Tracing growth patterns in cod (Gadus morhua L.) using bioenergetic modelling.

Understanding individual growth in commercially exploited fish populations is key to successful stock assessment and informed ecosystem-based fisheries management. Traditionally, growth rates in marine fish are estimated using otolith age-readings in combination with age-length relationships from field samples, or tag-recapture field experiments. However, for some species, otolith-based approaches have been proven unreliable and tag-recapture experiments suffer from high working effort and costs as well as low recapture rates. An important alternative approach for estimating fish growth is represented by bioenergetic modelling which in addition to pure growth estimation can provide valuable insights into the processes leading to temporal growth changes resulting from environmental and related behavioural changes. We here developed an individual-based bioenergetic model for Western Baltic cod (Gadus morhua), traditionally a commercially important fish species that however collapsed recently and likely suffers from climate change effects. Western Baltic cod is an ideal case study for bioenergetic modelling because of recently gained in-situ process knowledge on spatial distribution and feeding behaviour based on highly resolved data on stomachs and fish distribution. Additionally, physiological processes such as gastric evacuation, consumption, net-conversion efficiency and metabolic rates have been well studied for cod in laboratory experiments. Our model reliably reproduced seasonal growth patterns observed in the field. Importantly, our bioenergetic modelling approach implementing depth-use patterns and food intake allowed us to explain the potentially detrimental effect summer heat periods have on the growth of Western Baltic cod that likely will increasingly occur in the future. Hence, our model simulations highlighted a potential mechanism on how warming due to climate change affects the growth of a key species that may apply for similar environments elsewhere.

Recombinant Porphyromonas gingivalis W83 FimA alters immune response and metabolic gene expression in oral squamous carcinoma cells.

OBJECTIVES: The Gram-negative anaerobic rod Porphyromonas gingivalis (P. gingivalis) is regarded as a keystone pathogen in periodontitis and expresses a multitude of virulence factors iincluding fimbriae that are enabling adherence to and invasion in cells and tissues. The progression of periodontitis is a consequence of the interaction between the host immune response and periodontal pathogens. The aim of this study was to investigate the genome-wide impact of recombinant fimbrial protein FimA from P. gingivalis W83 on the gene expression of oral squamous carcinoma cells by transcriptome analysis. MATERIALS AND METHODS: Human squamous cell carcinoma cells (SCC-25) were stimulated for 4 and 24 h with recombinant FimA. RNA sequencing was performed and differential gene expression and enrichment were analyzed using gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and REACTOME. The results of transcriptome analysis were validated using quantitative real-time polymerase chain reaction (PCR) with selected genes. RESULTS: Differential gene expression after 4 and 24 h revealed upregulation of 464 (4 h) and 179 genes (24 h) and downregulation of 69 (4 h) and 312 (24 h) genes. GO, KEGG, and REACTONE enrichment analysis identified a strong immunologic transcriptomic response signature after 4 h. After 24 h, mainly those genes were regulated, which belonged to cell metabolic pathways and replication. Real-time PCR of selected genes belonging to immune response and signaling demonstrated strong upregulation of CCL20, TNFAIP6, CXCL8, TNFAIP3, and NFkBIA after both stimulation times. CONCLUSIONS: These data shed light on the RNA transcriptome of human oral squamous carcinoma epithelial cells following stimulation with P. gingivalis FimA and identify a strong immunological gene expression response to this virulence factor. The data provide a base for future studies of molecular and cellular interactions between P. gingivalis and oral epithelium to elucidate basic mechanisms that may provide new prospects for periodontitis therapy and give new insights into the development and possible treatments of cancer.

Porphyromonas gingivalis W83 Membrane Components Induce Distinct Profiles of Metabolic Genes in Oral Squamous Carcinoma Cells.

