Dagny Sandnes

Response to transforming growth factor α (TGFα) and epidermal growth factor (EGF) in hepatocytes: Lower EGF receptor affinity of TGFα is associated with more sustained activation of p42/p44 mitogen-activated protein kinase and greater efficacy in stimulation of DNA synthesis

The epidermal growth factor (EGF) receptor mediates the effects of both EGF and transforming growth factor α (TGFα). Recent data suggested that EGF acts as a partial agonist/antagonist in hepatocytes, TGFα exerting a larger maximal stimulation of DNA synthesis than EGF. To further study the mechanisms involved in mediating the different effects of EGF and TGFα, we have examined receptor binding of the two growth factors and their action on the p42/p44 mitogen‐activated protein (MAP) kinase activity in hepatocytes. Single‐ligand concentration curves and competition experiments showed that the binding affinity to a common population of surface binding sites was about 20‐fold lower for TGFα than for EGF. MAP kinase activity responded to EGF and TGFα with different kinetics. While the two agents produced almost identical acute (5 min) stimulation (peak about fivefold), TGFα produced a more sustained MAP kinase activity than EGF. The difference between EGF and TGFα was still detectable 24 h after growth factor addition. The results show that in hepatocytes a lower receptor affinity of TGFα, as compared to EGF, is associated with a more sustained activation of the MAP kinase and a greater efficacy in the stimulation of DNA synthesis. This suggests that differential interaction of these two agents with the EGF receptor results in differences in the downstream events elicited at a given level of receptor occupancy. The data also are compatible with a role of a prolonged MAP kinase activity in the mitogenic effects of EGF and TGFα. J. Cell. Physiol. 175:10–18, 1998.

Activation of p42/p44 mitogen-activated protein kinase by angiotensin II, vasopressin, norepinephrine, and prostaglandin F2α 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) F2α, 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 PGF2α. 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. J. Cell. Physiol. 175:348–358, 1998.

Prostaglandin E2 upregulates EGF-stimulated signaling in mitogenic pathways involving Akt and ERK in hepatocytes.

Prostaglandins (PGs) such as PGE2 enhance proliferation in many cells, apparently through several distinct mechanisms, including transactivation of the epidermal growth factor (EGF) receptor (EGFR) as well as EGFR‐independent pathways. In this study we found that in primary cultures of rat hepatocytes PGE2 did not induce phosphorylation of the EGFR, and the EGFR tyrosine kinase blockers gefitinib and AG1478 did not affect PGE2‐stimulated phosphorylation of ERK1/2. In contrast, PGE2 elicited EGFR phosphorylation and EGFR tyrosine kinase inhibitor‐sensitive ERK phosphorylation in MH1C1 hepatoma cells. These findings suggest that PGE2 elicits EGFR transactivation in MH1C1 cells but not in hepatocytes. Treatment of the hepatocytes with PGE2 at 3 h after plating amplified the stimulatory effect on DNA synthesis of EGF administered at 24 h and advanced and augmented the cyclin D1 expression in response to EGF in hepatocytes. The pretreatment of the hepatocytes with PGE2 resulted in an increase in the magnitude of EGF‐stimulated Akt phosphorylation and ERK1/2 phosphorylation and kinase activity, including an extended duration of the responses, particularly of ERK, to EGF in PGE2‐treated cells. Pertussis toxin abolished the ability of PGE2 to enhance the Akt and ERK responses to EGF. The results suggest that in hepatocytes, unlike MH1C1 hepatoma cells, PGE2 does not transactivate the EGFR, but instead acts in synergism with EGF by modulating mitogenic mechanisms downstream of the EGFR. These effects seem to be at least in part Gi protein‐mediated and include upregulation of signaling in the PI3K/Akt and the Ras/ERK pathways. J. Cell. Physiol. 214: 371–380, 2008.

Elevated level of β-adrenergic receptors in hepatocytes from regenerating rat liver: Time study of [125I]iodocyanopindolol binding following partial hepatectomy and its relationship to catecholamine-sensitive adenylate cyclase

Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of beta-adrenergic receptors, measured by binding of [125I]iodocyanopindolol ([125I]CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approximately 1 800 to approximately 10 500 sites per cell). Thereafter, the number of beta-adrenergic receptors decreased gradually. Competition experiments indicated beta 2-type receptors. Parallelism was found between the change in the number of beta 2-adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of alpha 1-adrenergic receptors, determined by binding of [3H]prazosin, was transiently lowered by about 35% at 18-24 h, with no significant change in Kd. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of beta 2-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver.

