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

Background It 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. Methods The 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. Results In 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. Conclusion The 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)

The Cardiac Ventricular 5-HT4 Receptor Is Functional in Late Foetal Development and Is Reactivated in Heart Failure

A positive inotropic responsiveness to serotonin, mediated by 5-HT4 and 5-HT2A receptors, appears in the ventricle of rats with post-infarction congestive heart failure (HF) and pressure overload-induced hypertrophy. A hallmark of HF is a transition towards a foetal genotype which correlates with loss of cardiac functions. Thus, we wanted to investigate whether the foetal and neonatal cardiac ventricle displays serotonin responsiveness. Wistar rat hearts were collected day 3 and 1 before expected birth (days -3 and -1), as well as day 1, 3, 5 and 113 (age matched with Sham and HF) after birth. Hearts from post-infarction HF and sham-operated animals (Sham) were also collected. Heart tissue was examined for mRNA expression of 5-HT4, 5-HT2A and 5-HT2B serotonin receptors, 5-HT transporter, atrial natriuretic peptide (ANP) and myosin heavy chain (MHC)-α and MHC-β (real-time quantitative RT-PCR) as well as 5-HT-receptor-mediated increase in contractile function ex vivo (electrical field stimulation of ventricular strips from foetal and neonatal rats and left ventricular papillary muscle from adult rats in organ bath). Both 5-HT4 mRNA expression and functional responses were highest at day -3 and decreased gradually to day 5, with a further decrease to adult levels. In HF, receptor mRNA levels and functional responses reappeared, but to lower levels than in the foetal ventricle. The 5-HT2A and 5-HT2B receptor mRNA levels increased to a maximum immediately after birth, but of these, only the 5-HT2A receptor mediated a positive inotropic response. We suggest that the 5-HT4 receptor is a representative of a foetal cardiac gene program, functional in late foetal development and reactivated in heart failure

Animal characteristics of Sham and HF rats.

<p>Animal characteristics are given as mean values ± SEM. LVEDP, left ventricular end diastolic pressure; LVSP, left ventricular systolic pressure; LVDd, left ventricular diameter diastole; LVFS, left ventricular fractional shorting; LAD, left atrial diameter;</p>*<p>HF vs. Sham p<0.05;</p>***<p>HF vs. Sham p<0.001.</p

Correlation between the MHC-β/MHC-α mRNA expression ratio and the 5-HT₄ mRNA expression level in ventricles from foetal and neonatal hearts and left ventricular papillary muscles from 113-day-old, Sham and HF rats.

<p>The ratio of MHC-β to MHC-α mRNA levels and 5-HT₄ mRNA level showed a proportional relationship with a slope which significantly deviated from zero, p<0.0001 (linear regression analysis).</p

Inotropic responses to serotonin and to isoproterenol in ventricles from foetal and neonatal hearts and left ventricular papillary muscles from 113-day-old, Sham and HF rats.

<p>Positive inotropic response to serotonin (10 µM) in ventricles from foetal and neonatal rats and papillary muscles from normal adult (day 113), Sham and HF rats driven at 1 Hz in the presence of prazosine (0.1 µM), timolol (1 µM) and atropine (1 µM); ketanserin (0.1 µM; A) or GR113808 (1 µM; B) and inotropic response to subsequent addition of isoproterenol (100 µM; C). The contractile response to serotonin or isoproterenol was measured after stabilisation at its maximum within 2–7 minutes as previously demonstrated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045489#pone.0045489-Qvigstad1" target="_blank">[13]</a>. Panels show maximum inotropic response in % above basal. *<i>vs</i>. group indicated p<0.05; #HF <i>vs</i>. Sham p<0.05.</p

Expression profiles of mRNA markers of cardiac phenotypes.

<p>Messenger RNA expression in cardiac ventricles from foetal (day 3 and 1 before expected birth; days -3 and -1), neonatal (day 1, 3 and 5 after birth; days 1, 3 and 5), adult (day 113), Sham and HF rats. (A) Expression of ANP mRNA increases at birth and in HF; (B) MHC-α mRNA expression increases at birth and during transition from neonatal to adult and decreases in HF; (C) MHC-β mRNA expression demonstrates a transitional increase at birth and an increase in HF; (D) The ratio of MHC-β over MHC-α mRNA expression confirms transition in phenotype from foetal to adult and from Sham to HF. The results are normalised to the geometric mean of Arbp, Tbp, Rpl4 and Rpl32 and presented relative to day 113 (assigned value 1) for foetal and neonatal, and to Sham (assigned value 1) for HF. *<i>vs</i>. day 113 p<0.05; **<i>vs</i>. day 113 p<0.01; ***<i>vs</i>. day 113 p<0.001; ##HF <i>vs</i>. Sham p<0.01.</p

Expression of 5-HT_4(b), 5-HT_2A, 5-HT_2B and 5-HTT mRNA in left ventricular myocardium.

<p>Messenger RNA expression in cardiac ventricles from foetal (day 3 and 1 before expected birth; days -3 and -1), neonatal (day 1, 3 and 5 after birth; days 1, 3 and 5), adult (day 113), Sham and HF rats. (A) 5-HT_4(b) mRNA levels decreased with foetal and neonatal development and the level in adult myocardium was one tenth of that at day -3 but reappeared in HF; (B) 5-HT_2A mRNA expression increased at birth (day 1), decreased during neonatal development and was unaltered in HF; (C) 5-HT_2B mRNA expression was transiently increased at birth (day 1) compared to adult (day 113) and not changed in HF; (D) 5-HTT mRNA expression was lower in foetal hearts compared to neonatal, adult, Sham and HF which all showed a similar expression level. The results are normalised to the geometric mean of Arbp, Tbp, Rpl4 and Rpl32 and presented relative to day 113 (assigned value 1) for foetal and neonatal or to Sham (assigned value 1) for HF. *<i>vs</i>. day 113 p<0.05; **<i>vs</i>. day 113 p<0.01; ***<i>vs</i>. day 113 p<0.001; ##HF <i>vs</i>. Sham p<0.01.</p