Diversity of protists and bacteria determines predation performance and stability

Predation influences prey diversity and productivity while it effectuates the flux and reallocation of organic nutrients into biomass at higher trophic levels. However, it is unknown how bacterivorous protists are influenced by the diversity of their bacterial prey. Using 456 microcosms, in which different bacterial mixtures with equal initial cell numbers were exposed to single or multiple predators (Tetrahymena sp., Poterioochromonas sp. and Acanthamoeba sp.), we showed that increasing prey richness enhanced production of single predators. The extent of the response depended, however, on predator identity. Bacterial prey richness had a stabilizing effect on predator performance in that it reduced variability in predator production. Further, prey richness tended to enhance predator evenness in the predation experiment including all three protists predators (multiple predation experiment). However, we also observed a negative relationship between prey richness and predator production in multiple predation experiments. Mathematical analysis of potential ecological mechanisms of positive predator diversity—functioning relationships revealed predator complementarity as a factor responsible for both enhanced predator production and prey reduction. We suggest that the diversity at both trophic levels interactively determines protistan performance and might have implications in microbial ecosystem processes and services.     

Feeding In Planktonic Protozoans: Evidence For Non-Random Acquisition of Prey

ABSTRACT The literature on discriminant feeding by planktonic protozoans using geometric and nongeometric criteria is reviewed with emphasis on recent studies that indicate phagotrophic protists can use information other than particle size or shape to sort among potential prey. Sufficient data are available for ciliates, aplastidic microflagellates, and phagotrophic dinoflagellates. Numerous representative taxa of all three groups have chemosensory capabilities, either to specific chemicals or to prey exudates, that modify their motility patterns resulting in aggregation or dispersal. Representatives of all three groups also have specific prey preferences. These considerations imply, but do not prove, selectivity in feeding through use of chemical cues. Although prey geometry is clearly a first-order determinant of ingestion through passive mechanical selection, recent studies illustrate that planktonic ciliates and flagellates can use other criteria to discriminate among prey. the evidence clearly implicates use of chemical cues, most likely perceived through contact chemoreception. Filter feeders as well as raptors have such abilities indicating that feeding mechanisms per se do not imply limitations on feeding behavior. Evidence of considerable flexibility and complexity in chemoperceptive feeding suggests that we have only glimpsed the more detailed features of feeding behavior in aquatic protozoans.     

Distinctive regulation and function of PI 3K/Akt and MAPKs in doxorubicin-induced apoptosis of human lung adenocarcinoma cells

Regulation and function of PI 3K/Akt and mitogen-activated protein kinases (MAPKs) in doxorubicin-induced cell death were investigated in human lung adenocarcinoma cells. Doxorubicin induced dose-dependent apoptosis of human lung adenocarcinoma NCI-H522 cells. Prior to cell death, both Akt and the MAPK family members (MAPKs: ERK1/2, JNK, and p38) were activated in response to the drug treatment. The kinetics of the inductions for Akt and MAPKs are, however, distinct. The activation of Akt was rapid and transient, activated within 30 min of drug addition, then declined after 3 h, whereas the activations of three MAPKs occurred later, 4 h after addition of the drug and sustained until cell death occurred. Inhibition of PI 3K/Akt activation had no effect on MAPKs' activation, suggesting that the two pathways are independently activated in response to the drug treatment. Inhibition of PI 3K/Akt and p38 accelerated and enhanced doxorubicin-induced cell death. On the contrary, inhibition of ERK1/2 or JNK had no apparent effect on the cell death. Taken together, these results suggest that PI 3K/Akt and MAPKs signaling pathways are all activated, but with distinct mechanisms, in response to doxorubicin treatment. Activation of PI 3K/Akt and p38 modulates apoptotic signal pathways and inhibits doxorubicin-induced cell death. These responses of tumor cells to cancer drug treatment may contribute to their drug resistance. Understanding of the mechanism and function of the responses will be beneficial for the development of novel therapeutic approaches for improvement of drug efficacy and circumvention of drug resistance.     