Periodontitis, a chronic inflammatory disease is caused by a bacterial biofilm, affecting all periodontal tissues and structures. This chronic disease seems to be associated with cancer since, in general, inflammation intensifies the risk for carcinoma development and progression. Interactions between periodontal pathogens and the host immune response induce the onset of periodontitis and are responsible for its progression, among them Porphyromonas gingivalis (P. gingivalis), a Gram-negative anaerobic rod, capable of expressing a variety of virulence factors that is considered a keystone pathogen in periodontal biofilms. The aim of this study was to investigate the genome-wide impact of P. gingivalis W83 membranes on RNA expression of oral squamous carcinoma cells by transcriptome analysis. Human squamous cell carcinoma cells (SCC-25) were infected for 4 and 24 h with extracts from P. gingivalis W83 membrane, harvested, and RNA was extracted. RNA sequencing was performed, and differential gene expression and enrichment were analyzed using GO, KEGG, and REACTOME. The results of transcriptome analysis were validated using quantitative real-time PCR with selected genes. Differential gene expression analysis resulted in the upregulation of 15 genes and downregulation of 1 gene after 4 h. After 24 h, 61 genes were upregulated and 278 downregulated. GO, KEGG, and REACTONE enrichment analysis revealed a strong metabolic transcriptomic response signature, demonstrating altered gene expressions after 4 h and 24 h that mainly belong to cell metabolic pathways and replication. Real-time PCR of selected genes belonging to immune response, signaling, and metabolism revealed upregulated expression of CCL20, CXCL8, NFkBIA, TNFAIP3, TRAF5, CYP1A1, and NOD2. This work sheds light on the RNA transcriptome of human oral squamous carcinoma cells following stimulation with P. gingivalis membranes and identifies a strong metabolic gene expression response to this periodontal pathogen. The data provide a base for future studies of molecular and cellular interactions between P. gingivalis and oral epithelium to elucidate the basic mechanisms of periodontitis and the development of cancer.

Predation risk triggers copepod small-scale behavior in the Baltic Sea.

Predators not only have direct impact on biomass but also indirect, non-consumptive effects on the behavior their prey organisms. A characteristic response of zooplankton in aquatic ecosystems is predator avoidance by diel vertical migration (DVM), a behavior which is well studied on the population level. A wide range of behavioral diversity and plasticity has been observed both between- as well as within-species and, hence, investigating predator-prey interactions at the individual level seems therefore essential for a better understanding of zooplankton dynamics. Here we applied an underwater imaging instrument, the video plankton recorder (VPR), which allows the non-invasive investigation of individual, diel adaptive behavior of zooplankton in response to predators in the natural oceanic environment, providing a finely resolved and continuous documentation of the organisms' vertical distribution. Combing observations of copepod individuals observed with the VPR and hydroacoustic estimates of predatory fish biomass, we here show (i) a small-scale DVM of ovigerous Pseudocalanus acuspes females in response to its main predators, (ii) in-situ observations of a direct short-term reaction of the prey to the arrival of the predator and (iii) in-situ evidence of pronounced individual variation in this adaptive behavior with potentially strong effects on individual performance and ecosystem functioning.

Alternative reproductive tactics are associated with sperm performance in invasive round goby from two different salinity environments.

During male-male competition, evolution can favor alternative reproductive tactics. This often results in a dominant morph that holds a resource, such as a nest for egg laying, which competes with a smaller sneaker morph that reproduces by stealing fertilizations. The salinity environment can influence male growth rates, for example, via osmoregulatory costs, which in turn may influence the use of sneaker tactics for small males competing for mating opportunities. Salinity can also affect sperm directly; however, little is known of how salinity influences sneaker tactics through sperm performance. We sampled males of the invasive round goby (Neogobius melanostomus) from two environments, a freshwater river and a brackish estuary. This fish has two male morphs: nest-holding dark males and non-nest-holding light males. We examined the role of water salinity of 0, 8, and 16 on sperm performance and found that for estuarine males, a salinity of 0 reduced sperm velocity compared to a salinity of 8 and 16. Riverine males had low velocity in all salinities. Sperm viability also decreased by over 30% in 0 salinity, compared to 8 and 16, for fish from both environments. Gobies produce ejaculate contents in specialized glands that could in theory shield sperm in an adverse environment. However, gland contents did not improve sperm performance in our tests. Body mass and age estimates indicate that riverine males invested more in somatic growth compared to estuarine males. Estuarine light morph males had a high enough gonadosomatic index to indicate sneaker tactics. We propose that when sperm performance is low, such as for the riverine males, sneaker tactics are ineffective and will be selected against or phenotypically suppressed. Instead, we interpret the increased investment in somatic growth found in riverine males as a life-history decision that is advantageous when defending a nest in the next reproductive season.

