Gastroenterology
Volume 128, Issue 7 , Pages 2131-2146, June 2005

The Role of Protein Kinase C in Gastrointestinal Function and Disease

Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas, USA

Received 16 June 2004; accepted 23 September 2004.

Article Outline

Abbreviations used in this paper:  PDK-1, phosphoinositide-dependent kinase-1 , PK, protein kinase

 

Externally triggered signaling pathways allow cells to adapt to their environment through diverse cellular responses. In a given milieu, depending on cell and stimulus specificity, the activation of these pathways may induce survival, proliferation, differentiation, or death. These responses may be triggered by receptor-mediated or independent mechanisms and frequently involve alterations in protein phosphorylation. The activation state of many intracellular second messengers is dependent on their phosphorylation state, and thus kinases and phosphatases are often key regulators of cell response. The phosphorylation state of a protein can control cellular location, enzymatic activity, susceptibility to protease degradation, and the ability of the protein to interact with substrates and other proteins.1 The kinases involved in this signaling are divided into 2 categories, those that phosphorylate tyrosine residues and those that phosphorylate serine and threonine residues. The serine/threonine kinases include protein kinase A (PKA), protein kinase B (more often referred to as Akt), protein kinase C (PKC), and the protein kinase D (PKD) family members. This review will focus on the regulation of PKCs and their role in the development, function, and pathology of the gastrointestinal tract.

Back to Article Outline

The Protein Kinase C Family 

Classification and Structure 

PKC is a multigene family whose members are involved in a plethora of cellular signaling cascades and divergent biologic functions. A role for PKCs has been demonstrated in development, differentiation, homeostasis, migration, contraction, secretion, and immunity.2, 3, 4, 5 PKCs are activated via tyrosine kinase and G-protein-coupled receptors, as well as by nonreceptor-mediated signaling cascades. At least 11 mammalian PKC isozymes have been isolated, and these are divided into 3 subfamilies consisting of the classical, novel, and atypical PKC isozymes (Table 1) based on their specific requirements for Ca+2, phosphatidyl-L-serine (PS), and 1,2-sn-diacyglycerol (DAG) for activity.2, 4, 6, 7

Table 1. Subfamilies of Protein Kinase C
Subfamily NameIncluded isoformsRequired cofactors
Conventional PKC (cPKC)αCalcium
βI, βIIDiacyglycerol
γPhosphatidylserine
Novel PKC (nPKC)δDiacyglycerol
εPhosphatidylserine
η
θ
Atypical PKC (aPKC)ζPhosphatidylserine
ι/λ

PKCs are a single polypeptide chain consisting of several conserved regions (C1–C4) interrupted by variable regions (Figure 1). Similar to other inducible enzymes, the PKCs contain both a regulatory (20–70 kilodaltons) and catalytic (45 kilodaltons) domain.8 The regulatory domain spans the amino terminus up to the adenosine triphosphate (ATP)-binding site (C3). It is responsible for the dependence on cofactors and includes an amino proximal pseudosubstrate (autoinhibitory) motif in all of the PKC isozymes. The regulatory regions of the classical PKCs (cPKC) contain a C1 domain that binds phosphatidyl-L-serine, DAG, and phorbol esters; a C2 domain that mediates Ca+2 dependency; and a C3 domain that binds ATP that is required for substrate phosphorylation (Figure 1). The novel PKCs (nPKC) have a similar structure, but they lack the calcium-dependent C2 domain. The regulatory domain of the atypical PKCs (aPKC) is even further truncated and only contains phosphatidylserine and ATP-binding sites. Thus, the dependence of each subfamily on cofactors is due to the presence or absence of these regulatory domains.

  • View full-size image.
  • Figure 1. 

    Structure of PKC isoforms. The structure for classical (cPKC), novel (nPKC), and atypical (aPKC) isoforms is shown schematically. They are divided into their regulatory and catalytic domains as shown. The N-terminal pseudosubstrate (PSEUDO), the phophatidyl-L-serine (PS), and the ATP (C3) binding domains and the C4 catalytic domain are common to all 3 subfamilies.70 The cPKC and the nPKCs both contain functional 1,2-sn-diacylglycerol (DAG) binding domains (C1) and the cPKC contains a Ca+2-binding domain that is nonfunctional and more amino terminal in the nPKCs and completely absent in the aPKCs.

Regulation 

For simplification, the activation of the cPKCs will be used as a model for PKC regulation in general (Figure 2). De novo-translated PKC associates with the cytoskeleton in an “immature” form2, 3, 8, 9 that is phosphorylated into its “mature” form.9 In 1989, approximately 10 years after the discovery that PKCs are lipid-regulated enzymes, Fabbro et al demonstrated that PKCs are phosphorylated in vivo.10 After synthesis, the immature PKCs are then phosphorylated at 3 sites.6, 11 The mechanism of phosphorylation during maturation has best been described for cPKCβII, and the series of events seems fairly well conserved among all PKCs. During maturation, as shown in Figure 2, an initial phosphorylation of the active loop is performed by phosphoinositide-dependent kinase-1 (PDK-1).2, 7, 12, 13, 14 In cPKCβII, this rate-limiting phosphorylation occurs in the C4 domain at Thr500, which is highly conserved among PKCs. Modeling studies suggest that this amino acid is blocked by binding of the pseudosubstrate and that binding of DAG or other lipids may be required to make the activation loop accessible to PDK-1.6 Once PDK-1-dependent phosphorylation occurs, 2 autophosphorylations then occur in the catalytic domain at Thr641 and Ser660 in cPKCβII, and these 2 residues are also highly conserved in most, but not all, mammalian PKCs. For example, aPKCζ and λ have a Glu residue at this position, which, because of its negative charge, may confer the same effect as phosphorylation.7

  • View full-size image.
  • Figure 2. 

    Model of PKC activation. (1) Newly synthesized PKC (PKCβII depicted here) binds to the cytoskeleton in its “immature” form. (2 and 3) During maturation, PDK-1 phosphorylates at Thr500 of C1, which makes the activation loop accessible for binding of the pseudosubstrate to the catalytic domain. This permits autophosphorylation at Thr641 and Ser660. (3) Once the phosphorylation is complete, the “mature” inactive PKC can then bind to the plasma membrane (4) via weak interactions with negatively charged lipid head groups. This binding is transient, and the PKC fluctuates between membrane bound and a free cytosolic equilibrium. During agonist activation, phospholipase type C (PLC) hydrolyzes lipid, releasing Ca+2 and membrane associated 1,2-sn-diacylglycerol (DAG). (5) Ca+2 binding to the C2 domain tethers the PKC to the membrane, allowing it to scan for DAG. (6) Upon interaction with DAG, the C1 domain of PKC forms a strong bond with phosphatidyl-L-serine (PS) in the membrane, resulting in an active, open PKC enzyme that is able to phosphorylate its appropriate substrate.

