Differential effects of epidermal growth factor (EGF) receptor ligands on receptor binding, downstream signalling pathways and DNA synthesis in hepatocytes.

Hepatocytes are responsive to mitogenic effects of several ligands acting via EGFR. Studying primary cultures of rat hepatocytes, we found that, as compared to EGF, HB-EGF had a markedly higher affinity of the EGFR, while AR and TGFα had lower affinity. HB-EGF was also more potent compared to the other growth factors regarding phosphorylation of EGFR, Shc, ERK1/2 and Akt. All ligands induced phosphorylation of ErbB2, indicating receptor heterodimerization. TGFα, despite having much lower receptor affinity, was about equally potent and efficacious as HB-EGF as a stimulator of DNA synthesis. In contrast, EGF had relatively high affinity but markedly lower efficacy in stimulation of DNA synthesis. The results suggest that amplifying and/or inhibitory mechanisms may modulate the mitogenic responses downstream of the initial signalling steps, and that this may affect the effects of the EGFR ligands differentially.

Metabolic switching of human myotubes is improved by n-3 fatty acids.

The aim of the present study was to examine whether pretreatment with different fatty acids, as well as the liver X receptor (LXR) agonist T0901317, could modify metabolic switching of human myotubes. The n-3 FA eicosapentaenoic acid (EPA) increased suppressibility, the ability of glucose to suppress FA oxidation. Substrate-regulated flexibility, the ability to increase FA oxidation when changing from a high glucose, low fatty acid condition ("fed") to a high fatty acid, low glucose ("fasted") condition, was increased by EPA and other n-3 FAs. Adaptability, the capacity to increase FA oxidation with increasing FA availability, was enhanced after pretreatment with EPA, linoleic acid (LA), and palmitic acid (PA). T0901317 counteracted the effect of EPA on suppressibility and adaptability, but it did not affect these parameters alone. EPA per se accumulated less, however, EPA, LA, oleic acid, and T0901317 treatment increased the number of lipid droplets (LD) in myotubes. LD volume and intensity, as well as mitochondrial mass, were independent of FA pretreatment. Microarray analysis showed that EPA regulated more genes than the other FAs and that specific pathways involved in carbohydrate metabolism were induced only by EPA. The present study suggests a favorable effect of n-3 FAs on skeletal muscle metabolic switching and glucose utilization.

LXR{beta} is the dominant LXR subtype in skeletal muscle regulating lipogenesis and cholesterol efflux.

Liver X receptors (LXRs) are important regulators of cholesterol, lipid, and glucose metabolism and have been extensively studied in liver, macrophages, and adipose tissue. However, their role in skeletal muscle is poorly studied and the functional role of each of the LXRalpha and LXRbeta subtypes in skeletal muscle is at present unknown. To study the importance of each of the receptor subtypes, myotube cultures derived from wild-type (WT) and LXRalpha and LXRbeta knockout (KO) mice were established. The present study showed that treatment with the LXR agonist T0901317 increased lipogenesis and apoA1-dependent cholesterol efflux in LXRalpha KO and WT myotubes but not in LXRbeta KO cells. The functional studies were confirmed by T0901317-induced increase in mRNA levels of LXR target genes involved in lipid and cholesterol metabolism in myotubes established from WT and LXRalpha KO mice, whereas only minor changes were observed for these genes in myotubes from LXRbeta KO mice. Gene expression analysis using microarrays showed that very few genes other than the classical, well-known LXR target genes were regulated by LXR in skeletal muscle. The present study also showed that basal glucose uptake was increased in LXRbeta KO myotubes compared with WT myotubes, suggesting a role for LXRbeta in glucose metabolism in skeletal muscle. In conclusion, LXRbeta seems to be the main LXR subtype regulating lipogenesis and cholesterol efflux in skeletal muscle.

Electrical pulse stimulation of cultured skeletal muscle cells as a model for in vitro exercise - possibilities and limitations.