Refractory neutrophil activation in type 2 diabetics with chronic periodontitis.

BACKGROUND: Inflammation increases diabetes mellitus type 2 (T2DM) progression and severity. T2DM patients are at high risk of the rapid development of chronic periodontitis (CP). Topical presence, high numbers, and bactericidal effects of immune cells are challenged by augmented antigen-induced inflammation, which promotes both diseases. OBJECTIVES: To investigate gingival cellular inflammatory responses in individuals with previously undiagnosed T2DM with CP or CP alone and in systemically and periodontally healthy controls (H) in vivo and to establish an ex vivo technique permitting quantitative and qualitative assessments of gingival crevicular immune cells. MATERIALS AND METHODS: T2DM + CP, CP, and H individuals (n = 10, each) received a 2-week oral hygiene regimen (OHR). Afterwards, a noninvasive sampling technique was performed to evaluate gingival inflammation induced under standardized conditions in vivo, that is, in the absence of severe periodontal destruction and inflammation at clinically healthy sites. Stimuli (casein/test or phosphate-buffered saline w/o. Ca2+ or Mg2+ , PBS(-/-) /control) were randomly applied contralaterally in the gingival sulci of participants' upper dentes canini. One day after completion of the OHR, gingival crevicular fluid (GCF) was kinetically assayed between the time of the baseline (BL) measurement and 55 minutes. Polymorphonuclear leukocyte (PMN) content (PMNGCF ) was quantitated at an optimum time of 35 minutes. PMNGCF counts reflect local inflammation. Ex vivo samples were fluorimetrically labeled, gated according to the donor's peripheral blood polymorphonuclear neutrophils (PMNPB ), and then counted, employing flow cytometry. RESULTS: PMNGCF counts in unstimulated gingival crevices (at BL) in the T2DM + CP group were higher than those in the CP and H groups. PMNGCF counts were elevated in casein vs PBS(-/-) -stimulated gingival crevices in all groups. Patients with T2DM + CP showed increased PMNGCF counts compared to those with CP (P = .035) according to scatter plots. CD45+ counts in the stimulated sites in T2DM + CP patients were higher than those in CP and H patients (P = .041). Under stimulation conditions, the CD45+ counts differed from those under placebo conditions (P = .019), indicating augmented, inducible inflammatory leukocyte infiltrate in T2DM + CP patients. CONCLUSIONS: This noninvasive technique permits quantitative assessment of (experimental) gingival inflammation in vivo, revealing an influence of T2DM + CP on the number of primary immune cells in the gingival crevice. Patients who are challenged with (local) leukocytosis are likely at risk of collateral damage to the gingival crevice neighboring tissues, favoring the severity and progression of CP and consequently T2DM (www.clinicaltrials.gov NCT01848379).

Metabolic costs of spontaneous swimming in Sprattus sprattus L., at different water temperatures.

Oxygen uptake (MO2; mgO2 fish-1h-1) of fish groups was measured at temperatures between 10-19°C in an intermittent-flow respirometer to quantify the metabolic costs of spontaneous swimming patterns in the small clupeid Sprattus sprattus. Movements of individual fish within the school were tracked automatically during respirometry. Oxygen uptake was then related to mean swimming speeds and the number of sharp turns (>90°), which are common behavioural elements of spontaneous swimming in clupeid fish. Different possible model formulations for describing the relationship between respiration and swimming patterns were compared via the AIC. The final model revealed that costs for sharp turns at a frequency of 1 s-1 doubled the metabolic costs compared to those with zero turns but with likewise a moderate swimming speed of 0.28 body length -1. The cost for swimming doubled if the swimming speed was doubled from 0.28 to 0.56 BLs-1 but increased by a factor of 4.5 if tripled to 0.84 BLs-1. Costs for transport were minimal at a speed of 0.4 body lengths s-1 at all temperatures. New basic input parameters to estimate energy losses during spontaneous movements, which occur typically during foraging in this small pelagic fish, are provided.

Genetic analyses reveal complex dynamics within a marine fish management area.

