Mitochondrial complex I deficiency stratifies idiopathic Parkinson's disease.

Idiopathic Parkinson's disease (iPD) is believed to have a heterogeneous pathophysiology, but molecular disease subtypes have not been identified. Here, we show that iPD can be stratified according to the severity of neuronal respiratory complex I (CI) deficiency, and identify two emerging disease subtypes with distinct molecular and clinical profiles. The CI deficient (CI-PD) subtype accounts for approximately a fourth of all cases, and is characterized by anatomically widespread neuronal CI deficiency, a distinct cell type-specific gene expression profile, increased load of neuronal mtDNA deletions, and a predilection for non-tremor dominant motor phenotypes. In contrast, the non-CI deficient (nCI-PD) subtype exhibits no evidence of mitochondrial impairment outside the dopaminergic substantia nigra and has a predilection for a tremor dominant phenotype. These findings constitute a step towards resolving the biological heterogeneity of iPD with implications for both mechanistic understanding and treatment strategies.

Early Forms of α-Synuclein Pathology Are Associated with Neuronal Complex I Deficiency in the Substantia Nigra of Individuals with Parkinson's Disease.

Idiopathic Parkinson's disease (iPD) is characterized by degeneration of the dopaminergic substantia nigra pars compacta (SNc), typically in the presence of Lewy pathology (LP) and mitochondrial respiratory complex I (CI) deficiency. LP is driven by α-synuclein aggregation, morphologically evolving from early punctate inclusions to Lewy bodies (LBs). The relationship between α-synuclein aggregation and CI deficiency in iPD is poorly understood. While studies in models suggest they are causally linked, observations in human SNc show that LBs preferentially occur in CI intact neurons. Since LBs are end-results of α-synuclein aggregation, we hypothesized that the relationship between LP and CI deficiency may be better reflected in neurons with early-stage α-synuclein pathology. Using quadruple immunofluorescence in SNc tissue from eight iPD subjects, we assessed the relationship between neuronal CI or CIV deficiency and early or late forms of LP. In agreement with previous findings, we did not observe CI-negative neurons with late LP. In contrast, early LP showed a significant predilection for CI-negative neurons (p = 6.3 × 10-5). CIV deficiency was not associated with LP. Our findings indicate that early α-syn aggregation is associated with CI deficiency in iPD, and suggest a double-hit mechanism, where neurons exhibiting both these pathologies are selectively lost.

The TGFß-SMAD3 pathway inhibits IL-1α induced interactions between human pancreatic stellate cells and pancreatic carcinoma cells and restricts cancer cell migration.

BACKGROUND: The most abundant cells in the extensive desmoplastic stroma of pancreatic adenocarcinomas are the pancreatic stellate cells, which interact with the carcinoma cells and strongly influence the progression of the cancer. Tumor stroma interactions induced by IL-1α/IL-1R1 signaling have been shown to be involved in pancreatic cancer cell migration. TGFß and its receptors are overexpressed in pancreatic adenocarcinomas. We aimed at exploring TGFß and IL-1α signaling and cross-talk in the stellate cell cancer cell interactions regulating pancreatic adenocarcinoma cell migration. METHODS: Human pancreatic stellate cells were isolated from surgically resected pancreatic adenocarcinomas and cultured in the presence of TGFß or pancreatic adenocarcinoma cell lines. The effects of TGFß were blocked by inhibitors or amplified by silencing the endogenous inhibitor of SMAD signaling, SMAD7. Pancreatic stellate cell responses to IL-1α or to IL-1α-expressing pancreatic adenocarcinoma cells (BxPC-3) were characterized by their ability to stimulate migration of cancer cells in a 2D migration model. RESULTS: In pancreatic stellate cells, IL-1R1 expression was found to be down-regulated by TGFß and blocking of TGFß signaling re-established the expression. Endogenous inhibition of TGFß signaling by SMAD7 was found to correlate with the levels of IL-1R1, indicating a regulatory role of SMAD7 in IL-1R1 expression. Pancreatic stellate cells cultured in the presence of IL-1α or in co-cultures with BxPC-3 cells enhanced the migration of cancer cells. This effect was blocked after treatment of the pancreatic stellate cells with TGFß. Silencing of stellate cell expression of SMAD7 was found to suppress the levels of IL-1R1 and reduce the stimulatory effects of IL-1α, thus inhibiting the capacity of pancreatic stellate cells to induce cancer cell migration. CONCLUSIONS: TGFß signaling suppressed IL-1α mediated pancreatic stellate cell induced carcinoma cell migration. Depletion of SMAD7 upregulated the effects of TGFß and reduced the expression of IL-1R1, leading to inhibition of IL-1α induced stellate cell enhancement of carcinoma cell migration. SMAD7 might represent a target for inhibition of IL-1α induced tumor stroma interactions.