The beneficial health-related effects of exercise are well recognized, and numerous studies have investigated underlying mechanism using various in vivo and in vitro models. Although electrical pulse stimulation (EPS) for the induction of muscle contraction has been used for quite some time, its application on cultured skeletal muscle cells of animal or human origin as a model of in vitro exercise is a more recent development. In this review, we compare in vivo exercise and in vitro EPS with regard to effects on signalling, expression level and metabolism. We provide a comprehensive overview of different EPS protocols and their applications, discuss technical aspects of this model including critical controls and the importance of a proper maintenance procedure and finally discuss the limitations of the EPS model.

Inhibition of hepatocyte DNA synthesis by transforming growth factor beta 1 and cyclic AMP: effect immediately before the G1/S border.

Previous studies have shown that both transforming growth factor beta (TGF-beta) and cyclic AMP (cAMP) inhibit hepatocyte DNA synthesis. While cAMP (in addition to being stimulatory in G0/early G1) exerts its inhibition on hepatocytes late in G1, the point where TGF-beta inhibits has not been precisely defined. We have now examined further the inhibitory effects of cAMP and TGF-beta 1 on DNA synthesis in primary rat hepatocyte cultures and, in particular, tried to determine where in the prereplicative period the cells are sensitive to these agents. Although a transient exposure to TGF-beta 1 (but not glucagon) during the first hours of the cell culturing led to inhibition of DNA synthesis, the cells were more sensitive at a point late in G1, where they also were inhibited by cAMP. Thus, exposure to TGF-beta 1, glucagon, or the cAMP analogue 8-chlorophenylthio-cAMP at a time when there was a continuous recruitment of cells to S phase strongly decreased the rate of S-phase entry. For both TGF-beta 1 and cAMP the inhibition was established within 1-2 h, the lag time being indistinguishable for the two agents. No evidence was found for a synergism between TGF-beta 1 and cAMP. Treatment with TGF-beta 1 did not detectably alter basal or glucagon-stimulated cAMP concentrations. The results suggest that in hepatocytes there is a process immediately before the G1/S border which is sensitive to both TGF-beta 1 and cAMP and which appears to represent a major point of inhibition.

Fluoride-induced apoptosis in epithelial lung cells involves activation of MAP kinases p38 and possibly JNK.

Exposure to fluorides can induce inflammatory reactions, cell cycle arrest, and apoptosis in different experimental systems. Fluorides are known G-protein activators, but less is known about fluoride effects downstream of G-protein activation. The aim of this study was to elucidate whether the induction of apoptosis by fluorides and inhibition of proliferation is mediated by MAP kinases in primary rat lung, alveolar type 2 cells and the human epithelial lung cell line A549. Sodium fluoride (NaF) induced apoptosis in both cell types but at different concentrations, with the primary cells being more sensitive to NAF: Proliferation of the type 2 cells and A549 cells was inhibited in the presence of NAF: NaF induced a prolonged activation of MAP kinase ERK. NaF also activated p38 and JNK in A549 cells for several hours (maximally 6-fold and 3-fold increase, respectively). Inhibition of ERK with the MEK1,2 inhibitor PD98059 increased apoptosis 2-fold, whereas the inhibitor of p38, SB202190, decreased the level of apoptotic cells by approximately 40%. SB202190 also inhibited apoptosis by almost 40% when ERK activity was reduced in the presence of PD98059. Neither PD98059 nor SB202190 did affect the NaF-induced inhibition of proliferation. These observations indicate that activation of MAP kinases p38 and possibly JNK are involved in NaF-induced apoptosis of epithelial lung cells, whereas ERK activation seems to counteract apoptosis in epithelial lung cells. In contrast, activation of ERK and p38 are not involved in NaF-induced inhibition of cell proliferation.

Mechanisms in fluoride-induced interleukin-8 synthesis in human lung epithelial cells.