Genetic data have great potential for improving fisheries management by identifying the fundamental management units-that is, the biological populations-and their mixing. However, so far, the number of practical cases of marine fisheries management using genetics has been limited. Here, we used Atlantic cod in the Baltic Sea to demonstrate the applicability of genetics to a complex management scenario involving mixing of two genetically divergent populations. Specifically, we addressed several assumptions used in the current assessment of the two populations. Through analysis of 483 single nucleotide polymorphisms (SNPs) distributed across the Atlantic cod genome, we confirmed that a model of mechanical mixing, rather than hybridization and introgression, best explained the pattern of genetic differentiation. Thus, the fishery is best monitored as a mixed-stock fishery. Next, we developed a targeted panel of 39 SNPs with high statistical power for identifying population of origin and analyzed more than 2,000 tissue samples collected between 2011 and 2015 as well as 260 otoliths collected in 2003/2004. These data provided high spatial resolution and allowed us to investigate geographical trends in mixing, to compare patterns for different life stages and to investigate temporal trends in mixing. We found similar geographical trends for the two time points represented by tissue and otolith samples and that a recently implemented geographical management separation of the two populations provided a relatively close match to their distributions. In contrast to the current assumption, we found that patterns of mixing differed between juveniles and adults, a signal likely linked to the different reproductive dynamics of the two populations. Collectively, our data confirm that genetics is an operational tool for complex fisheries management applications. We recommend focussing on developing population assessment models and fisheries management frameworks to capitalize fully on the additional information offered by genetically assisted fisheries monitoring.

Evaluation of enterotoxin gene expression and enterotoxin production capacity of the probiotic strain Bacillus toyonensis BCT-7112T.

The aim of the present study was to evaluate the safety of the probiotic strain Bacillus toyonensis BCT-7112T (active ingredient of Toyocerin) in relation to the enterotoxins haemolysin BL (Hbl) and the non-haemolytic enterotoxin (Nhe) by performing a quantitative reverse transcription (RT) real-time polymerase chain reaction (PCR) and a Western blot assay. The expression levels of the enterotoxin genes hblA, hblD, nheA, nheB and nheC, determined by means of RT real-time PCR in B. toyonensis, were lower than those in B. cereus reference strains. No expression of hblC was detected. The Western blot assays of native and 25-fold concentrated supernatants from B. toyonensis, using monoclonal antibodies directed against the Hbl component L1 and the Nhe component NheB, showed weak bands. The NheC component was not detected in the native supernatant, but weakly in the 25-fold concentrated supernatant. According to the results of the present study, the enterotoxin expression and protein levels of B. toyonensis BCT-7112T were absent or clearly lower compared to the B. cereus reference strains. Thus, their ability to form functional enterotoxins can also be considered to be lower or unlikely compared to the B. cereus reference strains. This experimental approach can be implemented when studying the health and safety as well as harmlessness of probiotic microorganisms.

Degree of SGLT1 phosphorylation is associated with but does not determine segment-specific glucose transport features in the porcine small intestines.

Glucose-induced electrogenic ion transport is higher in the porcine ileum compared with the jejunum despite equal apical abundance of SGLT1. The objective of this study was a detailed determination of SGLT1 and GLUT2 expressions at mRNA and protein levels along the porcine small intestinal axis. Phosphorylation of SGLT1 at serine 418 was assessed as a potential modulator of activity. Porcine intestinal tissues taken along the intestinal axis 1 h or 3 h after feeding were analyzed for relative mRNA (RT-PCR) and protein levels (immunoblot) of SGLT1, pSGLT1, GLUT2, (p)AMPK, ß2 -receptor, and PKA substrates. Functional studies on electrogenic glucose transport were done (Ussing chambers: short circuit currents (Isc )). Additionally, effects of epinephrine (Epi) administration on segment-specific glucose transport and pSGLT1 content were examined. SGLT1 and GLUT2 expression was similar throughout the small intestines but lower in the duodenum and distal ileum. pSGLT1 abundance was significantly lower in the ileum compared with the jejunum associated with significantly higher glucose-induced Isc . SGLT1 phosphorylation was not inducible by Epi. Epi treatment decreased glucose-induced Isc and glucose flux rates in the jejunum but increased basal Isc in the ileum. Epi-induced PKA activation was detectable in jejunal tissue. These results may indicate that SGLT1 phosphorylation at Ser418 represents a structural change to compensate for certain conditions that may decrease glucose transport (unfavorable driving forces/changed apical membrane potential) rather than being the cause for the overall differences in glucose transport characteristics between the jejunum and ileum.