Prey location, recognition and ingestion by the phagotrophic marine dinoflagellate Oxyrrhis marina

The initial ingestion rates of Isochrysis galbana and Dunaliella primolecta by Oxyrrhis populations precultured separately on these phytoplanktonic prey were quantified and related to the chemosensory responses elicited in Oxyrrhis by the filtrate from live and heat killed prey cells. Despite evidence to suggest that Oxyrrhis shows specific distaste towards Isochrysis (but not Dunaliella) such that consumption of N-deplete Isochrysis halted in grazing experiments, positive chemotaxis was observed towards the cell-free filtrate from both species. These results suggest that while tactile cues encountered upon contact with Isochrysis and Dunaliella may enable Oxyrrhis to recognise differences between the two species, the chemosensory responses observed towards dissolved chemical cues derived from potential prey items are non-specific. That chemosensory and ingestion behaviours do not appear to be tightly coupled raises important questions concerning the ecological implications of chemotaxis in Oxyrrhis. Chemotaxis may enhance the overall efficiency of prey detection; however, when confronted with a variety of chemical stimuli (i.e. from a mixed-prey assemblage) Oxyrrhis may be unable to discern the difference between cues that originate from high quality, poor quality (or even toxic) prey items. The positive chemosensory responses observed towards a range of synthetic amino acid, amino sugar and ammonium solutions suggest that chemotaxis could facilitate the detection of solute gradients in prey deplete environments for direct exploitation via osmotrophy. Furthermore, the positive chemotaxis elicited by regenerated ammonium and compounds derived from heat killed conspecifics suggests that Oxyrrhis may release chemical cues which induce cannibalistic behaviour as a ‘life boat mechanism’ when no other suitable (non-self) prey items are available. Further work is required to explore the nature of the chemosensory apparatus and signal transduction pathways that mediate responses to dissolved chemical stimuli in Oxyrrhis and to investigate other sensory mechanisms that enable cells to recognise and differentiate between potential prey items.     

Hepatocyte growth factor induced human trophoblast motility involves phosphatidylinositol-3-kinase,mitogen-activated protein kinase,and inducible nitric oxide synthase

Hepatocyte growth factor (HGF) increases human trophoblast motility and invasion, an effect which is abrogated when inducible nitric oxide synthase (iNOS) is inhibited. In this study we have investigated the pathways involved in the regulation of trophoblast motility. Both basal and HGF-stimulated motility of the extravillous trophoblast cell line, SGHPL-4, were inhibited in a dose-dependent manner by the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, LY294002. HGF-stimulated iNOS expression was also inhibited by LY294002 and direct activation of PI3-kinase, using the peptide 740Y-P, led to an increase in iNOS expression and cell motility. Pretreatment with rapamycin, which acts at a point distal to PI3-kinase activation, also inhibited basal and HGF-stimulated motility. Inhibition of the p42/p44 mitogen activated protein kinase (MAPK) pathway but not the p38 MAPK pathway had significant inhibitory effects on HGF-stimulated but not basal trophoblast motility. Inhibition of p42/p44 MAPK also inhibited HGF-induced iNOS expression. This data demonstrate that the PI3-kinase signaling pathway is involved in basal trophoblast motility and that both MAPK and PI3-kinase signaling pathways are important in HGF-stimulated motility and iNOS expression.     

Photoresponse in the heterotrophic marine dinoflagellate Oxyrrhis marina

Expressed rhodopsins were detected by proteomic analysis in an investigation of potential signal receptors in the cell membrane of the marine heterotrophic dinoflagellate Oxyrrhis marina (CCMP604). We inferred these to be sensory rhodopsins, a type of G-protein-coupled receptor trans-membrane signaling molecule. Because phototactic behavior based on sensory rhodopsins has been reported in other protists, we investigated the photosensory response of O. marina. This dinoflagellate exhibited strongest positive phototaxis at low levels (2-3 μE/m(2)/s) of white light when the cells were previously light adapted and well fed. Positive phototaxis was also found for blue (450 nm), green (525 nm), and red (680 nm) wavelengths. In a further test, O. marina showed significantly greater phototaxis toward concentrated algal food illuminated by blue light to stimulate red chlorophyll-a autofluorescence in the prey, compared with using bleached algae as prey. Concentration of a cytoplasmic downstream messenger molecule, cyclic adenosine monophosphate, a component of the signaling pathway of G-protein-coupled receptor molecules, rapidly increased in O. marina cells after exposure to white light. In addition, treatment with hydroxylamine, a rhodopsin signaling inhibitor, significantly decreased their phototactic response. Our results demonstrate that a heterotrophic marine dinoflagellate can orient to light based on rhodopsins present in the outer cell membrane and may be able to use photosensory response to detect algal prey based on chlorophyll autofluorescence.     