Sodium fluoride (NaF) has previously been reported to induce a strong IL-8 response in human epithelial lung cells (A549) via mechanisms that seem to involve the activation of G proteins. In the present study the signal pathways downstream of the G proteins have been examined. NaF induced a weak, but sustained increase in PKC activity. In contrast, the PKC activator TPA induced a relatively strong, but transient effect and augmented the NaF-induced PKC activity. TPA induced a marked IL-8 response compared to NaF. PDB, another PKC activator, was less effective, but augmented the IL-8 response to NaF. Pretreatment with TPA for 20 h, or the PKC inhibitor GF109203X for 1 h, abolished the basal and NaF-induced PKC activities and partially prevented the NaF-induced IL-8 response. Inhibition of the MAP kinase p38 by SB202190 partially reduced the IL-8 response to NaF, whereas a reduction in ERK activity by PD98059 led to an increased response. The NaF-induced IL-8 response was weakly augmented by the PKA stimulator forskolin and the G(i) inhibitor pertussis toxin. The PKA inhibitor H89 seemed to reduce the NaF-induced IL-8 response, but the measured effect was not statistically significant. BAPTA-AM, KN93 and W7, that inhibit Ca(2+)-linked effects, did not affect the IL-8 response. Furthermore, the tyrosine kinase inhibitor genestein, the PI-3 kinase inhibitor wortmannin and phosphatase inhibition were without effects. In conclusion, the data suggest that NaF-induced increase of IL-8 in A549 cells involved PKC- and p38-linked pathways, whereas an ERK-dependent pathway counteracted the response. Tyrosine kinases, Ca(2+)-linked pathways, PI-3 kinase, PKA and phosphatase inhibition seem to play no or minor roles in the fluoride-induced IL-8 response.

Benfotiamine increases glucose oxidation and downregulates NADPH oxidase 4 expression in cultured human myotubes exposed to both normal and high glucose concentrations.

The aim of the present work was to study the effects of benfotiamine (S-benzoylthiamine O-monophosphate) on glucose and lipid metabolism and gene expression in differentiated human skeletal muscle cells (myotubes) incubated for 4 days under normal (5.5 mM glucose) and hyperglycemic (20 mM glucose) conditions. Myotubes established from lean, healthy volunteers were treated with benfotiamine for 4 days. Glucose and lipid metabolism were studied with labeled precursors. Gene expression was measured using real-time polymerase chain reaction (qPCR) and microarray technology. Benfotiamine significantly increased glucose oxidation under normoglycemic (35 and 49% increase at 100 and 200 µM benfotiamine, respectively) as well as hyperglycemic conditions (70% increase at 200 µM benfotiamine). Benfotiamine also increased glucose uptake. In comparison, thiamine (200 µM) increased overall glucose metabolism but did not change glucose oxidation. In contrast to glucose, mitochondrial lipid oxidation and overall lipid metabolism were unchanged by benfotiamine. The expression of NADPH oxidase 4 (NOX4) was significantly downregulated by benfotiamine treatment under both normo- and hyperglycemic conditions. Gene set enrichment analysis (GSEA) showed that befotiamine increased peroxisomal lipid oxidation and organelle (mitochondrial) membrane function. In conclusion, benfotiamine increases mitochondrial glucose oxidation in myotubes and downregulates NOX4 expression. These findings may be of relevance to type 2 diabetes where reversal of reduced glucose oxidation and mitochondrial capacity is a desirable goal.

Metabolic switching of human skeletal muscle cells in vitro.

In this review we will focus on external factors that may modify energy metabolism in human skeletal muscle cells (myotubes) and the ability of the myotubes to switch between lipid and glucose oxidation. We describe the metabolic parameters suppressibility, adaptability and substrate-regulated flexibility, and show the influence of nutrients such as fatty acids and glucose (chronic hyperglycemia), and some pharmacological agents modifying nuclear receptors (PPAR and LXR), on these parameters in human myotubes. Possible cellular mechanisms for changes in these parameters will also be highlighted.

Liver X receptor antagonist reduces lipid formation and increases glucose metabolism in myotubes from lean, obese and type 2 diabetic individuals.

