1. ERK Belongs to the MAPK Family
The Extracellular Signal-Regulated Kinase (ERK) pathway is the best understood of the Mitogen Activated Protein Kinase (MAPK) cascades (29, 40). The pathways are named for the central kinase that affects cell function, ERK, JNK, p38 MAPK and ERK5. These MAPKs are synthesized by cytoplasmic ribosomes but can migrate into the nucleus when activated. Each MAPK pathway consists of a least three kinase components generically termed MAP3K, MAP2K and MAPK which sequentially activate the downstream component. For the ERK pathway these kinases are Raf, MEK and ERK (Figure 1). The ERK pathway is activated by growth factors, mitogens, hormones and some neurotransmitters which bind to tyrosine kinase and G protein coupled receptors. The JNK and p38 MAPK pathways are most often activated by cytokines and cell stress. At many levels of the three pathways there are multiple forms such as ERK1 and ERK2 (ERK1/2) and JNK1/2/3 (40). These multiple species are more closely related and in some cases have the same actions. For the ERK pathway Human ERK1 and ERK2 are 84% identical and all known stimuli activate both forms (3). By contrast, the p38 Map Kinase has four forms (α, β, γ, δ) which may have different regulation and actions. The MAPK pathways are often organized by scaffolding proteins (28, 42) For ERK the best studied scaffold is KSR1 (Kinase Suppressor of Ras-1) which binds all three members of the ERK kinase cascade. For JNK the cascade may be organized by the binding protein JIP-1. MAPK cascade components are all inactivated by phosphatases including pSer/Thr phosphatases such as pp2A, Tyr phosphatases or in the case of the MAPKs themselves by dual specificity phosphatases or DUSPS that dephosphorylate both Ser/Thr and Tyr (23, 26, 40). There are ten catalytically active DUSPS arranged in three families by their nuclear or cytosolic localization.
The MAPK cascades all have multiple actions in both the nucleus and cytosol. In the nucleus, ERK and its downstream effectors such as p90 Ribosomal S6 Kinase (RSK) phosphorylate ternary complex factors such as ELK-1 and thereby stimulate transcription of early response genes such as Fos and Egr1 and are important in initiating mitogenesis (43, 54). In the cytoplasm, ERK and another downstream kinase, MAPK-interacting protein kinase (MNK-1) phosphorylate specific translational factors including eIF4E and eIF4G as well as cPLA2 (cytoplasmic phospholipase A2) ERK also localizes to other organelles including endosomes, caveolae, Golgi and cytoskeleton (53).
This review will first consider what is known about activation of ERK1/2 in pancreatic acinar cells and then cover what is known regarding the actions of ERK in this cell.
Figure 1. Pancreatic Acinar Cell ERK activation.
2. Activation of ERK pathway in Pancreatic Acinar Cells (Figure 1)
ERK activation is usually monitored by following the dual phosphorylation of the Thr and Tyr residues in the Thr-Glu-Tyr activation sequence brought about by MEK as there are a number of good phosphospecific antibodies directed at this epitope. It can also be shown by phosphorylation of myelin basic protein either in a test tube or by an in gel technique following gel electrophoresis and renaturization. Both Western blots and the in gel kinase procedure reveal the two forms of ERK at approximately 44 and 42 kDa; in fact, the molecules were originally referred to as p42 and p44 MAPK with p42 being what is now referred to as ERK2 and p44 now being ERK1. Using isolated rat or mouse pancreatic acini in vitro, ERK1/2 is activated by CCK, bombesin, substance P, and carbachol, all of which activate G protein coupled receptors coupled to Gq and calcium mobilization but not by secretin or VIP which activate receptors coupled to Gs and cAMP formation (10, 12, 15, 16, 41). By contrast, human acini show an increase in pERK in response to cholinergic agonist but not CCK although this was ascribed to the absence of CCK receptors (24). ERK1/2 is also activated by EGF, HGF, IGF-1 and other growth factors which activate Tyr kinase containing receptors (1, 10, 51). Most mechanistic studies have utilized CCK and EGF as they induce the most robust activation. Both of these agonists activate ERK1/2 within minutes in vitro with the response sustained for at least an hour. CCK effects are seen at 3 or 10 pM which is slightly higher than that required to mobilize Ca2+ or stimulate digestive enzyme secretion. TGF-β has also been shown to activate ERK1/2 in pancreatic acini with the effect mediated by Smad4 (46). In vivo, there is little change in phospho ERK between fasting and refeeding chow but phospho ERK shows a large increase after refeeding chow with trypsin inhibitor that increases plasma CCK to around 10 pM and induces adaptive pancreatic growth (18, 47, 48). In another type of growth, p42 and p44 MAPK were shown to be activated during pancreatic regeneration following partial pancreatectomy (33).
ERK appears to be activated in pancreatic acinar cells by the canonical pathway of RAF – MEK – ERK as RafA, RafB and c-Raf1 as well as MEK1 and MEK2 are all present and activated by CCK and EGF (15, 16). EGF activates this pathway by activating Ras and is not blocked by inhibiting Protein Kinase C (PKC) (10). Whether CCK activates Ras is unclear with different reports indicating activation (13, 16) or lack of activation (10). The two studies showing activation used high concentrations of CCK. In addition the effects of CCK to activate ERK were blocked by a PKC inhibitor but not by dominant negative Ras (9, 35). It appears that the CCK1 receptor and receptors for other agonists that activate Gq primarily activate PKC via Ca2+ and diacylglycerol (DAG) and thereby activate the ERK pathway and the ERK mediator RSK (2). In some other cell types a pertussis toxin (PTx) sensitive G protein is involved in ERK activation. In AR42J cells derived from a rat pancreatic tumor, PTx partially inhibited ERK activation in response to CCK, EGF and phorbol ester (37). However, this was shown to be due to disinhibition of adenylyl cyclase signaling. Gastrin or CCK2 receptors have also been shown to be able to activate ERK (9, 14). In contrast to rodent pancreatic acini, in AR42J cells the action of CCK to induce ERK activation was mediated by the CCK2 receptor, the tyrosine kinase Yes and transactivation of the EGFR (36). This transactivation is known to occur in some cells but in rat pancreatic acinar cells there has been no CCK induced EGFR activation observed (11). Another mechanism for ERK activation in some cells is through G protein coupled receptors kinases that phosphorylate the receptor which then binds β-arrestins which recruit other signaling molecules and mediate the prolonged activation of ERK (32). However, a β-arrestin mechanism has not been described for pancreatic acini or the CCK1R.