Gastrointestinal transport of calcium and glucose in lactating ewes.

During lactation, mineral and nutrient requirements increase dramatically, particularly those for Ca and glucose. In contrast to monogastric species, in ruminants, it is rather unclear to which extend this physiological change due to increased demand for milk production is accompanied by functional adaptations of the gastrointestinal tract (GIT). Therefore, we investigated potential modulations of Ca and glucose transport mechanisms in the GIT of lactating and dried-off sheep. Ussing-chamber technique was applied to determine the ruminal and jejunal Ca flux rates. In the jejunum, electrophysiological properties in response to glucose were recorded. Jejunal brush-border membrane vesicles (BBMV) served to characterize glucose uptake via sodium-linked glucose transporter 1 (SGLT1), and RNA and protein expression levels of Ca and glucose transporting systems were determined. Ruminal Ca flux rate data showed a trend for higher absorption in lactating sheep. In the jejunum, small Ca absorption could only be observed in lactating ewes. From the results, it may be assumed that lactating ewes compensate for the Ca loss by increasing bone mobilization rather than by increasing supply through absorption from the GIT Presence of SGLT1 in the jejunum of both groups was shown by RNA and protein identification, but glucose uptake into BBMV could only be detected in lactating sheep. This, however, could not be attributed to electrogenic glucose absorption in lactating sheep under Ussing-chamber conditions, providing evidence that changes in jejunal glucose uptake may include additional factors, that is, posttranslational modifications such as phosphorylation.

Ultrafast Relaxation Dynamics of the Ethylene Cation C(2)H(4)+.

We present a combined experimental and computational study of the relaxation dynamics of the ethylene cation. In the experiment, we apply an extreme-ultraviolet-pump/infrared-probe scheme that permits us to resolve time scales on the order of 10 fs. The photoionization of ethylene followed by an infrared (IR) probe pulse leads to a rich structure in the fragment ion yields reflecting the fast response of the molecule and its nuclei. The temporal resolution of our setup enables us to pinpoint an upper bound of the previously defined ethylene-ethylidene isomerization time to 30 ± 3 fs. Time-dependent density functional based trajectory surface hopping simulations show that internal relaxation between the first excited states and the ground state occurs via three different conical intersections. This relaxation unfolds on femtosecond time scales and can be probed by ultrashort IR pulses. Through this probe mechanism, we demonstrate a route to optical control of the important dissociation pathways leading to separation of H or H2.

Hydrostatic pressure affects selective tidal stream transport in the North Sea brown shrimp (Crangon crangon).

The brown shrimp (Crangon crangon) is a highly abundant invertebrate in the North Sea, with its life cycle stages ranging from deep offshore spawning to shallow onshore nursery areas. To overcome the long distances between these two habitats, brown shrimp are suspected to use selective tidal stream transport (STST), moving with the cyclic tide currents towards their preferred water depths. However, it is not known which stimulus actually triggers STST behavior in brown shrimp. In this work, we determined the influence of different hyperbaric pressures on STST behavior of juvenile brown shrimp. Brown shrimp activity was recorded in a hyperbaric pressure chamber that supplied constant and dynamic pressure conditions simulating different depths, with and without a tidal cycle. Subsequent wavelet and Fourier analysis were performed to determine the periodicity in the activity data. The results of the experiments show that STST behavior in brown shrimp varies with pressure and therefore with depth. We further show that STST behavior can be initiated by cyclic pressure changes. However, an interaction with one or more other environmental triggers remains possible. Furthermore, a security ebb-tide activity was identified that may serve to avoid potential stranding in shallow waters and is 'remembered' by shrimp for about 1.5 days without contact with tidal triggers.

Neutrophil activation and periodontal tissue injury.