The mature (phosphorylated) PKC is still inactive and binds via low-affinity interactions to both plasma and cytosolic membranes containing a negatively charged phospholipid head group.8 The association with these membranes is short-lived, and an equilibrium establishes between cytosolic and membrane bound PKC. In its unstimulated state, the kinase is rendered inactive by association of the pseudosubstrate of the regulatory domain with the kinase core of the catalytic domain, which is structurally highly similar to that of the PKA domain, via electrostatic interaction with acidic residues.7, 15, 16 In quiescent cells, the majority of PKCs (90%) are believed to be in this inactive state in equilibrium between being weakly membrane bound and unbound in the cytosol.9

Activation of the kinase involves a series of conformational changes in the regulatory domain induced by binding to 1,2-sn-diacylglycerol (DAG) and phosphatidyl-L-serine (PS).2, 6, 7, 8, 17 In the classical model, agonists bind at the cell surface via receptors or membrane interaction and activate phospholipase type C (Figure 2). This leads to the generation of DAG and soluble inositol phosphates through the hydrolysis of membrane inositol phospholipids as well as the release of Ca+2 from intracellular stores. Ca+2 binds to the C2 domain of cytosolic PKC, allowing the C2 domain to tether to the plasma membrane upon subsequent binding to negative head groups.9 This tethered PKC then diffuses in the plane of the membrane at which plasma membrane-embedded DAG binds to the C1 domain. The energy of DAG binding is used to release the pseudosubstrate, relieving autoinhibition and thus activating the PKC, which binds and phosphorylates its substrate using the C3 bound ATP. This DAG-induced conformational change is mimicked by the addition of phorbol ester (PMA). Both PMA and DAG cap the hydrophilic ligand groove of PKC, thereby altering the hydrophobicity of the domain. Thus, DAG and PMA promote membrane interaction by dramatically increasing membrane affinity.6

Concomitant with DAG binding, conformational change in the activation of PKC is also mediated via binding the cofactor PS to the regulatory domain. Once DAG binds to the C1 domain, the specificity for PS dramatically increases. This affinity for PS binding is increased linearly with the concentration of intracellular Ca+2, which binds to the C2 domain.6, 7, 8 In the case of the cPKCs, Ca+2/PS synergize with DAG to induce a high-affinity interaction of PKC with the membrane. In both the classical and the novel PKCs, this binding of PS leads to activation and release of the pseudosubstrate. However, the dependence on Ca+2 is lost in the nPKCs because the classical C2 domain is absent in this subfamily. It has been reported that nPKCs have a C2-like domain at their N-termini.18 This domain does not appear to bind PS in a Ca+2-dependent manner, and it is possible that it remains in a conformation similar to the Ca+2-bound C2 domains of the cPKCs. The aPKCs have only 1 C1 domain and lack a C2 domain, yielding them unresponsive to DAG, PMA, or Ca+2.

Regulation Through Translocation 

Because cells express multiple isoforms of PKC, which regulate diverse functions within the cell, a mechanism must exist that confers specificity of activity and substrate to the PKCs. The current paradigm is that, prior to activation, PKCs are diffusely distributed throughout the cytosol or localized to distinct structures or regions of the cell cytoplasm.19 Upon activation, the PKCs are recruited to specific foci in the cell so that they are proximal to their appropriate substrates. Thus, the specific signal transduction pathway driven by PKCs is dependent on their appropriate recruitment to subcellular compartments of activation. This model is attractive because the substrate activities of the various PKCs, at least in vitro, appear to be redundant. The fact that PKCs are involved in so many diverse biologic functions and have a multitude of substrates suggests that their specificity is dependent on regulatory inputs that affect both their localization and substrate specificity. This hypothesis is further supported by the observations that a given PKC isozyme’s targeting is dependent, not only on cell type, but on the stage of differentiation, as well as the concentration of the agonist used.19, 20, 21, 22 This specific location targeting of PKCs appears to be governed, at least in part, via association with regulatory proteins such as the PKC-binding proteins receptors for activated C kinases (RACKs), substrates that interact with C kinases (STICKs), receptors for inactive C kinases (RICKs), and a kinase-anchoring protein (AKAP).3, 8, 23, 24, 25, 26 These proteins assist in positioning PKCs at specific cellular locations at which they can respond to second messengers and be accessible to the appropriate substrates. ϵRACK binding to nPKCϵ increases the translocation rate from the cytosol to the particulate fraction by inducing an open, active conformation in nPKCϵ.27 RACKs may also influence PKC translocation by mechanisms other than direct binding. RACK1 not only acts as a scaffold for PKC but also binds to inositol 1,4,5 triphosphate receptors, thereby increasing intracellular Ca+2 levels.28 The mechanisms by which these proteins interact and regulate PKC activation is an area of active investigation, and the details of the process are still unresolved.

Although it was initially believed that PKC activation only resulted in plasma membrane-associated PKC, it is now clear that nuclear targeting of PKC isozymes exists and that they play essential roles in the regulation of nuclear events.3, 4 Translocation of activated PKCs from the cytosol to the nucleus has been observed for all of the PKC isoforms. PKCs contain a nuclear localization sequence (NLS),29 which would allow them to shuttle through nuclear pores. Furthermore, PKCs also reside in the nucleus in their inactive conformation, suggesting that PKCs are in the nucleus awaiting activation signals.3 These observations have led to a new area of investigation in PKC research in which these kinases are not only viewed as cytoplasmic signal transducers but also appear to be involved in the phosphorylation of nuclear proteins as well.

Roles of PKC in the Gastrointestinal Tract 

Although many of the initial studies involving the role of PKCs have focused on cardiac development, as well as the role of PKC in regulating NF-κB during inflammation, accumulating data indicate the relevance of PKCs to gastroenterology. Herein, we summarize the roles of PKC in development, barrier formation and maintenance, inflammation, ion transport, protein secretion, peristalsis, and carcinogenesis of the gastrointestinal tract. Space constraints do not allow an encyclopedic review, but examples of PKC involvement in each of these areas are discussed in the text and listed in Table 2.