Mechanisms for the emergence of catecholamine-sensitive adenylate cyclase and β-adrenergic receptors in cultured hepatocytes: Dependence on protein and RNA synthesis and suppression by isoproterenol

Adult male rat hepatocytes, which normally respond poorly to β‐adrenergic agents, acquire such responsiveness during primary monolayer culture. We here show that the rise in catecholamine‐sensitive adenylate cyclase activity in hepatocytes in vitro is closely paralleled by an increase in the ability to bind the β‐adrenoceptor ligand [125I]cyanopindolol. The emergence of β‐adrenergic responsiveness did not require cell attachment or serum. Addition of dexamethasone, insulin, thyroxine or dihydortestosterone to the cultures, singly or in combination, did not prevent the augmented β‐adrenergic responsiveness. The increase in catecholamine‐sensitive adenylate cyclase activity and [125I]cyanopindolol binding could be blocked by cycloheximide or actinomycin D. Exposure of the cultures to isoproterenol at 3‐hourly intervals led to a dose‐dependent suppression of the rise in isoproterenol‐responsive adenylate cyclase and prevented the increase in β‐adrenoceptor binding.Adult male rat hepatocytes, which normally respond poorly to beta-adrenergic agents, acquire such responsiveness during primary monolayer culture. We here show that the rise in catecholamine-sensitive adenylate cyclase activity in hepatocytes in vitro is closely paralleled by an increase in the ability to bind the beta-adrenoceptor ligand [125I]cyanopindolol. The emergence of beta-adrenergic responsiveness did not require cell attachment or serum. Addition of dexamethasone, insulin, thyroxine or dihydrotestosterone to the cultures, singly or in combination, did not prevent the augmented beta-adrenergic responsiveness. The increase in catecholamine-sensitive adenylate cyclase activity and [125I]cyanopindolol binding could be blocked by cycloheximide or actinomycin D. Exposure of the cultures to isoproterenol at 3-hourly intervals led to a dose-dependent suppression of the rise in isoproterenol-responsive adenylate cyclase and prevented the increase in beta-adrenoceptor binding.

Effects of pertussis toxin on extracellular signal‐regulated kinase activation in hepatocytes by hormones and receptor‐independent agents: Evidence suggesting a stimulatory role of Gi proteins at a level distal to receptor coupling

It was previously found that pertussis toxin (PTX) pretreatment inhibits the activation of extracellular signal‐regulated kinases ERK1 (p44mapk) and ERK2 (p42mapk) in hepatocytes in response to either agonists that bind to heptahelical receptors or epidermal growth factor (EGF), suggesting a role of Gi proteins in stimulatory mechanisms for ERK1/2. The present work shows that ERK1/2 is activated in a PTX‐sensitive way not only by vasopressin, angiotensin II, prostaglandin (PG) F2α, α1‐adrenergic stimulation, and EGF but also by agents whose actions bypass receptors and stimulate protein kinase C (PKC) and/or elevate intracellular Ca2+, such as 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA), exogenous phosphatidylcholine‐specific phospholipase C (PC‐PLC, from Bacillus cereus), thapsigargin, and the Ca2+ ionophore A23187. Under the same conditions, PTX did not affect agonist stimulation of phosphoinositide‐specific phospholipase C (PI‐PLC) (IP3 generation), and did not reduce the activation by these agents of phospholipase D (PLD). The results suggest that in hepatocytes a PTX‐sensitive mechanism, presumably involving Gi proteins, exerts a stimulatory effect on ERK at a level distal to receptor coupling, acting either as an integral part of the signaling pathway(s) or by a permissive, synergistic regulation. J. Cell. Physiol. 184:27–36, 2000.