Atorvastatin Prevents Glutamate Uptake Reduction Induced by Quinolinic Acid Via MAPKs Signaling

Statins have been shown to promote neuroprotection in a wide range of neurological disorders. However, the mechanisms involved in such effects of statins are not fully understood. Quinolinic acid (QA) is a neurotoxin that induces seizures when infused in vivo and promotes glutamatergic excitotoxicity in the central nervous system. The aim of this study was to evaluate the putative glutamatergic mechanisms and the intracellular signaling pathways involved in the atorvastatin neuroprotective effects against QA toxicity. Atorvastatin (10 mg/kg) treatment for 7 days prevented the QA-induced decrease in glutamate uptake, but had no effect on increased glutamate release induced by QA. Moreover, atorvastatin treatment increased the phosphorylation of ERK1 and prevented the decrease in Akt phosphorylation induced by QA. Neither atorvastatin treatment nor QA infusion altered glutamine synthetase activity or the levels of phosphorylation of p38^MAPK or JNK1/2 during the evaluation. Inhibition of MEK/ERK signaling pathway, but not PI3K/Akt signaling, abolished the neuroprotective effect of atorvastatin against QA-induced decrease in glutamate uptake. Our data suggest that atorvastatin protective effects against QA toxicity are related to modulation of glutamate transporters via MAPK/ERK signaling pathway.     

The cytomegalovirus homolog of interleukin-10 requires phosphatidylinositol 3-kinase activity for inhibition of cytokine synthesis in monocytes

Human cytomegalovirus (CMV) has evolved numerous strategies for evading host immune defenses, including piracy of cellular cytokines. A viral homolog of interleukin-10, designated cmvIL-10, binds to the cellular IL-10 receptor and effects potent immune suppression. The signaling pathways employed by cmvIL-10 were investigated, and the classic IL-10R/JAK1/Stat3 pathway was found to be activated in monocytes. However, inhibition of JAK1 had little effect on cmvIL-10-mediated suppression of tumor necrosis factor alpha (TNF-alpha) production. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway had a more significant impact on TNF-alpha levels but did not completely relieve the immune suppression, demonstrating that cmvIL-10 stimulates multiple signaling pathways to modulate cell function.     

Intracellular events mediating insulin-like growth factor I-induced oligodendrocyte development: modulation by cyclic AMP

Insulin-like growth factor I (IGF-I) is a potent inducer of oligodendrocyte development and myelination. Although IGF-I intracellular signaling has been well described in several cell types, intracellular mechanisms for IGF-I-induced oligodendrocyte development have not been defined. By using specific inhibitors of intracellular signaling pathways, we report here that the MAPK and phosphatidylinositol 3-kinase signaling pathways are required for the full effect of IGF-I on oligodendrocyte development in primary mixed rat cerebrocortical cell cultures. The MAPK activation, but not the phosphatidylinositol 3-kinase activation, leads to phosphorylation of the cAMP response element-binding protein, which is necessary for IGF-I to induce oligodendrocyte development. cAMP, although it does not show any effect on oligodendrocyte development, has an inhibitory effect on IGF-I-induced oligodendrocyte development that is mediated by the cAMP-dependent protein kinase. Furthermore, cAMP also has an inhibitory effect on IGF-I-dependent MAPK activation. This is a cAMP-dependent protein kinase-independent effect and probably contributes to the cAMP action on IGF-I-induced oligodendrocyte development.     

What makes the mitochondria a killer? Can we condition them to be less destructive?

Cardioprotection, such as preconditioning and postconditioning, has been shown to result in a significant reduction in cell death. Many of the signaling pathways activated by cardioprotection have been elucidated, but there is still a lack of understanding of the mechanisms by which these signaling pathways reduce cell death. Mitochondria have been reported to be an important player in many types of apoptotic and necrotic cell death. If mitochondria play an important role in cell death, then it seems reasonable to consider that cardioprotective mechanisms might act, at least in part, by opposing mitochondrial cell death pathways. One of the major mechanisms of cell death in ischemia-reperfusion is suggested to be the opening of a large conductance pore in the inner mitochondrial membrane, known as the mitochondrial permeability transition pore. Inhibition of this mitochondrial pore appears to be one of the major mechanisms by which cardioprotection reduces cell death. Cardioprotection activates a number of signaling pathways that reduce the level of triggers (reactive oxygen species and calcium) or enhances inhibitors of the mitochondrial permeability transition pore at the start of reperfusion. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