Table 2. Known Functions of PKCs in Gastroenterology
FunctionPKCTypeOrgan/cell typePhysiologic effect
DevelopmentPKC-3aPKCIntestine and pharynx (C elegans)Formation and maintenance of epithelial tight junctions that are critical for polarization and mediating development30, 31, 32, 33, 37, 38, 39
PKCλ/ιaPKCIntestine, liver, pancreas (vertebrates)
Epithelial Barrier FormationPKCαcPKCGastrointestinal mucosal epitheliumTranslocation of claudin-1 and ZO-2 from the cytosol to membranes34
PKCδnPKC
PKCεnPKC
PKCεnPKC Phosphorylation of occludin, recruitment of Claudin-1 and ZO-2 to the tight junction, thereby increasing transepithelial resistance (TER)34
Disruption of the Epithelial BarrierNDNDHuman intestine/TNBS treated rat intestinePKC levels elevated during ulcerative colitis46, 52, 56
NDND
Human hepatocytesPKC levels elevated during bile salt-mediated injury53
PKCδnPKCIEC-18 cellsActivation is necessary for PMA or TNF-α-induced injury and caspase-3 activation26
PKCεnPKC
PKCαcPKCT84 cellLoss of TER, increase in paracellular mannitol flux, and redistribution of ZO-1 during EPEC, EHEC, and C deficile toxin A exposure34
PKCβ
Protectors of Epithelial BarrierNDNDT84 cellsBlocks disruption of barrier integrity during H pylori infection43
PKCεnPKCT84 cellsDiminishes loss of TER and increased mannitol flux associated with a TNF-α model of inflammation and Crohn’s disease by increasing shedding of the TNF-α receptor34
PKCαcPKCCaCo-2 cellsIncreased oxygen generation during inflammation disrupts barrier function. EGF maintains barrier function dependent on PKCα, PKCβI, and PKCζ activation. PKCζ partially blocks disruption by inhibiting iNOS generation and NF-κB activity57, 58, 59, 60, 61, 62
PKCβIcPKC
PKCζaPKC
Immune ModulationNDNDIntestinal epithelial cells the ratMediate LPS-induced cytotoxicity via iNOS up-regulation54
PKCδnPKCPancreatic islet cellsLeads to stabilization of iNOS mRNA, resulting in increased iNOS activity in type I diabetes55
PKCδnPKCEpithelial cells in Crohn’s disease and T-84 cellsPKCδ and PKCε mediate TNF-α activation and increased oxidant release associated with epithelial injury26, 34, 57
PKCδnPKC
PKCεnPKCRat pancreatic acinar cellsInvolved in CCK and ethanol-induced pancreatitis via NF-κB induction resulting in inflammation65
Electrolyte TransportPKCεnPKCT84 cellsInhibits basolateral Cl− secretion by increasing transporter endocytosis66
PKCεnPKCCaCo-2 cells, murine and rabbit parietal cellsDecreases apical uptake and secretion by inhibiting Cl−/OH− exchanger67, 70
PKCαcPKCCaCo-2 cellsPKCα inhibits NHE3 expression, thereby regulating vectoral transport79
Protein SecretionPKCεnPKCPancreatic β-cellsAssociates with insulin granules and mediates secretion83
PKCεnPKCT84 and HT29/A1Required for increased mRNA of mucins MUC2 and MUC5AC84
NDNDPancreatic aciniInvolved in cholecystokinin-induced amylase secretion by increasing RHOA p2185, 86
Gut MotilityPKCβcPKCEsophageal muscleRequired for acetylcholine induced contraction and sustained myosin light chain phosphorylation90, 95
PKCεnPKCLower esophageal sphincter
PKCαcPKCIleumLoss of PKCα, PKCβ, and PKCε is seen during colitis and results in loss of tonic and phasic responses91, 97
PKCβcPKCIleum
PKCεcPKCIleum
CarcinogenesisPKCβIIcPKCMurine and human intestineInduces epithelial hyperplasia by increasing nuclear levels of β-catenin and COX-2 regulation113, 120
PKCζaPKCHuman colonic myofibroblastsRequired for the up-regulation of stromal COX-2 expression during inflammation138

ND, not determined.

Development 

Because PKCs are key regulators of cell homeostasis, proliferation, and apoptosis, it is not surprising that they play a role in organogenesis and development. PKCs are involved in the development of such diverse organs as the cardiovascular system, appendages, kidney, and central nervous system in Caenorhabditis elegans, drosophila, zebra fish, and mammals.30, 31, 32, 33 One of the key developmental roles of PKCs is in the critical process of the formation and maintenance of epithelial sheets. Polarization of epithelial cells into separate apical and basolateral membrane domains results from the formation of junctional complexes such as the zonula adherens and the zonula occludens on the apical aspect of the intracellular space and then the planar association of separate epithelial cells.

Tight junctions, or zonula occludens, are composed of the proteins claudins; occludin; and zonula occludin proteins (ZO)-1, -2, and -334 as well as other adaptor proteins.35 The claudins and the occludins are the major structural components of the tight junction at which highly phosphorylated forms of occludin concentrate.36 ZO-1, -2, and -3 are members of the guanylate kinase family and act as a bridge between the occludins-claudins complex and the actin cytoskeleton.

An adaptor protein complex, highly conserved across species (PAR, named for the C elegans partition-deficient mutant), has been identified that is a critical regulator of cell polarity and is composed of the proteins Par-3 and Par-6 and an aPKC (PKCλ/ι and aPKCζ in vertebrates, PKC3 in C elegans, and DaPKC in Drosophila).30 Studies of vertebrate organogenesis have focused mainly on a role for aPKCs in cardiovascular, retinal, and renal development, but their role in the formation of the gastrointestinal tract in C elegans and zebra fish has been shown also.30, 31, 32 The entire C elegans intestine is composed of a single layer of 20 epithelial cells, and the pharynx has approximately 4 times that number. During C elegans organ morphogenesis, PAR-3 protein-mediated polarity31 helps form cell junctions and is critical for gut differentiation.37 PKC-3, the equivalent of aPKC in humans, localizes to the apical surface membrane within the adherens junctions in both the intestinal and pharyngeal primordia and colocalizes with PAR-3. Ablation of PAR-3 or PKC-3 expression in embryos by inhibitory RNA (iRNA) injection inhibits gut development by blocking cell differentiation, although complete ablation of PKC-3 could not be studied because it arrests development and prevents hatching.37, 38 A key regulator in zebra fish organogenesis, the heart and soul chromosomal locus, encodes aPKCλ.30 Loss of aPKCλ activity affects the formation and maintenance of the zonula adherens in the epithelia of several organs and results in lumenal reduplication of the intestine as well as incomplete formation of the liver and pancreas and improper migration to their appropriate position in the peritoneal cavity.30 In a diabetes disease model induced in pregnant mice, PKC activity was increased and resulted in gut defects in the pups, suggesting a role for PKC in intestinal organogenesis in mammals.39 Thus, it is clear that PKC, and more specifically aPKC, is involved in organogenesis in the gastrointestinal tract, and the regulation of cell polarity appears to be the initial and crucial step.