Neutrophilic polymorphonuclear leukocytes (PMNL) track, engage and eliminate foreign entities, including bacteria, fungi and subcellular particles. PMNL are the major host-cell line involved in the acute response during the early stages of infections, including those in the oral cavity. Rather short lived, they are among the fastest moving cells in the human body and travel great distances only to be immolated after encountering and neutralizing antigens. Although their role as the first line of host defense is well established, their role in chronic granulomatous inflammations, diseases and infections remains poorly understood, and many questions on the activation, motility, bactericidity and termination of PMNL in these conditions remain unanswered. This review aims to summarize our current understanding of the molecular mechanisms of PMNL activation and signaling events. Recent evidence indicates the presence of collateral tissue damage caused by poorly regulated PMNL pursuits of periodontal bacteria. Imbalances between the antigenic challenge and the primary host response may augment periodontal tissue breakdown. Thereafter, orchestrated regulation of the resolution of inflammation fails in the presence of a pathogenic periodontal biofilm.

Segmental diversity of electrogenic glucose transport characteristics in the small intestines of weaned pigs.

It has been shown in several species that the intestinal Na(+)-dependent glucose co-transporter 1 (SGLT1) is more abundant in the jejunum than in ileum. In contrast, the efficiency of intestinal glucose uptake rates in suckling piglets or weaned pigs is not clearly fitting with this segmental distribution. The aim of this study was to evaluate SGLT1 mediated glucose absorption in the jejunum and ileum of growing pigs (Sus scrofa) in more detail. In Ussing chambers, basal short-circuit currents were significantly more positive in the jejunum. It could be demonstrated that the electrogenic ileal glucose transport was significantly more pronounced in different breeds and occurred at 5 mmol∙L(-1) glucose 7 times faster in the ileum, although slightly higher jejunal expression of glycosylated SGLT1 was detected by Western blotting. This expression pattern was connected to significantly lower phlorizin sensitivity in the jejunum. As the more efficient ileal glucose absorption was also observable with glucose uptake studies into isolated brush-border membrane vesicles without differences in abundance and activity of the Na(+)/K(+)-ATPase in both segments, we conclude that the segmental differences in porcine glucose transport characteristics may be based on direct or indirect modulations of SGLT1 activity.

Transepithelial transport and intraepithelial metabolism of short-chain fatty acids (SCFA) in the porcine proximal colon are influenced by SCFA concentration and luminal pH.

Short-chain fatty acids (SCFA) are end products of bacterial fermentation in the colon and cecum of monogastric animals. As SCFA serve as relevant energy suppliers for colonocytes and various tissues, it is important to reveal fundamental mechanistic characteristics of their transepithelial transport subjected to transient variations of fermentations rates. We performed Ussing chamber studies with porcine (Sus scrofa) colon epithelium under physiological conditions and examined individual mucosal disappearance, metabolized loss, tissue concentrations and serosal release of acetate, propionate and butyrate by gas chromatography. Reduction of initial SCFA concentrations from 80 to 40 mmol/L resulted in diminished absolute flux rates, but the relative proportions of mucosal disappearance and intracellular metabolization of individual SCFA were slightly enhanced. Simulation of high fermentation rates by lowering the mucosal pH induced an increase in mucosal disappearance and serosal release of all SCFA, while their tissue contents trended to lower levels. With respect to the metabolization at lowered pH we found increased acetate concentrations and a decrease of propionate and butyrate. Our data indicate that the colon epithelium possesses a high adaptive capacity to ensure its energetic maintenance under various intraluminal fermentation rates by utilizing the unique features of individual SCFA as energy sources.

BMP-7 as antagonist of organ fibrosis.

Fibrosis is a scarring process that is a common feature of chronic organ injury. It is characterized by elevated activity of transforming growth factor-beta resulting in increased and altered deposition of extracellular matrix and other fibrosis-associated proteins. Recent work has demonstrated that bone morphogenetic protein-7 blocks transforming growth factor-beta signaling. Moreover, member of the CCN family, Endoglin, Sclerostin, Sclerostin domain-containing proteins, Gremlin, Noggin, Chordin, and Kielin/Chordin-like protein influence the biological activity of both cytokines. As a consequence, they modulate cellular proliferation, migration, adhesion and extracellular matrix production. This tight protein network consisting of transforming growth factor-betas, bone morphogenetic proteins and various binding partners includes potential novel molecular targets and biomarkers useful for prognostication, disease monitoring and therapy. We here summarize recent advances in understanding bone morphogenetic protein-7 function and signaling and the current attempts to use this critical modulator as a pharmacological device to reverse transforming growth factor-beta-induced fibrogenesis.