Profile of MMP and TIMP Expression in Human Pancreatic Stellate Cells: Regulation by IL-1α and TGFß and Implications for Migration of Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma is characterized by a prominent fibroinflammatory stroma with both tumor-promoting and tumor-suppressive functions. The pancreatic stellate cell (PSC) is the major cellular stromal component and the main producer of extracellular matrix proteins, including collagens, which are degraded by metalloproteinases (MMPs). PSCs interact with cancer cells through various factors, including transforming growth factor (TGF)ß and interleukin (IL)-1α. The role of TGFß in the dual nature of tumor stroma, i.e., protumorigenic or tumor suppressive, is not clear. We aimed to investigate the roles of TGFß and IL-1α in the regulation of MMP profiles in PSCs and the subsequent effects on cancer cell migration. Human PSCs isolated from surgically resected specimens were cultured in the presence of pancreatic cancer cell lines, as well as IL-1α or TGFß. MMP production and activities in PSCs were quantified by gene array transcripts, mRNA measurements, fluorescence resonance energy transfer-based activity assay, and zymography. PSC-conditioned media and pancreatic cancer cells were included in a collagen matrix cell migration model. We found that production of IL-1α by pancreatic cancer cells induced alterations in MMP and tissue inhibitors of matrix metalloproteinase (TIMP) profiles and activities in PSCs, upregulated expression and activation of MMP1 and MMP3, and enhanced migration of pancreatic cancer cells in the collagen matrix model. TGFß counteracted the effects of IL-1α on PSCs, reestablished PSC MMP and TIMP profiles and activities, and inhibited migration of cancer cells. This suggests that tumor TGFß has a role as a suppressor of stromal promotion of tumor progression through alterations in PSC MMP profiles with subsequent inhibition of pancreatic cancer cell migration.

Lysophosphatidic acid induces both EGFR-dependent and EGFR-independent effects on DNA synthesis and migration in pancreatic and colorectal carcinoma cells.

Lysophosphatidic acid (LPA) is a small glycerophospholipid ubiquitously present in tissues and plasma. It acts through receptors belonging to the G-protein-coupled receptor (GPCR) family, is involved in several biological processes, and is strongly implicated in different cancers. In this paper, we have investigated the effects of LPA on DNA synthesis and migration in a panel of pancreatic and colon cancer cells, with particular focus on the involvement of the epidermal growth factor (EGF) receptor (EGFR) in LPA-induced signaling. LPA stimulated DNA synthesis and/or migration in all the cell lines included in this study. In five of the six cell lines, LPA induced phosphorylation of the EGFR, and the effects on EGFR and Akt, and in some of the cells also ERK, were sensitive to the EGFR tyrosine kinase inhibitor gefitinib, strongly suggesting LPA-induced EGFR transactivation in these cells. In contrast, in one of the pancreatic carcinoma cell lines (Panc-1), we found no evidence of transactivation of the EGFR. In the pancreatic carcinoma cell lines where transactivation took place (BxPC3, AsPC1, HPAFII), gefitinib reduced LPA-induced DNA synthesis and/or migration. However, we also found evidence of transactivation in the two colon carcinoma cell lines (HT29, HCT116) although gefitinib did not inhibit LPA-induced DNA synthesis or migration in these cells. Taken together, the data indicate that in many gastrointestinal carcinoma cells, LPA uses EGFR transactivation as a mechanism when exerting such effects as stimulation of cell proliferation and migration, but EGFR-independent pathways may be involved instead of, or in concerted action with, the EGFR transactivation.