Nck and Crk mediate distinct VEGF-induced signaling pathways that serve overlapping functions in focal adhesion turnover and integrin activation

We have asked whether the Nck and Crk adaptor proteins play important roles in the vascular endothelial growth factor (VEGF)-induced signaling pathways that lead to an enhancement in cell migration. The introduction into human umbilical vein endothelial cells of a dominant-negative inhibitor for either Nck or Crk blocked the recruitment of both endogenous proteins to the KDR VEGF receptor subtype indicating that both proteins are recruited to the same docking site. The Nck and Crk dominant-negatives led to the formation of abnormally large focal adhesion, blocked VEGF-induced integrin activation, and blocked VEGF-induced actin dynamics. The dominant-negatives had no effects on these properties in cells expressing constitutively active Rac1 or RhoA. Since a DN to either Nck or Crk blocks the cellular responses mediated by both proteins, we performed experiments directed at clarifying signaling pathways specifically mediated by each protein. Inhibition of the interaction between Nck with its downstream effector PAK led to abnormally large focal adhesions, but had no effect on integrin activation or cell adhesiveness. Evidence is presented that Crk complexes with C3G in control cells, and VEGF treatment leads to the recruitment of the complex to the cell surface. Inhibition of the C3G downstream effector Rap1 leads to enlarged focal adhesions and blocks VEGF-induced integrin activation. We conclude that Nck and Crk mediate distinct VEGF-induced signaling pathways that serve overlapping functions in cell migration.     

Signaling crosstalk of FHIT,CHK2 and p38 in etoposide induced growth inhibition in MCF-7 cells

FHIT (Fragile Histidin Triad) is a tumor suppressor gene involved in regulating cell death during DNA damage conditions. The exact mechanism of DNA damage-induced FHIT signaling is not well understood. It is known that p38 kinase and CHK2 kinase are being activated during stress-induced conditions and DNA damage, resulting in cell death. Since both CHK2 and FHIT are being influenced by DNA damage, we have evaluated the interplay of p38, CHK2 and FHIT in response to etoposide-induced cell death. DNA damage was induced by etoposide in MCF-7 cells and viability was examined using MTT. FHIT expression was blocked using siRNA. Protein expression was measured using western blotting. Our results indicated that etoposide induced cytotoxicity in MCF-7. Block of FHIT expression, completely reversed etoposide cytotoxicity. Besides, etoposide induced p38 and CHK2 phosphorylation and reduced FHIT expression in a time-dependent manner. The time-course study indicated that CHK2 had been phosphorylated prior to p38 activation. Knockdown of FHIT expression reduced CHK2 phosphorylation but had no significant effect on p38 activation. Inhibition of p38 kinase and CHK2 prevented etoposide induced alteration in FHIT expression. Furthermore, p38 inhibitors augmented etoposide-induced CHK2 phosphorylation. These results indicate that etoposide lowers FHIT expression and induces cell death via p38 and CHK2 phosphorylation. These results demonstrate a time dependent complex crosstalk of FHIT, p38 and CHK2 pathways in response to etoposide. Moreover, our findings suggest signaling interaction for these pathways which can be targeted for manipulating cell proliferation.     

Cooccurrence of prey species alters the impact of predators on prey performance through multiple mechanisms

When prey are differentially affected by intra and interspecific competition, the cooccurrence of multiple prey species alters the per capita availability of food for a particular prey species which could alter how prey respond to the threat of predation, and hence the overall‐effect of predators. We conducted an experiment to examine the extent to which the nonconsumptive and overall effect of predatory water bugs on snail and tadpole traits (performance and morphology) depended on whether tadpoles and snails cooccurred. Tadpoles and snails differed in their relative susceptibility to intraspecific and interspecific competition, and predators affected both prey species via consumptive and nonconsumptive mechanisms. Furthermore, the overall effect of predators often depended on whether another prey species was present. The reasoning for why the overall effect of predators depended on whether prey species cooccurred, however, differed for each of the response variables. Predators affected snail body growth via nonconsumptive mechanisms, but the change in the overall effect of predators on snail body growth was attributable to how snails responded to competition in the absence of predators, rather than a change in how snails responded to the threat of predation. Predators did not affect tadpole body growth via nonconsumptive mechanisms, but the greater vulnerability of competitively superior prey (snails) to predators increased the strength of consumptive mechanisms (and hence the overall effect) through which predators affected tadpole growth. Predators affected tadpole morphology via nonconsumptive mechanisms, but the greater propensity for predators to kill competitively superior prey (snails) enhanced the ability of tadpoles to alter their morphology in response to the threat of predation by creating an environment where tadpoles had a higher per capita supply of food available to invest in the development of morphological defenses. Our work indicates that the mechanisms through which predators affect prey depends on the other members of the community.     