Epithelial Barrier Integrity 

The gastrointestinal epithelial barrier is necessary for the prevention of the passage of bacteria, endotoxins, and other harmful agents from the outside world into the mucosa and the systemic circulation. Loss of barrier function can lead to the initiation and perpetuation of mucosal inflammation and injury. As described below, the role of PKCs in maintaining barrier function during inflammation seems paradoxical: PKCs appear to be both the culprits in loss of mucosal integrity as well as key regulators in maintaining the intestinal barrier.

Because PKCs play a role in the maintenance of epithelial tight junctions during embryogenesis, it is not surprising that they also play a role in the maintenance of the epithelial barrier between the gastrointestinal lumen and the inside of the body. PKCs are known to affect barrier function by regulating both phosphorylation states and localization of several tight junction proteins. Bryostatin-1, a nonphorbol ester PKC agonist that selectively activates PKC-α, -ϵ, and -δ in T84 cells, induces translocation of claudin-1 and ZO-2 from a detergent soluble (cytosol) to a detergent insoluble (membrane) fraction of cell lysates.34 In addition, nPKCϵ activation results in phosphorylation of occludin, and this is accompanied by increasing transepithelial resistance (TER), an electrical measure of epithelial barrier (predominantly tight junction) integrity, in T84 monolayers. Presumably, this occurs by recruiting claudin-1, ZO-2, and occludin into the tight junctional complex.34 Therefore, PKCs are vital in regulating the assembly of tight junctions.

The intestinal barrier can also be compromised during bacterial infection. Both Clostridium difficile and enteropathogenic (EPEC) and enterohemorrhagic Escherichia coli (EHEC) have been shown to lower TER by tight junction disruption.40 C difficile toxin A induces increased cPKCα and cPKCβ activity, which leads to a redistribution of the tight junction protein ZO-1.36, 40 EHEC lowers TER, increases paracellular flux of mannitol, and alters ZO-1 distribution via a cPKC-dependent pathway.40 EPEC also disrupts tight junctions; however, this process was found to be independent of cPKC activity.41 Besides affecting barrier function, EPEC also elicits an inflammatory response by inducing interleukin (IL)-8 secretion via an NF-κB-dependent pathway that is partially dependent on the aPKCζ.41 In addition, EPEC infection results in watery diarrhea because of a PKC-mediated secretion of ions and fluids into the intestinal lumen.42

Almost paradoxical to the observations above, PKC activation has also been shown to maintain barrier function during intestinal infection and injury. Infection with Helicobacter pylori reduces gastric epithelial TER, and this effect was reversed by PKC activation,43 suggesting that H pylori interferes with the normal PKC signaling pathways of the host. As discussed below in more detail, PKC activation augments TNF-induced cytotoxicity in intestinal epithelial monolayers. However, activation of PKC, most likely nPKCϵ, also prevents the tumor necrosis factor (TNF)-α-induced decrease in TER by increasing TNF-receptor 1 (TNF-R1) shedding, thereby decreasing the availability of surface expressed TNF-R1.34

Inflammation 

General role 

PKC mediation of inflammation has been an active area of investigation. Although early studies focused on the role of PKC in lymphocyte function, more recent studies demonstrate its importance in nonprofessional immune cells. Traditionally, the focus was on the proinflammatory effect of PKC via NF-κB activation; however, this pathway also regulates cellular transformation, oncogenesis, and apoptosis.44 NF-κB activation is a double-edged sword that can be both proinflammatory/antiapoptotic, or it may induce an anti-inflammatory/proapoptotic cascade. As is often the case with PKCs, which of these events occurs is cell type and stimulus specific. Activation of cPKCα and the atypical PKCs mediates a cell survival response during inflammation and injury, whereas the activation of nPKCδ and nPKCθ results in apoptosis.45

During inflammation, PKCs regulate the biosynthesis of proinflammatory cytokines, nitric oxide (NO), and reactive oxygen species (ROS) as well as activate phospholipase A2.46 In a classic model of inflammation, such as T-cell activation, receptor engagement induces phospholipase C activation, resulting in both DAG and Ca+2 release, which in turn activates PKC and downstream NF-κB.47 Phospholipases are also involved in the inflammatory response of fibroblasts in which secretory phospholipase A2 acts in an autocrine fashion, resulting in cytosolic phospholipase A2 production via a PKC-dependent mechanism.48 However, as mentioned above, PKC activation is not always proinflammatory. The resolution of inflammation is believed to depend partly on the induction of apoptosis of neutrophils as well as cells resident to the affected organs, ie, fibroblasts, that mediate inflammation.45, 49, 50

Environmental triggers, such as cytokine deprivation, result in caspase-3 and -8 activation.45 Once activated, these caspases cleave nPKCδ and aPKCζ45, 51 as well as other cell regulatory molecules. Cleavage of aPKCζ inactivates the kinase, thereby blocking its ability to activate NF-κB and induce a survival response. The cleavage of nPKCδ results in a constitutively active molecule consisting of the nPKCδ catalytic domain that is known to induce apoptosis by inducing the translocation and activation of annexin 1.51

PKC activity is increased in inflammatory disease of the skin, cartilage, respiratory tract, liver, and colon,46, 52, 53 and its involvement in gastrointestinal inflammation is described below in more detail.