Gab1 amplifies signaling in response to low-intensity stimulation by HGF.

The receptor tyrosine kinases EGFR and Met induce phosphorylation of the docking protein Gab1, and there is evidence that Gab1 may have a role in the signaling from these receptors. Studying hepatocytes, we previously found that although Gab1 mechanistically interacted in different ways with EGFR and Met, it was involved in mitogenic signaling induced by both EGF and HGF. It has been reported that in EGFR, Gab1 is required particularly at a low dose of EGF. Whether this also applies to HGF/Met signaling has not been investigated. We have studied the role of Gab1 in activation of the Akt and ERK pathways at low- and high-intensity stimulation with EGF and HGF in cultured hepatocytes. In cells where Gab1 was depleted by a specific Gab1-directed siRNA, the EGF-induced phosphorylation of ERK was lowered and HGF-induced phosphorylation of both ERK and Akt was substantially reduced. These effects were more marked at low-dose HGF stimulation. The inhibitory consequence of Gab1 depletion was particularly pronounced for HGF-induced Akt phosphorylation. The results suggest that Gab1 is an important signal amplifier for low-intensity stimulation by HGF.

Bone marrow stroma-derived PGE2 protects BCP-ALL cells from DNA damage-induced p53 accumulation and cell death.

BACKGROUND: B cell precursor acute lymphoblastic leukaemia (BCP-ALL) is the most common paediatric cancer. BCP-ALL blasts typically retain wild type p53, and are therefore assumed to rely on indirect measures to suppress transformation-induced p53 activity. We have recently demonstrated that the second messenger cyclic adenosine monophosphate (cAMP) through activation of protein kinase A (PKA) has the ability to inhibit DNA damage-induced p53 accumulation and thereby promote survival of the leukaemic blasts. Development of BCP-ALL in the bone marrow (BM) is supported by resident BM-derived mesenchymal stromal cells (MSCs). MSCs are known to produce prostaglandin E(2) (PGE(2)) which upon binding to its receptors is able to elicit a cAMP response in target cells. We hypothesized that PGE(2) produced by stromal cells in the BM microenvironment could stimulate cAMP production and PKA activation in BCP-ALL cells, thereby suppressing p53 accumulation and promoting survival of the malignant cells. METHODS: Primary BCP-ALL cells isolated from BM aspirates at diagnosis were cocultivated with BM-derived MSCs, and effects on DNA damage-induced p53 accumulation and cell death were monitored by SDS-PAGE/immunoblotting and flow cytometry-based methods, respectively. Effects of intervention of signalling along the PGE(2)-cAMP-PKA axis were assessed by inhibition of PGE(2) production or PKA activity. Statistical significance was tested by Wilcoxon signed-rank test or paired samples t test. RESULTS: We demonstrate that BM-derived MSCs produce PGE(2) and protect primary BCP-ALL cells from p53 accumulation and apoptotic cell death. The MSC-mediated protection of DNA damage-mediated cell death is reversible upon inhibition of PGE(2) synthesis or PKA activity. Furthermore our results indicate differences in the sensitivity to variations in p53 levels between common cytogenetic subgroups of BCP-ALL. CONCLUSIONS: Our findings support our hypothesis that BM-derived PGE(2), through activation of cAMP-PKA signalling in BCP-ALL blasts, can inhibit the tumour suppressive activity of wild type p53, thereby promoting leukaemogenesis and protecting against therapy-induced leukaemic cell death. These novel findings identify the PGE(2)-cAMP-PKA signalling pathway as a possible target for pharmacological intervention with potential relevance for treatment of BCP-ALL.

Inhibitory effects of prostaglandin E2 on collagen synthesis and cell proliferation in human stellate cells from pancreatic head adenocarcinoma.