AIMS/HYPOTHESIS: Liver X receptors (LXRs) play important roles in lipid and carbohydrate metabolism. The purpose of the present study was to evaluate effects of the endogenous LXR agonist 22-R-hydroxycholesterol (22-R-HC) and its stereoisomer 22-S-hydroxycholesterol (22-S-HC), in comparison with the synthetic agonist T0901317 on lipid and glucose metabolism in human skeletal muscle cells (myotubes). METHODS: Myotubes established from lean and obese control volunteers and from obese type 2 diabetic volunteers were treated with LXR ligands for 4 days. Lipid and glucose metabolisms were studied with labelled precursors, and gene expression was analysed using real-time PCR. RESULTS: Treatment with T0901317 increased lipogenesis (de novo lipid synthesis) and lipid accumulation in myotubes, this increase being more pronounced in myotubes from type 2 diabetic volunteers than from lean volunteers. Furthermore, 22-S-HC efficiently counteracted the T0901317-induced enhancement of lipid formation. Moreover, synthesis of diacylglycerol, cholesteryl ester and free cholesterol from acetate was reduced below baseline by 22-S-HC, whereas glucose uptake and oxidation were increased. Both 22-S-HC and 22-R-HC, in contrast to T0901317, decreased the expression of genes involved in cholesterol synthesis, whereas only 22-R-HC, like T0901317, increased the expression of the gene encoding the reverse cholesterol transporter ATP-binding cassette subfamily A1 (ABCA1). CONCLUSIONS/INTERPRETATION: T0901317-induced lipogenesis and lipid formation was more pronounced in myotubes from type 2 diabetic patients than from lean individuals. 22-S-HC counteracted these effects and reduced de novo lipogenesis below baseline, while glucose uptake and oxidation were increased.

Leukaemia inhibitory factor stimulates glucose transport in isolated cardiomyocytes and induces insulin resistance after chronic exposure.

AIMS/HYPOTHESIS: Hypertrophic and failing hearts have increased utilisation of glucose, but also develop insulin resistance and reduced ability to produce ATP. Increased levels of the IL-6-related cytokine leukaemia inhibitory factor (LIF) are found in failing hearts, and we have recently shown that LIF reduces ATP production in isolated cardiomyocytes. In the present study we investigated effects of LIF on glucose metabolism, and how LIF-treated cells respond to insulin stimulation. METHODS: Cardiomyocytes were isolated from adult Wistar rats by collagen digestion, maintained in culture for 48 h, and then treated with 1 nmol/l LIF. RESULTS: Acute LIF treatment increased deoxyglucose uptake compared with controls, but no additive effect was observed in cardiomyocytes treated with LIF and insulin. The phosphatidylinositol 3-kinase inhibitor wortmannin did not affect LIF-induced glucose uptake. LIF had no effect on AMP-activated protein kinase phosphorylation. Cardiomyocytes treated with LIF for 48 h did not respond to insulin by increasing deoxyglucose uptake and showed a reduced insulin-mediated uptake of oleic acid and formation of complex lipids compared with control cells. Chronic LIF treatment increased gene expression of the suppressor of cytokine signalling (Socs) 3 and reduced expression of solute carrier family 2, member 4 (Slc2a4, previously known as glucose transporter 4 [Glut4]). In line with these observations, chronic LIF treatment reduced insulin-mediated phosphorylation of both Akt/protein kinase B (PKB) and glycogen synthase kinase (GSK)-3. CONCLUSIONS/INTERPRETATION: Acute LIF treatment increased glucose uptake in isolated cardiomyocytes by a pathway different from that of insulin. Chronic LIF treatment induced insulin resistance, possibly mediated by altered expression of Socs3 and Slc2a4, and impaired insulin-mediated phosphorylation of GSK-3 and Akt/PKB.

Lipid metabolism in human skeletal muscle cells: effects of palmitate and chronic hyperglycaemia.