Lipopolysaccharide 

Intravenous lipopolysaccharide (LPS) administration is associated with cytotoxic effects on small and large intestinal epithelium of the rat,54 at least in part through generation of considerable quantities of NO. Elevated PKC activity preceded inducible nitric oxide synthase (iNOS) enzymatic activation after LPS treatment, and inhibition of PKC prevented LPS-induced iNOS activity, indicating its role in LPS-induced injury. Inflammation also occurs in type I diabetes and results in the apoptosis of pancreatic β-cells. Exposure of pancreatic islets to a key proinflammatory cytokine (IL-1β) resulted in NO production and type II iNOS expression, which could be attenuated by inhibiting nPKCδ.55 nPKCδ appears to play a role in stabilizing the iNOS mRNA, thereby leading to greater iNOS expression.

Inflammatory bowel disease 

Trinitrobenzene sulfonic acid (TNBS)-induced colitis, a model of ulcerative colitis in the rat, demonstrated increased mucosal PKC levels,46, 56 and the severity of injury was ameliorated by treatment with PKC inhibitors.46 PMA alone also induced mucosal damage.46, 56 Antineutrophil serum-induced neutropenia had no significant effect on TNBS-induced injury or on PKC activation, demonstrating that both processes were independent of white cell infiltration.

High levels of injurious oxidants are also seen in the colonic mucosa of patients with Crohn’s disease, ulcerative colitis, and infectious colitis, as well as in animal models of intestinal epithelial insult, and are believed to disrupt barrier integrity.57, 58, 59, 60, 61, 62 This elevated level of oxidants is believed to result in the initiation and/or perpetuation of inflammation and injury of the mucosa. Keshavarzian et al have presented several reports in which epidermal growth factor (EGF) is employed to protect and maintain GI barrier function.57, 58, 60, 61 Treatment of epithelial monolayers with EGF resulted in a rapid redistribution of cPKCα, cPKCβI, and aPKCζ to cell membranes and protected against oxidant-induced microtubule disruption and loss of barrier integrity.58, 60 The classical PKCβI and atypical PKCζ were the 2 isozymes necessary for EGF-conferred protection.59, 61 cPKCβI activation alone resulted in a 50% protection of cytoskeleton disruption and loss of barrier function. Although the mechanism of cPKCβI-induced protection still remains unresolved, it may occur as a result of decreases in oxidative stress, normalization of cytosolic Ca+2, or phosphorylation of tubulin.60 The protection conferred by aPKCζ activation is believed to be the result of blocking oxidant-induced NF-κB activity by stabilizing I-κBα, thus preventing NF-κB-mediated disruption of the cytoskeleton and barrier function.59 Furthermore, aPKCζ was found to inhibit oxidant-induced iNOS activation, a key mediator of barrier function disruption.61 This inhibition of iNOS reduced NO generation, oxidative stress, tubulin oxidation, and nitration, and this was accompanied by maintenance of the cytoarchitecture. Thus, cPKCβI and aPKCζ protect intestinal epithelial cells from oxidant injury.

During intestinal inflammation such as Crohn’s disease, a loss in barrier function occurs via increased TNF and oxidant release,34, 57 which are believed to contribute to inflammation, diarrhea, and increased epithelial permeability.34, 57 TNF induces a dose- and time-dependent decrease in TER accompanied by an increased transepithelial mannitol flux in a PKC-independent manner in T84 epithelial monolayers.34 Furthermore, although TNF-α and PMA decreased viability and increased apoptosis in IEC-18 cells, inhibition of nPKCϵ and nPKCδ reduced the severity of injury as measured by 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays and nuclear condensation. The inhibition of these PKCs also reduced the activation of caspase-3, a mediator of apoptosis.26 These data demonstrate that PKCs play a role in intestinal inflammation, both as mediators of cell injury and through alterations in tight junctional permeability.

Pancreatitis 

Experimental pancreatitis can be induced by excessive secretion of cholecystokinin (CCK). CCK is expressed in neurons and intestinal endocrine cells and is released after a meal. Supraphysiologic levels of CCK inhibit the secretion of digestive enzymes from pancreatic acinar cells.49 Activation of the intracellular enzymes or of secreted enzymes within the pancreas is thought to be the cause of the pancreatic inflammation. Although ethanol consumption, the most common cause of acute and chronic pancreatitis in humans, does not induce pancreatitis in rats, it does cause pancreatitis in animals given an otherwise benign dose of CCK-8.63 The ability of ethanol to sensitize animals to CCK-8-induced pancreatitis is due, at least in part, to augmention of CCK-8-induced NF-κB activation, a key inflammatory regulator in pancreatitis.63 The activation of NF-κB by CCK-8 involves the intracellular mobilization of Ca+2 as well as the activation of PKC.64 Ethanol inhibited Ca+2 mobilization, but augmented PMA-induced PKC activation, suggesting that NF-κB activation occurs via Ca+2-independent PKCs.63 This is supported by the findings that CCK-8-induced NF-κB activation is sensitive to broad-spectrum PKC inhibitors but not to inhibitors of conventional PKCs.63 A recent report has identified aPKC-δ -ϵ, and -ζ as the nonconventional PKCs activated by CCK and that NF-κB activation is dependent on the nPKCs (δ, ϵ).65 Thus, CCK and ethanol may induce pancreatitis by PKC-mediated NF-κB activation.

In addition to CCK inducing pancreatitis as an “inflammatory mediator,” it also may play a paradoxically protective role by inducing apoptosis in acinar cells. Both apoptosis and necrosis occur in models of pancreatitis, and an inverse correlation exists between the degree of apoptosis on one hand and the level of necrosis and the severity of disease on the other.49 This may be due to caspase activation not only inducing apoptosis but inhibiting trypsin activation and necrosis in acinar cells.49 CCK-8 treatment results in apoptosis of acinar cells via the activation of caspase-3 and caspase-8 that initiate a cascade that results in mitochondrial alteration and dysfunction including the release of cytochrome c. Once activated, these caspases cleave nPKCδ, rendering it active, a process that has been shown to result in apoptosis in other cell types.50 Thus, the activation of novel PKCs may be involved in both the apoptotic and inflammatory aspects of pancreatitis.