BACKGROUND: Several studies have described an increased cyclooxygenase-2 (COX-2) expression in pancreatic cancer, but the role of COX-2 in tumour development and progression is not clear. The aim of the present study was to examine expression of COX-2 in cancer cells and stromal cells in pancreatic cancer specimens, and to explore the role of PGE2 in pancreatic stellate cell proliferation and collagen synthesis. METHODS: Immunohistochemistry and immunofluorescence was performed on slides from whole sections of tissue blocks using antibodies against COX-2 and α-smooth muscle actin (αSMA). Pancreatic stellate cells (PSC) were isolated from surgically resected tumour tissue by the outgrowth method. Cells were used between passages 4 and 8. Collagen synthesis was determined by [(3)H]-proline incorporation, or by enzyme immunoassay measurement of collagen C-peptide. DNA synthesis was measured by incorporation of [(3)H]-thymidine in DNA. Cyclic AMP (cAMP) was determined by radioimmunoassay. Collagen 1A1 mRNA was determined by RT-qPCR. RESULTS: Immunohistochemistry staining showed COX-2 in pancreatic carcinoma cells, but not in stromal cells. All tumours showed positive staining for αSMA in the fibrotic stroma. Cultured PSC expressed COX-2, which could be further induced by interleukin-1ß (IL-1ß), epidermal growth factor (EGF), thrombin, and PGE2, but not by transforming growth factor-ß1 (TGFß). Indirect coculture with the adenocarcinoma cell line BxPC-3, but not HPAFII or Panc-1, induced COX-2 expression in PSC. Treatment of PSC with PGE2 strongly stimulated cAMP accumulation, mediated by EP2 receptors, and also stimulated phosphorylation of extracellular signal-regulated kinase (ERK). Treatment of PSC with PGE2 or forskolin suppressed both TGFß-stimulated collagen synthesis and PDGF-stimulated DNA synthesis. CONCLUSIONS: The present results show that COX-2 is mainly produced in carcinoma cells and suggest that the cancer cells are the main source of PGE2 in pancreatic tumours. PGE2 exerts a suppressive effect on proliferation and fibrogenesis in pancreatic stellate cells. These effects of PGE2 are mediated by the cAMP pathway and suggest a role of EP2 receptors.

Role of LPAR3, PKC and EGFR in LPA-induced cell migration in oral squamous carcinoma cells.

BACKGROUND: Oral squamous cell carcinoma is an aggressive neoplasm with serious morbidity and mortality, which typically spreads through local invasive growth. Lysophosphatidic acid (LPA) is involved in a number of biological processes, and may have a role in cancer cell migration and invasiveness. LPA is present in most tissues and can activate cells through six different LPA receptors (LPAR1-6). Although LPA is predominantly promigratory, some of the receptors may have antimigratory effects in certain cells. The signalling mechanisms of LPA are not fully understood, and in oral carcinoma cells the specific receptors and pathways involved in LPA-stimulated migration are unknown. METHODS: The oral carcinoma cell lines E10, SCC-9, and D2 were investigated. Cell migration was studied in a scratch wound assay, and invasion was demonstrated in organotypic three dimensional co-cultures. Protein and mRNA expression of LPA receptors was studied with Western blotting and qRT-PCR. Activation of signalling proteins was examined with Western blotting and isoelectric focusing, and signalling mechanisms were further explored using pharmacological agents and siRNA directed at specific receptors and pathways. RESULTS: LPA stimulated cell migration in the two oral carcinoma cell lines E10 and SCC-9, but was slightly inhibitory in D2. The receptor expression profile and the effects of specific pharmacological antagonist and agonists indicated that LPA-stimulated cell migration was mediated through LPAR3 in E10 and SCC-9. Furthermore, in both these cell lines, the stimulation by LPA was dependent on PKC activity. However, while LPA induced transactivation of EGFR and the stimulated migration was blocked by EGFR inhibitors in E10 cells, LPA did not induce EGFR transactivation in SCC-9 cells. In D2 cells, LPA induced EGFR transactivation, but this was associated with slowing of a very high inherent migration rate in these cells. CONCLUSION: The results demonstrate LPA-stimulated migration in oral carcinoma cells through LPAR3, mediated further by PKC, which acts either in concert with or independently of EGFR transactivation.

The involvement of the docking protein Gab1 in mitogenic signalling induced by EGF and HGF in rat hepatocytes.

