathway, but not RhoA, is required for LPA-stimulated c-Fos expression in Rat-1 cells. C3 toxin stimulated the stress-activated protein kinases JNK and p38 and potentiated c-Jun expression and phosphorylation; these properties were shared by another cellular stress agonist the protein kinase C inhibitor Ro-31-8220. However, C3 toxin alone or in combination with growth factors did not stimulate AP-1:Luc activity and actually antagonized the synergistic activation of AP-1:Luc observed in response to co-stimulation with growth factors and Ro-31-8220. These data indicate that C3 toxin is a cellular stress which antagonizes activation of AP-1 at a point downstream of stress-activated kinase activation or immediate-early gene induction.

Regulation of mitogen-activated protein kinase phosphatase-1 expression by extracellular signal-related kinase-dependent and Ca2+-dependent signal pathways in Rat-1 cells.

Stimulation of Rat-1 cells with lysophosphatidic acid (LPA) or epidermal growth factor (EGF) results in a biphasic, sustained activation of extracellular signal-regulated kinase 1 (ERK1). Pretreatment of Rat-1 cells with either cycloheximide or sodium orthovanadate had little effect on the early peak of ERK1 activity but potentiated the sustained phase. Cycloheximide also potentiated ERK1 activation in Rat-1 cells expressing DeltaRaf-1:ER, an estradiol-regulated form of the oncogenic, human Raf-1. Since cycloheximide did not potentiate MEK activity but abrogated the expression of mitogen-activated protein kinase phosphatase (MKP-1) normally seen in response to EGF and LPA, we speculated that the level of MKP-1 expression may be an important regulator of ERK1 activity in Rat-1 cells. Inhibition of LPA-stimulated MEK and ERK activation with PD98059 and pertussis toxin, a selective inhibitor of Gi-protein-coupled signaling pathways, reduced LPA-stimulated MKP-1 expression by only 50%, suggesting the presence of additional MEK- and ERK-independent pathways for MKP-1 expression. Specific activation of the MEK/ERK pathway by DeltaRaf-1:ER had little or no effect on MKP-1 expression, suggesting that activation of the Raf/MEK/ERK pathway is necessary but not sufficient for MKP-1 expression in Rat-1 cells. Activation of PKC played little part in growth factor-stimulated MKP-1 expression, but LPA- and EGF-induced MKP-1 expression was blocked by buffering [Ca2+]i, leading to a potentiation of the sustained phase of ERK1 activation without potentiating MEK activity. In Rat-1DeltaRaf-1:ER cells, we observed a strong synergy of MKP-1 expression when cells were stimulated with estradiol in the presence of ionomycin, phorbol 12-myristate 13-acetate, or okadaic acid under conditions where these agents did not synergize for ERK activation. These results suggest that activation of the Raf/MEK/ERK pathway is insufficient to induce expression of MKP-1 but instead requires other signals, such as Ca2+, to fully reconstitute the response seen with growth factors. In this way, ERK-dependent and -independent signals may regulate MKP-1 expression, the magnitude of sustained ERK1 activity, and therefore gene expression.

Differential regulation of extracellular signal-regulated protein kinase 1 and Jun N-terminal kinase 1 by Ca2+ and protein kinase C in endothelin-stimulated Rat-1 cells.

The extracellular signal-regulated protein kinase (ERK) and Jun N-terminal kinase (JNK) signalling cascades transduce signals from the cell cytoplasm to the nucleus, where they regulate gene expression. The activation of ERK1 by lysophosphatidic acid (LPA) and endothelin 1 (Et-1) was compared in Rat-1 cells. Both stimulated DNA synthesis to a similar degree but, in contrast with LPA, Et-1 did not stimulate sustained ERK1 activation, a signal that is thought to be important for the proliferation of fibroblasts. Et-1, but not LPA, was able to activate JNK1; pharmacological analysis revealed that the same EtA receptor mediates DNA synthesis, ERK1 and JNK1 activation. However, activation of JNK1 required higher concentrations of Et-1 than was required for stimulation of ERK1 or DNA synthesis. Signalling to ERK1 and JNK1 was partly inhibited by pertussis toxin, suggesting that both pathways are regulated in part by Gi or G0 proteins. Activation of JNK1 by Et-1 lagged behind ERK1 activation but was not dependent on it because PD98059, an inhibitor of mitogen-activated protein kinase (or ERK) kinase, was without effect on JNK1 activation. In contrast with recent studies, activation of protein kinase C (PKC) or Ca2+ fluxes inhibited activation of JNK1 but not ERK1; furthermore inhibition of PKC or sequestration of Ca2+ potentiated JNK1 activation by Et-1 but not by anisomycin, and again had little effect on ERK1 activation. These results demonstrate that the same G-protein-coupled receptor can activate both the ERK and JNK signal pathways but the two kinase cascades seem to be separate, parallel pathways that are differentially regulated by PKC and Ca2+. The results are discussed in terms of the role of ERK and JNK in proliferative signalling.