This review focuses on the effect of exogenous factors known to be of importance for the development of insulin resistance in differentiated human myotubes. Recent data from our laboratory on the effects of fatty acid pre-treatment and chronic glucose oversupply on fatty acid and glucose metabolism, without and with acute insulin are presented, and discussed in the context of other recent publications in the field. Pre-treatment of myotubes with palmitate, chronic hyperglycaemia, and acute high concentrations of insulin changed fatty acid metabolism in favour of accumulation of intracellular lipids. Acute insulin exposure increased (14)C-oleate uptake and levels of free fatty acids (FFA) and triacylglycerol (TAG). Palmitate pre-treatment further increased oleate uptake, both under basal conditions and in the presence of insulin, with a marked increase in the phospholipid (PL) fraction, with a concomitant reduction in oleate oxidation. Chronic hyperglycaemia also promoted increased lipogenesis and elevated levels of cellular lipids. Changes in fatty acid metabolism in human muscle, in particular fatty acid oxidation, are probably crucial for the molecular mechanism behind skeletal muscle insulin resistance and impaired glucose metabolism. Differentiated human skeletal muscle cells may be an ideal system to further explore the mechanisms regulating lipid metabolism.

Transforming growth factor beta 1 increases the phosphoenolpyruvate carboxykinase mRNA level in cultured rat hepatocytes.

Transforming growth factor beta 1 (TGF beta 1) elevated the phosphoenolpyruvate carboxykinase (PEPCK) mRNA abundance in primary cultures of rat hepatocytes. Although this increase was not as large as the rise in PEPCK gene expression induced by the cAMP-elevating agents glucagon or isoproterenol, the effect of TGF beta 1 was several-fold and concentration-dependent, with ED50 at about 2.5 pM, which is in the same concentration range as the previously found growth-inhibitory effect of TGF beta. The data show that the level of mRNA for PEPCK, an enzyme typically expressed in the liver, can be regulated in the same direction by TGF beta 1 and cAMP.

Effects of cAMP on ERK mitogen-activated protein kinase activity in hepatocytes do not parallel the bidirectional regulation of DNA synthesis.

Previous studies have indicated that cAMP has bidirectional effects on epidermal growth factor (EGF)-induced DNA synthesis in cultured hepatocytes, acting to stimulate soon after plating (early G(1)) and to inhibit at later stages (nearer the G(1)/S transition). In this study we examined the role of the extracellular signal-regulated kinase (ERK) subgroup (p42/p44) of the mitogen activated protein (MAP) kinases both at growth-stimulatory and growth-inhibitory conditions. When added at low concentrations early during culturing, glucagon and 8-chlorophenylthio-cAMP (8-CPT-cAMP) did not increase MAP kinase activity, but enhanced the subsequent DNA synthesis. However, when administered at 24 h, glucagon and 8-CPT-cAMP decreased basal and EGF-induced MAP kinase activity and also inhibited EGF-induced DNA synthesis. Thus, although MAP kinase might play a role in the growth-inhibitory effect, it does not seem to be involved in growth-promoting regulation by cAMP in hepatocytes.

Alpha1-adrenergic activation of myocardial Na-K-2Cl cotransport involving mitogen-activated protein kinase.

The translocation mechanisms involved in the alpha1-adrenoceptor-stimulated efflux of the potassium analog 86Rb+ were studied in isolated rat hearts. Phenylephrine (in the presence of a beta-blocker) increased the efflux of 86Rb+ and 42K+, and the Na-K-2Cl (or K-Cl) cotransport inhibitor bumetanide reduced the response by 42 +/- 11%. Furosemide inhibited the response with a lower potency than that of bumetanide. The bumetanide-insensitive efflux was largely sensitive to the K+ channel inhibitor 4-aminopyridine. Inhibitors of the Na+/H+ exchanger or the Na+-K+ pump had no effect on the increased 86Rb+ efflux. The activation of the Na-K-2Cl cotransporter was dependent on the extracellular signal-regulated kinase (ERK) subgroup of the mitogen-activated protein (MAP) kinase family. Phenylephrine stimulation increased ERK activity 3.4-fold. PD-98059, an inhibitor of the ERK cascade, reduced both the increased 86Rb+ efflux and ERK activity. Specific inhibitors of protein kinase C and Ca2+/calmodulin-dependent kinase II had no effect. In conclusion, alpha1-adrenoceptor stimulation increases 86Rb+ efflux from the rat heart via K+ channels and a Na-K-2Cl cotransporter. Activation of the Na-K-2Cl cotransporter is apparently dependent on the MAP kinase pathway.