Grb2-associated binder (Gab) family proteins are docking molecules that can interact with receptor tyrosine kinases (RTKs) and cytokine receptors and bind several downstream signalling proteins. Studies in several cell types have shown that Gab1 may have a role in signalling mediated by the two RTKs epidermal growth factor (EGF) receptor (EGFR) and Met, the receptor for hepatocyte growth factor (HGF), but the involvement of Gab1 in EGFR and Met signalling has not been directly compared in the same cell. We have studied mechanisms of activation and role in mitogenic signalling of Gab1 in response to EGF and HGF in cultured rat hepatocytes. Gab1, but not Gab2, was expressed in the hepatocytes and was phosphorylated upon stimulation with EGF or HGF. Depletion of Gab1, using siRNA, decreased the ERK and Akt activation, cyclin D1 expression, and DNA synthesis in response to both EGF and HGF. Studies of mechanisms of recruitment to the receptors showed that HGF induced co-precipitation of Gab1 and Met while EGF induced binding of Gab1 to Grb2 but not to EGFR. Gab1 activation in response to both EGF and HGF was dependent on PI3K. While EGF activated Gab1 and Shc equally, within the same concentration range, HGF very potently and almost exclusively activated Gab1, having only a minimal effect on Shc. Collectively, our results strongly suggest that although Gab1 interacts differently with EGFR and Met, it is involved in mitogenic signalling mediated by both these growth factor receptors in hepatocytes.

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-HT(4) and 5-HT(2A) 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-HT(4), 5-HT(2A) and 5-HT(2B) serotonin receptors, 5-HT transporter, atrial natriuretic peptide (ANP) and myosin heavy chain (MHC)-α and MHC-ß (real-time quantitative RT-PCR) as well as 5-HT-receptor-mediated increase in contractile function exvivo (electrical field stimulation of ventricular strips from foetal and neonatal rats and left ventricular papillary muscle from adult rats in organ bath). Both 5-HT(4) 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-HT(2A) and 5-HT(2B) receptor mRNA levels increased to a maximum immediately after birth, but of these, only the 5-HT(2A) receptor mediated a positive inotropic response. We suggest that the 5-HT(4) receptor is a representative of a foetal cardiac gene program, functional in late foetal development and reactivated in heart failure.

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 PLCß. 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, PLCß, Ca2+, Src, and proteinase-mediated release of membrane-associated EGFR ligand(s).

Prostanoid-mediated inotropic responses are attenuated in failing human and rat ventricular myocardium.

Prostanoid-modulatory approaches in heart failure patients have displayed effects which may seem to be mutually incompatible. Both treatment with prostanoids and inhibition of prostanoid synthesis have resulted in increased mortality in heart failure patients. Currently, it is unknown if prostanoids mediate contractile effects in failing human heart and if this can explain some of the clinical effects seen after prostanoid modulatory treatments. Therefore, the objectives of this study were to determine if prostanoids could elicit direct inotropic responses in human ventricle, and if so to determine if they are modified in failing ventricle. Contractile force was measured in left ventricular strips from non-failing or failing human and rat hearts. The ratio of phosphorylated to non-phosphorylated myosin light chain 2 (MLC-2) was measured by Western blotting in myocardial strips, and the levels of prostanoid FP receptor mRNA and protein were measured in rat by real-time RT-PCR and receptor binding assays. In non-failing human hearts, prostanoids evoked a positive inotropic effect and an increase of MLC-2 phosphorylation which was absent in failing human hearts. In failing rat heart, the prostanoid FP receptor-mediated inotropic response and prostanoid FP receptor-density was reduced by ~40-50% compared to non-failing rat heart. Prostanoids mediate a sustained positive inotropic response in non-failing heart, which appears to be down regulated in failing heart. The pathophysiological significance of changes in prostanoid-mediated inotropic support in the failing heart remains to be determined.

Role of ErbB2 in the prostaglandin E2-induced enhancement of the mitogenic response to epidermal growth factor in cultured hepatocytes.