Electrolyte Transport 

Na+-K+-2Cl− transporter 

One of the best studied areas of PKC biology is the activation or inhibition of ion channels and transporters. Mucosal surface hydration, absorption, and secretion are maintained by epithelial chloride secretion, and dysregulation of Cl− transport is the sine qua non of diseases such as cystic fibrosis and secretory diarrhea. PKC activation is an important regulator of Cl−.66 In vitro studies of epithelial monolayers using phorbol esters, a component of croton oil that years ago was used as a laxative, to induce PKC activation have demonstrated that PKC activity inhibits chloride secretion.66, 67, 68 In T84 cells, phorbol esters induced the translocation of nPKCϵ basolaterally, which inhibited chloride secretion, and cPKCα apically, which decreased TER.66 The mechanism for the decreased chloride secretion is believed to be nPKCϵ-mediated increased basolateral endocytosis of the transmembrane Na+-K+-2Cl− transporter protein,68 suggesting that diarrhea may be caused by decreased basolateral Cl− uptake. This effect of nPKCϵ on increased endocytosis appears to be opposed by cPKCα, thus demonstrating that various PKC isoforms can work in opposition to one another.69

Cl−/HCO3− exchanger 

In addition to affecting basolateral Cl− uptake, PKCs have been shown to regulate apical Cl− uptake and secretion. The dual ion exchangers Na+/H+ and Cl−/HCO3− have been suggested as being the main mechanisms for Na+ and Cl− enterocyte uptake and absorption in the mammalian ileum and colon,67, 70 and loss of regulation of Cl−, Na+, and HCO3−4 absorption results in diarrhea. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the apical Cl−/OH− exchanger activity in CaCo-2 cells via phosphatidylinositol 3-kinase-induced nPKCϵ activation.67 In rat hepatocytes, the Na+-independent Cl−/HCO3− exchanger allows hepatocytes to load acid in defense of an alkaline exposure, and activation of PKCs is known to inhibit canalicular Cl−/HCO3− exchange activity.71

Cystic fibrosis transmembrane conductance regulator 

PKCs regulate chloride secretion in the murine colonic mucosa and rabbit parietal cells.72, 73 In the disease transmissible murine colonic hyperplasia (TMCH), an increase in cystic fibrosis transmembrane conductance regulator (CFTR) ion channel expression and accumulation on the apical plasma membrane are observed along with increased cAMP-dependent Cl− secretion. Both increased Cl− secretion and colonocyte hyperproliferation were found to be dependent on cPKC activation because the alterations depended on an increase in intracellular Ca+2 and DAG levels.73 Colonic crypts isolated from Citrobacter-induced TMCH in mice have elevated cPKC α, βI, and βII expression and membrane translocation; however, βI was identified as the isoform involved in both the increased CFTR expression and the hyperplasia.73

Parietal cell HCl secretion 

A hallmark of gastric acid secretion is the dramatic morphologic alteration of the parietal cell because of intracellular canalicula formation prior to agonist-induced HCl secretion.72 These alterations are mediated by phosphorylation of the cytoskeletal linker protein ezrin. Although there are conflicting reports as to whether PKC activation stimulates or inhibits HCl secretion,74, 75, 76, 77 recent studies on enriched rabbit parietal cells suggest that PKCϵ inhibits the process. The mechanism in parietal cells appears to be phosphorylation of pp66, which colocalizes with ezrin and interferes with cytoskeletal cross-linking during cholinergic agonist (carbachol) exposure. Of interest, this inhibition was not seen in intact glands using histamine as a secretory agonist.72, 77 Thus, PKCs can act both apically as well as basolaterally and either increase or inhibit chloride secretion, depending on the cell and stimulus specificity.

Gastric and duodenal HCO3− secretion 

Bicarbonate secretion is an important defensive mechanism of the stomach and duodenal epithelium for protecting the mucosa against acid-peptic damage. Bicarbonate secretion is regulated by a host of factors including prostaglandin E2, cAMP, guanylin, and acetylcholine. Studies in murine duodenal mucosa by Tuo et al demonstrate that nPKCϵ is involved in this process.78 Although nPKCϵ alone does not alter basal bicarbonate secretion in the murine duodenal mucosa, it does potentiate db-cAMP-induced secretion. The mechanism of this augmented secretion is unknown, although the authors speculate that the CFTR channel may be involved. Coupled with the studies above, these findings demonstrate a role of PKC and more specifically of nPKCϵ in chloride, acid, and bicarbonate secretions in the stomach and duodenum.

Na+/H+ exchanger 

In addition to regulating chloride channels and bicarbonate secretion, PKC affects Na+/H+ exchange (NHE). NHEs are a family of membrane transport proteins that mediate the electroneutral exchange of extracellular Na+ with intracellular H+. Although 6 NHEs have been identified, only NHE1, NHE2, and NHE3 are expressed in the human intestine.79 The “housekeeping” NHE1 is ubiquitously expressed and localizes to the basolateral surface of virtually all mammalian epithelial cells and is believed to regulate cell volume and pH.79, 80 NHE2 and NHE3 have a more restricted tissue distribution (intestine and kidney) and are predominantly located on the apical epithelial membrane. They are probably involved in brush border Na+ absorption as well as bicarbonate and water reabsorption.79, 80 In CaCo-2 cells, cPKCα activation results in reduced apical Na+ transport, whereas inhibition leads to increased apical transport.79 Although NHE1 and NHE2 expression is unaffected, the NHE3 transcription rate is inhibited by cPKCα activation.79 These results are in agreement with those of Kandasamy et al, who demonstrated that PKC activation increases NHE1 and NHE2 activity while inhibiting that of NHE3,81 and those of Donowitz in which the Ca2+-dependent inhibition of NHE3 requires cPKCα binding to NHE3 kinase A regulatory protein (E3KARP).82 These data demonstrate that PKCs regulate vectoral transport by down-regulating NHE3.

Protein Secretion 

Insulin 

Glucose stimulates insulin secretion in pancreatic β-cells through a variety of signals that lead to exocytosis of insulin granules. nPKCϵ associates with these insulin granules and also changes its cellular distribution during glucose stimulation of murine β-cells.83 The functional importance of nPKCϵ was confirmed in a nPKCϵ dominant-negative trangenic mouse in which glucose-induced insulin secretion is abolished.83

Mucin 

Mucins are high-molecular-weight glycoproteins that are the major components of mucus, which plays a key role in protecting epithelia against mechanical damage, acid, and pepsin autodigestion, detergent bile salts, as well as stabilizing the luminal microenvironment. nPKCϵ activation is a key regulator on intestinal mucin expression. nPKCϵ activity is required for PMA-induced mRNA expression of the mucins MUC2 and MUC5AC but has no effect on MUC1, MUC5B, or MUC6 expression in HT-29/A1 and T84 monolayers.84

Amylase 

Amylase has been used as a measure for stimulus-induced secretion pathways in pancreatic acinar cells. Although the specific isozyme has not been identified, the role of PKC activation in (CCK)-induced amylase secretion by pancreatic acini is quite clear.85, 86 CCK induces the PKC cascade in acini, thereby increasing the intracellular level of the small GTP-binding protein RhoA p21.86 Elevated RhoA p21 levels then lead to increased exocytosis of pancreatic enzymes, possibly by interacting with focal adhesion kinase, phosphatidylinositol-3 kinase, and the actin cytoskeleton.