There is only a little information on the inactivation of ERK in acinar cells. In one study, pancreatic DUSP mRNA levels were very low but DUSP 5, -6, and -10 were induced by caerulein hyperstimulation such that they could be considered early response genes (21).
3. Actions of ERK1/2 in pancreas cells
ERK1/2 as protein kinases have broad substrate specificity for Ser or Thr residues upstream from Pro and can phosphorylate a large number of proteins with 659 target sites listed in a recent compendium (49). There are also 296 reported ERK-interacting proteins whose function may thereby be influenced by ERK (50). These include a number of molecules known to be important in pancreatic function including Stim1 which regulates Ca2+ entry channels (38) and Raptor (4) which promotes activation of the mTOR complex 1 (TORC1). However, most of the cellular responses of ERK signaling cascade include proliferation, differentiation, angiogenesis, survival, and metastasis. Many of the experimental studies have used MEK inhibitors as MEK is the only known activator of ERK1/2. The first MEK inhibitor was PD98059 which is a flavone and highly insoluble in water (17). Another MEK inhibitor U0126 was discovered shortly thereafter and is slightly more potent. These inhibitors have been used for in vitro studies of ERK signaling but were not very useful for in vivo studies due to poor solubility and short half-life. This led to the development of PD325901 and later Trametinib (GSK1120212) which are longer acting and effective experimentally in vivo (45).
In pancreas, as in other cell types, most studies of ERK action have focused on growth, proliferation and regeneration as ERK1/2 is recognized as a master regulator of the cell cycle focused on the G1 to S phase transition (31). To study adaptive growth mediated by CCK, Holtz et al fed mice trypsin inhibitor and showed that both PD325901 and Trametinib blocked acinar cell mitogenesis and pancreatic growth (22). The drugs were effective when fed orally by gavage or mixed into food and a single bolus dose was effective for at least 12 hours. Moreover, the drugs had no effect on other signaling pathways including mTOR, JNK, and STAT3. Cell cycle proteins including cyclin D1, D3 and E as well as PCNA and BrdU incorporation into DNA were inhibited. In vitro, inhibiting ERK with U0126 or PD98059 blocked proliferation of acinar cell monolayer cultures (19).
ERK activation has also been shown to play a role in cytokine production by pancreatic acinar cells. In isolated mouse acinar cells, PD98059 decreased production of MCP-1, MCP1α and MIP-2 induced by Substance P (41) and in rat pancreatic fragments, PD98059 reduced production of TNF-α and IL-1β induced by cerulein (44). The stimulation of cytokine production in both cases involved AP-1 transcription factor that is also blocked by inhibitors of JNK. EGR-1 is another early response gene whose expression in AR42J cells was shown to be blocked by ERK inhibitor PD98059 or overexpression of DUSP-1 (MKP-1) (25). MEK inhibition by either targeted shRNA or Trametanib has also been shown to reduce inflammatory cytokines in cerulein induced chronic pancreatitis (20). Some of this activation of MAPKs may be mediated by reactive oxygen species as hydrogen peroxide and menadione strongly activated all three MAPKs and the activation by CCK was reduced by antioxidants (8).
4. ERK1/2 and Pancreatic Disease
Active ERK1/2 has been observed in both pancreatitis and pancreatic cancer and localized to acinar cells, inflammatory cells and PanINs, the precursor lesion to PDAC (Pancreatic ductal adenocarcinoma). Early studies evaluated the role of ERK1/2 in acute pancreatitis utilized PD 98059 and U0126 dissolved in DMSO that was administered IP to rats or mice; in both studies modest to moderate inhibition of cerulein-induced pancreatitis was observed (5, 30, 34). Inhibition of ERK in isolated acinar cells with PD98059 blocked the upregulation of the Neurokinin 1 receptor induced by cerulein (27). More recent studies in vivo using the much longer acting and water soluble inhibitors PD 325901 or Trametanib both of which block ERK activity by the oral route had no effect on acute pancreatitis but could reverse chronic pancreatitis (6, 20). The ERK inhibitors also prevented the regeneration that occurs after pancreatitis through mitogenesis of acinar cells. These studies are complicated by the fact that ERK is present in more than one cell type. The evidence is clearer for a role in JNK in acute pancreatitis with ROS being one cause of activation.
Studies in PDAC are clearer as in mouse models with active Ras or with chronic pancreatitis, inhibition of ERK by PD 325901 or targeted shRNA to MEK prevented the development of ADM (Acinar Ductal Metaplasia) and PanINs (6, 20). ERK activation has also been reported to play a role in epithelial to mesenchymal transition induced by TGF-β (39). MEK inhibitors block growth of some but not all PDAC derived cell lines (7). Unfortunately, MEK inhibitors by reducing ERK feedback on Ras signaling have also activated other pathways including PI-3K - Akt which serve to maintain carcinogenesis (52). Current clinical trials of MEK inhibitors also often include comparison to combined therapy with both ERK and AKT inhibitors.
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