TLR3- and MyD88-Dependent Signaling Differentially Influences the Development of West Nile Virus-Specific B Cell Responses in Mice following Immunization with RepliVAX WN,a Single-Cycle Flavivirus Vaccine Candidate

Recognition of conserved pathogen-associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) results in the activation of innate signaling pathways that drive the innate immune response and ultimately shape the adaptive immune response. RepliVAX WN, a single-cycle flavivirus (SCFV) vaccine candidate derived from West Nile virus (WNV), is intrinsically adjuvanted with multiple PAMPs and induces a vigorous anti-WNV humoral response. However, the innate mechanisms that link pattern recognition and development of vigorous antigen-specific B cell responses are not completely understood. Moreover, the roles of individual PRR signaling pathways in shaping the B cell response to this live attenuated SCFV vaccine have not been established. We examined and compared the role of TLR3- and MyD88-dependent signaling in the development of anti-WNV-specific antibody-secreting cell responses and memory B cell responses induced by RepliVAX WN. We found that MyD88 deficiency significantly diminished B cell responses by impairing B cell activation, development of germinal centers (GC), and the generation of long-lived plasma cells (LLPCs) and memory B cells (MBCs). In contrast, TLR3 deficiency had more effect on maintenance of GCs and development of LLPCs, whereas differentiation of MBCs was unaffected. Our data suggest that both TLR3- and MyD88-dependent signaling are involved in the intrinsic adjuvanting of RepliVAX WN and differentially contribute to the development of vigorous WNV-specific antibody and B cell memory responses following immunization with this novel SCFV vaccine.     

Injury-stimulated Hedgehog signaling promotes regenerative proliferation of Drosophila intestinal stem cells

Many adult tissues are maintained by resident stem cells that elevate their proliferation in response to injury. The regulatory mechanisms underlying regenerative proliferation are still poorly understood. Here we show that injury induces Hedgehog (Hh) signaling in enteroblasts (EBs) to promote intestinal stem cell (ISC) proliferation in Drosophila melanogaster adult midgut. Elevated Hh signaling by patched (ptc) mutations drove ISC proliferation noncell autonomously. Inhibition of Hh signaling in the ISC lineage compromised injury-induced ISC proliferation but had little if any effect on homeostatic proliferation. Hh signaling acted in EBs to regulate the production of Upd2, which activated the JAK–STAT pathway to promote ISC proliferation. Furthermore, we show that Hh signaling is stimulated by DSS through the JNK pathway and that inhibition of Hh signaling in EBs prevented DSS-stimulated ISC proliferation. Hence, our study uncovers a JNK–Hh–JAK–STAT signaling axis in the regulation of regenerative stem cell proliferation.     

The mechanism whereby heat shock induces apoptosis depends on the innate sensitivity of cells to stress

The cellular response to heat shock (HS) is a paradigm for many human diseases collectively known as “protein conformation diseases” in which the accumulation of misfolded proteins induces cell death. Here, we analyzed how cells having a different apoptotic threshold die subsequent to a treatment with HS. Cells with a low apoptotic threshold mainly induced apoptosis through activation of conventional stress kinase signaling pathways. By contrast, cells with a high apoptotic threshold also died by apoptosis but likely after the accumulation of heat-aggregated proteins as revealed by the formation of aggresomes in these cells, which were associated with the generation of atypical nuclear deformations. Inhibition of the proteasome or expression of an aggregation prone protein produced similar nuclear alterations. Furthermore, elevated levels of chaperones markedly suppressed both HS-induced nuclear deformations and apoptosis induced upon protein aggregation whereas they had little effect on stress kinase-mediated apoptosis. We conclude that the relative contribution of stress signaling pathways and the accumulation of protein aggregates to cell death by apoptosis is related to the innate sensitivity of cells to deadly insults.     

Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways

Neurotrophins protect neurons against glutamate excitotoxicity, but the signaling mechanisms have not been fully elucidated. We studied the role of the phosphatidylinositol 3-kinase (PI3-K) and Ras/mitogen-activated protein kinase (MAPK) pathways in the protection of cultured hippocampal neurons from glutamate induced apoptotic cell death, characterized by nuclear condensation and activation of caspase-3-like enzymes. Pre-incubation with the neurotrophin brain-derived neurotrophic factor (BDNF), for 24 h, reduced glutamate-evoked apoptotic morphology and caspase-3-like activity, and transiently increased the activity of the PI3-K and of the Ras/MAPK pathways. Inhibition of the PI3-K and of the Ras/MAPK signaling pathways abrogated the protective effect of BDNF against glutamate-induced neuronal death and similar effects were observed upon inhibition of protein synthesis. Moreover, incubation of hippocampal neurons with BDNF, for 24 h, increased Bcl-2 protein levels. The results indicate that the protective effect of BDNF in hippocampal neurons against glutamate toxicity is mediated by the PI3-K and the Ras/MAPK signaling pathways, and involves a long-term change in protein synthesis.     

Transforming growth factor-beta1 regulation of growth zone chondrocytes is mediated by multiple interacting pathways

Transforming growth factor beta 1 (TGF-beta1) affects growth plate chondrocytes through Smad-mediated mechanisms and has been shown to increase protein kinase C (PKC). This study determined if PKC mediates the physiological response of rat costochondral growth zone (GC) chondrocytes to TGF-beta1; if the physiological response occurs via type II or type III TGF-beta receptors, and, if so, which receptor mediates the increase in PKC; and the signal transduction pathways involved. Treatment of confluent GC cells with TGF-beta1 stimulated [(3)H]thymidine and [(35)S]sulfate incorporation as well as alkaline phosphatase (ALPase) and PKC specific activities. Inhibition of PKC with chelerythrine, staurosporine, or H-7 caused a dose-dependent decrease in these parameters, indicating that PKC signaling was involved. TGF-beta1-dependent PKC and the physiological response of GC cells to TGF-beta1 was reversed by anti-type II TGF-beta receptor antibody and soluble type II TGF-beta receptor, showing that TGF-beta1 mediates these effects through the type II receptor. The increase in [3H]thymidine incorporation and ALPase specific activity were also regulated by protein kinase A (PKA) signaling, since the effects of TGF-beta1 were partially blocked by the PKA inhibitor H-8. The mechanism of TGF-beta1 activation of PKC is through phospholipase A(2) (PLA(2)) and not through phospholipase C (PLC). Arachidonic acid increased PKC in control cultures and was additive with TGF-beta1. Prostanoids are required, as indomethacin blocked the effect of TGF-beta1, and Cox-1, but not Cox-2, is involved. TGF-beta1 stimulates prostaglandin E(2) (PGE(2)) production and exogenous PGE(2) stimulates PKC, but not as much as TGF-beta1, suggesting that PGE(2) is not sufficient for all of the prostaglandin effect. In contrast, TGF-beta1 was not regulated by diacylglycerol; neither dioctanoylglycerol (DOG) nor inhibition of diacylglycerol kinase with R59022 had an effect. G-proteins mediate TGF-beta1 signaling at different levels in the cascade. TGF-beta1-dependent increases in PGE(2) levels and PKC were augmented by the G protein activator GTP gamma S, whereas inhibition of G-protein activity via GDP beta S, pertussis toxin, or cholera toxin blocked stimulation of PKC by TGF-beta1, indicating that both G(i) and G(s) are involved.Inhibition of PKA with H-8 partially blocked TGF-beta1-dependent PKC, suggesting that PKA inhibition on the physiological response was via PKA regulation of PKC signaling. This indicates that multiple interacting signaling pathways are involved: TGF-beta1 stimulates PLA(2) and prostaglandin release via the action of Cox-1 on arachidonic acid. PGE(2) activates the EP2 receptor, leading to G-protein-dependent activation of PKA. PKA signaling results in increased PKC activity and PKC signaling regulates proliferation, differentiation, and matrix synthesis.