Stimulation of hepatocyte DNA synthesis by prostaglandin E2 and prostaglandin F2 alpha: additivity with the effect of norepinephrine, and synergism with epidermal growth factor.

Previous data obtained in vivo and in vitro suggest that both prostaglandins (PGs) and catecholamines may have a role in promoting hepatocyte proliferation, and PGE2 and PGF2 alpha have also been implicated as mediators of the mitogenic actions of epidermal growth factor (EGF) (and transforming growth factor alpha [TGF alpha]). We have studied the effects of PGs and norepinephrine on DNA synthesis in serum-free primary cultures of rat hepatocytes, and compared the PG effects with those of norepinephrine. PGE2, PGF2 alpha, PGD2, and the synthetic analog dimethyl-PGE2 markedly enhanced the DNA synthesis. A more quantitative analysis of the effects of PGE2 and PGF2 alpha on the DNA synthesis, in the presence and absence of EGF, indicated that these PGs interacted in an essentially multiplicative manner with the effect of EGF. The effects of PGE2 and PGF2 alpha showed almost complete additivity with the stimulation of DNA synthesis produced by maximally effective concentrations of norepinephrine. The data suggest a) that PGE2 and PGF2 alpha facilitate and synergize with, rather than mediate, the actions of EGF in hepatocytes, and b) that this effect of the PGs occurs by mechanisms that are at least partly distinct from those of norepinephrine.

Activation of p42/p44 mitogen-activated protein kinase by angiotensin II, vasopressin, norepinephrine, and prostaglandin F2alpha in hepatocytes is sustained, and like the effect of epidermal growth factor, mediated through pertussis toxin-sensitive mechanisms.

Several agents that act through G-protein-coupled receptors and also stimulate phosphoinositide-specific phospholipase C (PI-PLC), including angiotensin II, vasopressin, norepinephrine, and prostaglandin (PG) F2alpha, activated the ERK1 (p44mapk) and ERK2 (p42mapk) members of the mitogen-activated protein (MAP) kinase family in primary cultures of rat hepatocytes, measured as phosphorylation of myelin basic protein (MBP) by a partially purified enzyme, immunoblotting, and in-gel assays. All these agonists induced a peak activation (two to threefold increase in MBP-phosphorylation) at 3-5 min, followed by a brief decrease, and then a sustained elevation or a second increase of the MAP kinase activity that lasted for several hours. Although all the above agents also stimulated PI-PLC, implicating a Gq-dependent pathway, the elevations of the concentration of inositol (1,4,5)-trisphosphate did not correlate well with the MAP kinase activity. Furthermore, pretreatment of the cells with pertussis toxin markedly reduced the MAP kinase activation by angiotensin II, vasopressin, norepinephrine, or PGF2alpha. In addition, hepatocytes pretreated with pertussis toxin showed a diminished MAP kinase response to epidermal growth factor (EGF). The results indicate that agonists acting via G-protein-coupled receptors have the ability to induce sustained activation of MAP kinase in hepatocytes, and suggest that Gi-dependent mechanisms are required for full activation of the MAP kinase signal transduction pathway by G-protein-coupled receptors as well as the EGF receptor.

EGF-induced activation of Stat1, Stat3, and Stat5b is unrelated to the stimulation of DNA synthesis in cultured hepatocytes.