Growth‐promoting effects of Ca2+‐mobilizing agents in hepatocytes: Lack of correlation between the acute activation of phosphoinositide‐specific phospholipase C and the stimulation of DNA synthesis by angiotensin II, vasopressin, norepinephrine, and prostaglandin F2α

Although several hormones that promote hepatocyte proliferation also activate phosphoinositide‐specific phospholipase C (PI‐PLC) and mobilize Ca2+, the role of PI‐PLC in the growth‐stimulating effect of these agents is not clear. We have investigated this issue further, by exposing freshly isolated adult rat hepatocytes to vasopressin, angiotensin II, norepinephrine (in the presence of the β‐adrenoceptor blocker timolol) or PGF2α, and examined both acute responses and the subsequent DNA synthesis when the cells were grown in monolayer culture. All the agonists elevated the level of inositol 1,4,5‐trisphosphate (InsP3) and enhanced the DNA synthesis, amplifying the response to epidermal growth factor (EGF), and this comitogenic effect could be exerted by a single exposure of the cells 24 h prior to the addition of EGF. The acute activation of PI‐PLC, measured as the early rise (peak 15–60 s) in InsP3, was 8–10‐fold with vasopressin or angiotensin II, 3–4‐fold with norepinephrine, and ∼︁2‐fold with PGF2α. For all the agonists, a rise in cytosolic free Ca2+ in 100% of the cells and a maximal increase in glycogen phosphorylase activity were evoked at concentrations that approximately doubled the level of InsP3. However, the growth‐stimulatory effects of these agonists showed a different order of efficacy as compared to the activation of PI‐PLC; in terms of the maximal stimulation of DNA synthesis, the effects were: norepinephrine ≈︂ PGF2α > angiotensin II > vasopressin. Also, norepinephrine, PGF2α, and angiotensin II, but not vasopressin, further enhanced the DNA synthesis when their concentrations were increased above those yielding maximal elevation of InsP3. In experiments where vasopressin and angiotensin II were combined, their effects on the DNA synthesis were additive while the InsP3 responses were not. The results show that the extent of the initial activation of PI‐PLC is not the determinant for the magnitude of the growth effects of Ca2+‐mobilizing hormones in hepatocytes. This suggests either (a) that the proliferative response to these agents is determined by the activity of PI‐PLC at a later time, or its integral over an extended part of the prereplicative period, rather than by the acute activation, or (b) that additional, PI‐PLC‐independent, mechanisms are required.

Ca2+-mediated activation of ERK in hepatocytes by norepinephrine and prostaglandin F2α: role of calmodulin and src kinases

BackgroundPrevious studies have shown that several agents that stimulate heptahelical G-protein coupled receptors activate the extracellular signal regulated kinases ERK1 (p44mapk) and ERK2 (p42mapk) in hepatocytes. The molecular pathways that convey their signals to ERK1/2 are only partially clarified. In the present study we have explored the role of Ca2+ and Ca2+-dependent steps leading to ERK1/2 activation induced by norepinephrine and prostaglandin (PG)F2α.ResultsPretreatment of the cells with the Ca2+ chelators BAPTA-AM or EGTA, as well as the Ca2+ influx inhibitor gadolinium, resulted in a partial decrease of the ERK response. Furthermore, the calmodulin antagonists W-7, trifluoperazine, and J-8 markedly decreased ERK activation. Pretreatment with KN-93, an inhibitor of the multifunctional Ca2+/calmodulin-dependent protein kinase, had no effect on ERK activation. The Src kinase inhibitors PP1 and PP2 partially diminished the ERK responses elicited by both norepinephrine and PGF2α.ConclusionThe present data indicate that Ca2+ is involved in ERK activation induced by hormones acting on G protein-coupled receptors in hepatocytes, and suggest that calmodulin and Src kinases might play a role in these signaling pathways.