Activation of TGF-beta within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor.

Recently, synthesis and secretion of connective tissue growth factor (CTGF)/CYR61/CTGF/NOV-family member 2 (CCN2) in cultures of hepatocytes were shown, which are sensitively up-regulated by exogenous TGF-beta. In this study TGF-beta-dependent CTGF/CCN2 expression in hepatocytes cultured under completely TGF-beta-free conditions was analysed by Western-blots, metabolic labelling, and CTGF-reporter gene assays. In alkaline phosphatase monoclonal anti-alkaline phosphatase complex (APAAP)-staining of cultured hepatocytes it was demonstrated that latent TGF-beta within the hepatocytes becomes rapidly detectable during culture indicating an intracellular demasking of the mature TGF-beta antigen. Subsequent signaling to theCTGF/CCN2 promoter occurs via p-Smad2, whereas p-Smad3 does not seem to be involved. Cycloheximide did not abolish the rapid immunocytochemical appearance of mature TGF-beta, but calpain inhibitors partially suppressed intracellular TGF-beta activation and subsequently CTGF up-regulation. Calpain treatment had the reverse effect. None of the inhibitors of extracellular TGF-beta signalling was effective in the reduction of spontaneous CTGF synthesis, but intracellularly acting Alk 4-/Alk 5-specific inhibitor SB-431542 was able to diminish CTGF expression. The assumption that latent intracellular TGF-beta is activated by calpains during culture-induced stress or injurious conditions in the liver in vivo was further validated by a direct effect of calpains on the activation of recombinant latent TGF-beta. In conclusion, these data are the first to suggest the possibility of intracrine TGF-beta signalling due to calpain-dependent intracellular proteolytic activation leading to transcriptional activation of CTGF/CCN2 as a TGF-beta-sensitive reporter gene. This mechanism might be deleterious for keeping long-term hepatocyte cultures due to TGF-beta-induced apoptosis and, further, might be of relevance for induction of apoptosis or epithelial-mesenchymal transition of hepatocytes in injured liver.

TGF-beta up-regulates serum response factor in activated hepatic stellate cells.

In differentiated smooth muscle cells (SMC) the regulation of SMC marker genes (e.g. alpha-smooth muscle actin) is mainly conducted by the serum response factor (SRF) and accessory co-factors like myocardin. A number of SMC markers are also expressed in activated hepatic stellate cells which are the main cellular effectors in liver fibrogenesis. In the present study we found that during cellular activation and transdifferentiation the SRF transcription factor is up-regulated by transforming growth factor-beta, accumulated in the nucleus, and exhibited increased DNA-binding activity. These observations were accompanied by a forced expression of the SRF co-activator myocardin. Specific targeting of SRF by small interference RNA resulted in diminished contents of alpha-smooth muscle actin. Therefore, we conclude that hepatic stellate cells retain differentiation capacity to evolve characteristics that are typical for cells of the cardiac and smooth muscle lineages.

Immortal hepatic stellate cell lines: useful tools to study hepatic stellate cell biology and function?

At the cellular level, the activation and transdifferentiation of quiescent hepatic stellate cells (HSC) into myofibroblasts is the key process involved in hepatic fibrogenesis that is associated with an increased and altered deposition of extracellular matrix components in the liver. The temporal sequence of molecular events associated with stellate cell activation turned out to be appropriately mimicked when HSC isolated from normal livers are cultured on uncoated plastic surface. Therefore, cultured primary cells isolated from rodents and human beings are common in vitro models in investigations addressing these issues of hepatic stellate biology and function. However, the limited supply, cost-effective isolation procedure and the ever growing need have resulted in efforts to establish immortalized stellate cell lines having the advantage of virtually unlimited access. They allow rapid screening for disease-associated factors and restrict the necessary number of animal experiments. From the first description of an immortal HSC line in 1986, a huge number of studies were conducted with these established cell lines. However, differences in morphology, growth characteristics and anomalies of chromosome number and structure make the applications of these models questionable. Here, we summarize the history and cellular characteristics of respective cell lines and discuss the differences of continuous HSC lines and their primary counterparts.