Transcription factors of the STAT family have been implicated in regulation of cell proliferation. EGF activates several STAT proteins in liver. We have studied the relationship between STAT activation and the growth-stimulatory effect of EGF in rat hepatocytes, assessing specific DNA-binding activity of STAT proteins in electrophoretic mobility-shift and supershift assays. In freshly isolated hepatocytes, EGF activated Stat1, Stat3, and, particularly, Stat5b. However, the ability of EGF to produce this activation was rapidly attenuated when the cells were cultured, while the activation by IFN-gamma (Stat1) and IL-6 (Stat3) was sustained. Hepatocytes cultured for 24-48 h are highly sensitive to the stimulatory effect of EGF on S phase entry. In these cells EGF did not detectably activate Stat1, Stat3, or Stat5b but markedly stimulated MAP kinase (Erk1/2). Thus, although EGF has the ability to activate several STAT proteins, this did not seem to be part of the mitogenic mechanisms used by the EGF receptor in hepatocytes.

Dexamethasone inversely regulates DNA synthesis and phosphoenolpyruvate carboxykinase mRNA levels in cultured rat hepatocytes: interactions with insulin, glucagon, and transforming growth factor beta 1.

In hepatocytes, glucocorticoids control the expression of several genes and exert significant, but complex, regulation of the proliferation. To shed more light on the growth responses to glucocorticoids in these cells, we treated adult rat hepatocytes in primary culture with dexamethasone, in various combinations with other hormones (insulin, glucagon, transforming growth factor beta 1 (TGF beta 1)), and examined the relationship between the effects on the DNA synthesis and the mRNA level of phosphoenolpyruvate carboxykinase, a gene typically expressed in differentiated hepatocytes. Insulin exhibited the previously observed suppressing effect on the glucocorticoid-induced phosphoenolpyruvate carboxykinase mRNA level, and also reversed growth-inhibitory effects of the glucocorticoid. Dexamethasone and glucagon (via cAMP) acted strongly synergistically both in enhancing the phosphoenolpyruvate carboxykinase expression and inhibiting the growth, the inhibitory effect of glucagon on DNA synthesis being totally dependent on dexamethasone. The effects of dexamethasone plus glucagon on both the phosphoenolpyruvate carboxykinase mRNA abundance and the DNA synthesis were partially counteracted by insulin. Dexamethasone is permissive for a promoting effect of TGF beta 1 on phosphoenolpyruvate carboxykinase expression, and was found to increase the maximal inhibitory effect of (but reduced the sensitivity to) TGF beta 1 on the DNA synthesis. The results indicate that there is an inverse glucocorticoid-induced regulation of the DNA synthesis and the expression of a liver-typical gene.

Persistent versus transient map kinase (ERK) activation in the proliferation of lung epithelial type 2 cells.

Type 2 pneumocytes are progenitor cells of alveolor epithelium and important for reepithelialization following lung injury. This study examined the role of persistent versus transient mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinase; ERK) in type 2 cell proliferation. Three different types of agents; epidermal growth factor (EGF), 12-O-tetradecanoylphorbol-13-acetate (TPA), and fetal bovine serum (FBS) induced different patterns of ERK activation. FBS induced a strong and persistent MAP kinase response, whereas the effect of EGF was transient with a strong activation at 5 minutes and only a slight stimulation at 4 hours. The TPA response was more prolonged than the EGF response, but not by far as strong and persistent as the FBS response. Activation by EGF and TPA and the early response induced by FBS were strongly reduced by the MEK inhibitor PD98059. The sustained FBS-induced ERK activation was inhibited by approximately 50%. The total number of cell, the percentage of cells in S and G2/M phase of the cell cycle and the incorporation of 3H-thymidine into DNA were strongly increased in response to FBS, whereas EGF and TPA were without effect. The proliferation was reduced by approximately 50% after pretreatment with PD98059. The results indicate that a persistent ERK activation of a critical size leads to type 2 cell proliferation, and that the proliferative response may also depend on a MEK-independent ERK activation.