Role of protein kinase C and epidermal growth factor receptor signalling in growth stimulation by neurotensin in colon carcinoma cells

BackgroundNeurotensin has been found to promote colon carcinogenesis in rats and mice, and proliferation of human colon carcinoma cell lines, but the mechanisms involved are not clear. We have examined signalling pathways activated by neurotensin in colorectal and pancreatic carcinoma cells.MethodsColon carcinoma cell lines HCT116 and HT29 and pancreatic adenocarcinoma cell line Panc-1 were cultured and stimulated with neurotensin or epidermal growth factor (EGF). DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA. Levels and phosphorylation of proteins in signalling pathways were assessed by Western blotting.ResultsNeurotensin stimulated the phosphorylation of both extracellular signal-regulated kinase (ERK) and Akt in all three cell lines, but apparently did so through different pathways. In Panc-1 cells, neurotensin-induced phosphorylation of ERK, but not Akt, was dependent on protein kinase C (PKC), whereas an inhibitor of the β-isoform of phosphoinositide 3-kinase (PI3K), TGX221, abolished neurotensin-induced Akt phosphorylation in these cells, and there was no evidence of EGF receptor (EGFR) transactivation. In HT29 cells, in contrast, the EGFR tyrosine kinase inhibitor gefitinib blocked neurotensin-stimulated phosphorylation of both ERK and Akt, indicating transactivation of EGFR, independently of PKC. In HCT116 cells, neurotensin induced both a PKC-dependent phosphorylation of ERK and a metalloproteinase-mediated transactivation of EGFR that was associated with a gefitinib-sensitive phosphorylation of the downstream adaptor protein Shc. The activation of Akt was also inhibited by gefitinib, but only partly, suggesting a mechanism in addition to EGFR transactivation. Inhibition of PKC blocked neurotensin-induced DNA synthesis in HCT116 cells.ConclusionsWhile acting predominantly through PKC in Panc-1 cells and via EGFR transactivation in HT29 cells, neurotensin used both these pathways in HCT116 cells. In these cells, neurotensin-induced activation of ERK and stimulation of DNA synthesis was PKC-dependent, whereas activation of the PI3K/Akt pathway was mediated by stimulation of metalloproteinases and subsequent transactivation of the EGFR. Thus, the data show that the signalling mechanisms mediating the effects of neurotensin involve multiple pathways and are cell-dependent.

Mechanisms involved in PGE2-induced transactivation of the epidermal growth factor receptor in MH1C1 hepatocarcinoma cells

BackgroundIt is important to understand the mechanisms by which the cells integrate signals from different receptors. Several lines of evidence implicate epidermal growth factor (EGF) receptor (EGFR) in the pathophysiology of hepatocarcinomas. Data also suggest a role of prostaglandins in some of these tumours, through their receptors of the G protein-coupled receptor (GPCR) family. In this study we have investigated mechanisms of interaction between signalling from prostaglandin receptors and EGFR in hepatocarcinoma cells.MethodsThe rat hepatocarcinoma cell line MH1C1 and normal rat hepatocytes in primary culture were stimulated with EGF or prostaglandin E2 (PGE2) and in some experiments also PGF2α. DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA, phosphorylation of proteins in signalling pathways was assessed by Western blotting, mRNA expression of prostaglandin receptors was determined using qRT-PCR, accumulation of inositol phosphates was measured by incorporation of radiolabelled inositol, and cAMP was determined by radioimmunoassay.ResultsIn the MH1C1 hepatocarcinoma cells, stimulation with PGE2 or PGF2α caused phosphorylation of the EGFR, Akt, and ERK, which could be blocked by the EGFR tyrosine kinase inhibitor gefitinib. This did not occur in primary hepatocytes. qRT-PCR revealed expression of EP1, EP4, and FP receptor mRNA in MH1C1 cells. PGE2 stimulated accumulation of inositol phosphates but not cAMP in these cells, suggesting signalling via PLCβ. While pretreatment with EP1 and EP4 receptor antagonists did not inhibit the effect of PGE2, pretreatment with an FP receptor antagonist blocked the phosphorylation of EGFR, Akt and ERK. Further studies suggested that the PGE2-induced signal was mediated via Ca2+ release and not PKC activation, and that it proceeded through Src and shedding of membrane-bound EGFR ligand precursors by proteinases of the ADAM family.ConclusionThe results indicate that in MH1C1 cells, unlike normal hepatocytes, PGE2 activates the MEK/ERK and PI3K/Akt pathways by transactivation of the EGFR, thus diversifying the GPCR-mediated signal. The data also suggest that the underlying mechanisms in these cells involve FP receptors, PLCβ, Ca2+, Src, and proteinase-mediated release of membrane-associated EGFR ligand(s).