Prostaglandin E(2) (PGE(2)) enhances the mitogenic response to epidermal growth factor (EGF) in hepatocytes, but the underlying mechanisms are not clear. We previously observed that PGE(2) upregulates EGF-induced signalling in the MEK/ERK and PI3K/Akt pathways in hepatocytes. Other investigations have indicated that ErbB2 enhances the mitogenic effect of EGF in these cells. In the present study we found that treatment with PGE(2) increased ErbB2 and decreased ErbB3 expression at both the mRNA and protein level in cultured rat hepatocytes. Silencing of the ErbB2 expression with specific siRNA blocked the stimulation by PGE(2) and EGF of cyclin D1 expression and DNA synthesis. Both EGF and PGE(2) increased the expression of ERK and Akt, but while the effect of EGF was inhibited by ErbB2-directed siRNA, this did not affect the PGE(2)-induced upregulation of ERK and Akt. These data suggest that PGE(2) can enhance the mitogenic effect of EGF both by increasing ErbB2 expression and by ErbB2-independent mechanisms.

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

BACKGROUND: Neurotensin 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. METHODS: Colon 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. RESULTS: Neurotensin 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 ß-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. CONCLUSIONS: While 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.

Induction of LRF-1/ATF3 by vasopressin in hepatocytes: role of MAP kinases.

BACKGROUND/AIMS: Liver regeneration factor 1 (LRF-1/ATF3) is an early response gene which is rapidly induced upon partial hepatectomy in rats, and by growth factors and G protein-coupled receptor (GPCR) agonists in cultured rat hepatocytes. The aim of the present study was to examine the mechanisms involved in induction of LRF-1/ATF3 by the GPCR agonist vasopressin. METHODS: Primary cultures of rat hepatocytes were treated with vasopressin, TPA, and the Ca2+-elevating agents thapsigargin and A23187. LRF-1/ATF3 mRNA and protein were measured by Northern blot analysis or RT-PCR and immunoblotting. Signalling pathways were examined by immunoblots and kinase assays. RESULTS: While elevation of intracellular calcium induced LRF-1/ATF3 expression, treatment with TPA did not. Inhibition of phospholipase C, protein kinase C, or pretreatment with calcium chelators did not affect vasopressin-induced expression of LRF-1/ATF3. Inhibition of each of the MAP kinases ERK1/2, JNK or p38 did not affect vasopressin-induced LRF-1/ATF3 expression. Combined inhibition of JNK and p38, and of ERK1/2 and either JNK or p38 suppressed vasopressin-induced expression of LRF-1/ATF3. CONCLUSION: Vasopressin induces LRF-1/ATF3 expression by mechanisms that differ from those activated by Ca2+-elevating agents. The results suggest that partly redundant, complex MAP kinase networks are involved in induction of LRF-1/ATF3 by vasopressin in hepatocytes.

The activity of pregnancy-associated plasma protein A (PAPP-A) as expressed by immunohistochemistry in atherothrombotic plaques obtained by aspiration thrombectomy in patients presenting with a ST-elevation myocardial infarction: a brief communication.

BACKGROUND: The expression of pregnancy-associated plasma protein A (PAPP-A) was identified by immunohistochemistry (IHC) in culprit atherothrombotic plaque specimens harvested from patients admitted with ST-segment elevation myocardial infarction (STEMI). METHODS: The atherothrombotic samples were collected from a consecutive cohort consisting of 20 individuals admitted with STEMI to Stavanger University Hospital, Norway, from 2005-2006, presenting angiographically with an acute thrombotic occlusion of a coronary artery characterized by TIMI flow 0. The atherothrombotic plaques were obtained by aspiration thrombectomy during percutaneous coronary intervention within 12 hours from the onset of symptoms and prepared for IHC analysis. RESULTS: In the IHC analysis staining for PAPP-A occurred in the extracellular matrix of the plaques and no evidence of staining for PAPP-A was found in the thrombi. CONCLUSION: Our results indicate that in vivo PAPP-A is strongly expressed in atherothrombotic plaques harvested from patients admitted with STEMI, as documented by IHC. TRIAL REGISTRATION: biobankregisteret@fhi.no1846.

Prostanoid F receptors elicit an inotropic effect in rat left ventricle by enhancing myosin light chain phosphorylation.