Neurotensin 

Specialized endocrine cells of the gastrointestinal mucosa secrete hormones that regulate gastrointestinal secretion, motility, and growth.87 Neurotensin is a tridecapeptide predominantly localized in the enteroendocrine cells of the adult small bowel that affects gut motility, secretion, and fatty acid translocation.88 In BON cells, a human pancreatic carcinoid cell line, PMA induces neurotensin production and secretion. Neurotensin secretion was associated with and dependent on PKCα and PKCδ activation. Inhibition of cPKCα and nPKCδ blocked PMA-induced neurotensin secretion, demonstrating that these isozymes are critical for neurotensin release.89

PKC and Gut Motility 

Peristalsis is controlled by a series of circular muscle contractions and relaxations that occur in a synchronized manner. Three distinct types of contractions are observed in mammalian intestinal smooth muscle: rhythmic phasic contractions (RPCs), ultrapropulsive contractions, and sustained (tonic) contractions.90, 91 The current dogma is that the phasic response is elicited by activation of Gq-coupled receptors by ligand binding, eg, cholecystokinin-A, leading to phosphorylation of the regulatory light chain of myosin II via a Ca+2/calmodulin-dependent myosin light chain kinase (MLCK).90, 92, 93, 94 The agonist-mediated increase in Ca+2 level is transient, and intracellular free Ca+2 levels rapidly return to normal, thus rendering the increased MLCK activity short-lived. However, the phosphorylation of the myosin light chain, as well as the muscle contraction, persist in the tonic response. Experiments using smooth muscle contraction induced by acetylcholine (ACh) demonstrate that ACh not only activates MLCK but also activates PKC via DAG generation as a result of increased phospholipase D hydrolysis of phosphotidylcholine.90 PKC in turn phosphorylates CPI-17, an inhibitor of type 1 phosphatase. This augments CPI-17’s ability to bind to the myosin light chain phosphatase (MLCP), thereby inhibiting its ability to dephosphorylate the light chain, resulting in prolonged muscle contraction.90

Prior to swallowing, the esophageal muscle (ESO) is relaxed while that of the lower esophageal sphincter (LES) exhibits high resting tone. During swallowing, the ESO rapidly contracts, and the LES relaxes. This agonist-induced ESO contraction is dependent on the Ca+2-insensitive nPKCϵ.95 Furthermore, although contraction of the LES under high levels of ACh is calmodulin and MLCK dependent, spontaneous tone and contraction under low levels of ACh are mediated through the Ca+2-dependent cPKCβ and are calmodulin insensitive.95

Inflammation suppresses the tonic and phasic contractions in colonic circular smooth muscle via down-regulation of L-type Ca+2 channels.91, 96 These channels are one of the targets of PKCs. Experimental colitis was induced in canines using ethanol and acetic acid, and PKC expression, distribution, and activation were studied.91, 97 In normal canine ileum, the ultrapropulsive response is triggered even in the presence of PKC inhibitors; however, in the inflamed ileum, PKC inhibition completely blocks this response. In both normal and inflamed tissue, the RPCs are dependent on PKC activation. In this model, PKCα, β, and ϵ isozyme expression is down-regulated during colitis. Furthermore, ACh-induced translocation of these enzymes from the cytosol to the plasma membrane occurs in normal but not inflamed tissue. These data suggest that the loss of tonic and phasic contractions in ileitis and colitis is due to PKC down-regulation.

Carcinogenesis 

The role of PKCs in maintaining epithelial barrier function often intertwines with that of carcinogenesis because the tumor promoting phorbol esters are often used as agonists of PKC activation. Alterations in barrier function in transformed epithelial cells were demonstrated as early as 1970.98 Given their role in maintaining barrier function, regulating differentiation, and controlling apoptosis and cell proliferation as well as their activation by tumor promoters (phorbol esters), the hypothesis that PKCs play a role in carcinomas, as well as nonepithelial cancer, seems intuitive. The activation of PKCs by phorbol esters suggests that carcinogenesis may include aberrant PKC signaling during the initiation as well as progression of neoplasia and malignancy. A current thrust in understanding carcinogenesis has focused on identifying which specific PKC isozymes contribute to the transformation process, what their downstream targets may be, and what chemotherapeutic strategies could be employed to prevent the progression to malignancy.

PKC expression, activation, and function have been extensively studied in the intestinal epithelium. In normal colonic epithelia, the highest level of PKC expression is found at the nonproliferating tip of the crypt (the surface epithelium), with a decreasing gradient of expression extending to the base of the crypt at which normal proliferation occurs.99, 100, 101, 102 It is believed that PKCs may function as regulators of postmitotic events (ie, differentiation and inhibition of growth) during intestinal epithelial self-renewal, a concept supported by the fact that PKCs mediate a coordinated program of cell cycle withdrawal in intestinal cells.102, 103, 104, 105 Activation of cPKCα by itself is able to induce cell cycle arrest in IEC-18 cells,104 and nPKCδ inhibits cell growth, limits survival, and enhances differentiation in CaCo-2 cells.106 These results, coupled with those in other epithelial systems, suggest that PKC-dependent pathways may impart negative control of cell growth and drive differentiation.103 However, it should be noted that activation of PKCs by angiotensin II or neurotensin is required for a mitogenic response in IEC-18 and in PANC-1 cells, respectively, demonstrating that PKC activation does not always result in cell cycle arrest.107, 108

Epithelial cells from colon cancer demonstrate a marked decrease in PKCα, δ, ϵ, ζ, and η levels,99, 101, 102 and human colonic neoplasms, as well as preneoplastic mucosa and adenomas, have decreased PKC activity compared with normal colonic mucosa.99, 109, 110, 111, 112 However, although most PKC levels decrease, those of cPKCβII increase in several models of colon cancer.113, 114, 115 These data suggest that down-regulation of cPKCα and nPKCδ and up-regulation of cPKCβII may yield a growth advantage to colonic epithelial cells. The resulting hyperproliferation would make these cells more susceptible to mutagens and thus contribute to the process of carcinogenesis.