AIMS: The aims of this study were to determine if the prostanoid F receptor (FPR)-mediated inotropic effect in rat ventricle is mediated by increased phosphorylation of myosin light chain-2 (MLC-2) and to elucidate the signalling pathway(s) activated by FPRs to regulate MLC-2 phosphorylation. METHODS AND RESULTS: Contractility was measured in left ventricular strips from adult male rats. Strips were also snap-frozen, and changes in the phosphorylation level of both MLC-2 and myosin phosphatase targeting subunit-2 (MYPT-2) were quantified. FPR stimulation with fluprostenol increased contractility by approximately 100% above basal and increased phosphorylation of both MLC-2 (by approximately 30%) and MYPT-2 (by approximately 50%). The FPR-mediated inotropic effect and MLC-2 phosphorylation were reduced by a similar magnitude in the presence of the myosin light chain kinase (MLCK) inhibitor ML-7 (approximately 60-70%) and an inhibitor of Ca(2+)/calmodulin, W-7 (approximately 35%). Inhibition of Rho-associated kinase by Y-27632 reduced the FPR-mediated inotropic effect and MLC-2 phosphorylation by approximately 40-45% and MYPT-2 phosphorylation by approximately 70%. ML-7 and Y-27632 together reduced contractility and MLC-2 phosphorylation by approximately 70-80%. The FPR-mediated inotropic effect was only modestly affected by high concentrations of the inositol tris-phosphate (IP(3)) receptor blocker 2-APB, but not by the protein kinase C (PKC) inhibitor bisindolylmaleimide. CONCLUSION: The FPR-evoked inotropic effect is mediated by increasing the phosphorylation of MLC-2 through regulation of both MLCK and myosin light chain phosphatase activities. The second messenger IP(3) and PKC are unlikely to be involved in the signalling cascade of the FPR-mediated positive inotropic effect. Therefore, FPR signalling mechanism(s) regulating MLC-2 phosphorylation likely extend beyond those classically established for G(q/11)-coupled receptors.

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 G(i) protein-mediated and include upregulation of signaling in the PI3K/Akt and the Ras/ERK pathways.

Prostaglandins enhance epidermal growth factor-induced DNA synthesis in hepatocytes by stimulation of E prostanoid 3 and F prostanoid receptors.

Prostaglandins stimulate hepatocyte proliferation in vivo and in vitro. We have examined the role of E prostanoid (EP) and F prostanoid receptors (FP) in enhancing the growth-stimulatory effect of epidermal growth factor (EGF) in cultured hepatocytes. The EP2 receptor agonist butaprost had no significant effect on EGF-induced DNA synthesis. EP1 receptor-selective antagonists did not affect the enhancement by prostaglandin E(2) of EGF-stimulated DNA synthesis. Sulprostone, misoprostol, and fluprostenol strongly enhanced DNA synthesis and inhibited glucagon-stimulated cAMP accumulation, indicating that they all activated EP3 receptors. Sulprostone and fluprostenol, and to a lesser extent misoprostol, stimulated accumulation of inositol phosphates. The effects of fluprostenol and sulprostone on phospholipase C (PLC) were inhibited by the FP receptor antagonist AL-8810 [9 alpha, 15R-dihydroxy-11 beta-fluoro-15-(2,3-dihydro-1H-inden-2-yl)-16,17,18,19,20-pentanor-prosta-5Z, 13E-dien-1-oic acid], indicating that this effect was mediated by FP receptors. Inhibition of protein kinase C with GF109203X [2-[1-(3-dimetylaminopropyl)-1H-indol-3-yl]-maleimide] resulted in a partial reduction of the growth stimulation induced by fluprostenol, indicating a minor role of FP receptors. Combining fluprostenol with misoprostol, but not with sulprostone, resulted in partially additive effects on DNA synthesis, suggesting that both EP3 and FP receptors are involved. Combining sulprostone with misoprostol did not result in additive effects on DNA synthesis, suggesting that EP4 receptors were not involved. We conclude that, although a minor effect is exerted by FP receptors, the growth-stimulatory effects of prostaglandins in rat hepatocytes are mediated mainly by EP3 receptors. We have found no evidence of EP1 receptor involvement.