cPKCβII and colorectal cancer 

Colorectal adenocarcinoma (CRC) is the third leading cause of cancer-related death in the Western world. The molecular pathogenesis of this disease is also the most extensively studied. Mutation of the adenomatous polyposis coli (APC) gene, a key step in the adenoma-carcinoma sequence of this disease,116, 117 leads to nuclear accumulation of β-catenin, and dysregulation of β-catenin is seen in all stages of the adenoma-carcinoma sequence. APC binding to β-catenin and its phosphorylation by glycogen synthase kinase-3β (GSK-3β) leads to its degradation. APC mutations prevent the binding of cytosolic β-catenin, resulting in nuclear accumulation of β-catenin.118 Inhibition of GSK-3β phosphorylation of β-catenin because of mutations of β-catenin or of GSK-3β also leads to increased cytosolic and nuclear levels of β-catenin. cPKCβII, whose levels are raised in CRC tissue and in colonic mucosa after exposure to secondary bile acids (suspected carcinogens), has been shown to phosphorylate and inactivate GSK-3β (Figure 3A).119, 120

  • View full-size image.
  • Figure 3. 

    Involvement of PKC and colorectal cancer. (A) β-Catenin regulation. β-Catenin is released into the cytosol where it is bound by adenomatous polyposis coli (APC) protein and phosphorylated by GSK-3β. These 2 events lead to the ubiquitination and degradation of β-catenin. Activation of cPKCβII results in phosphorylation of GSK-3β by PKCβII, and this inactivates GSK-3βm, resulting in cytosolic and eventually nuclear accumulation of β-catenin. Once in the nucleus, β-catenin acts as a transcriptional regulator leading to loss of cell regulation and eventually malignancy. (B) COX-2 induction in colonic epithelia. Over-expression of cPKCβII results in induction of COX-2 expression, which, in turn, down-regulates expression of the TGF-βRII. This results in hyperproliferation caused by a loss of TGF-β regulation, as well as increased proliferative signals mediated by COX-2 activity (ie, prostaglandin secretion). This activity of cPKCβII is down-regulated by nonfermentable brans, cellulose, and ω-3 polyunsaturated fatty acids (PUFA) and is augmented by azoxymethane (AOM). (C) COX-2 induction in colonic myofibroblasts. IL-1 binds to its receptor, activating aPKCζ and NF-κB. This activation of aPKCζ results in generation of reactive oxygen species (ROS) as well as translocation of aPKCζ into the nucleus. Through an unknown mechanism, aPKCζ and ROS potentiate COX-2 transcription.

In the azoxymethane (AOM) model of CRC, cPKCβII levels increase in preneoplastic lesions and in colon tumors.113 Transgenic mice that over express PKCβII demonstrate hyperproliferation of the colonic epithelium.113, 120 Because hyperproliferation is a significant risk factor for CRC, the sensitivity of these mice to azoxymethane-induced CRC was examined. Although the cPKCβII transgenic mice showed a trend toward increased tumorigenesis and increased mean tumor area compared with normal mice, these changes were not statistically significant.113 Interestingly, in these studies, the implicated role of cPKCβII in the progression of colon cancer was not found to be via GSK-3β inhibition. The authors conclude from additional studies that over expression of cPKCβII represses transforming growth factor-β type II receptor (TGF-βRII) by inducing cyclooxygenase-2 (COX-2) expression.121 The roles of PKC and COX-2 in CRC are described in more detail below.

The observed over expression of cPKCβΙΙ in CRC led to several studies designed to reduce cPKCβII expression in the colon. Diet was found to affect the activation of cPKCβII. In the distal colon of rodents, diets rich in wheat bran, rye bran, cellulose, and ω-3 fatty acids reduce cPKCβII expression and prevent hyperproliferation.121, 122, 123, 124, 125, 126, 127, 128 When rye bran and beef were fed to multiple intestinal neoplasia (Min) mice, rye bran lowered cytosolic β-catenin levels without affecting cPKCβII levels,129 supporting the argument that cPKCβII may induce colon cancer in a GSK-3β/β-catenin-independent manner. However, it should be noted that Min mice may have altered regulation of β-catenin and PKC because of an underlying genetic predisposition to cancer.

Regulation of COX-2 

Nonsteroidal antiinflammatory drugs (NSAIDs) reduce the size and number of colorectal adenomas and carcinomas in patients with CRC and in animal models of colon cancer, suggesting a role for COX-2 in the progression of CRC.130, 131, 132, 133 As mentioned previously, cPKCβII induces COX-2 expression in transgenic mice that over express cPKCβII, as well as in rat intestinal epithelial cells in vitro.121 Elevated COX-2 activity in the epithelium inhibits TGF-βRII expression rendering the cells insensitive to TGF-β, allowing hyperproliferation (Figure 3B). Celecoxib, a COX-2-specific inhibitor, restores TGF-βRII expression. Inhibition of cPKCβII by administration of the ω-3 fatty acid eicosapentaenoic acid blocked COX-2 expression and reestablished TGF-βRII expression.121 Thus, cPKCβII induces COX-2 expression, which in turn inhibits TGF-βRII expression and prevents TGF-β-inhibited growth.

Although the above demonstrates a role for cPKCβII in epithelial hyperproliferation via COX-2 activation, there is evidence that PKCs may be a permissive but not causal pathway initiating carcinogenesis. In this model, cPKCβII over expression leads to hyperproliferation but not to the formation of aberrant crypt foci or malignancy unless AOM is added. Second, work from our laboratory134 and others135, 136 localizes COX-2 expression early on in polyposis to stromal cells, not the epithelium. In both rodent models of colon cancer135, 136 as well as in sections from normal human colonic mucosa, hyperplastic polyps, and sporadic adenomas, COX-2 expression is limited to the subepithelial myofibroblasts, fibroblasts, macrophages, and endothelial cells.137 Expression in myofibroblasts or fibroblasts is the differentiating feature between normal mucosa and polyps. Thus, COX-2 expression does not appear in the epithelium until after the adenoma-carcinoma transition occurs. Using IL-1 as a model system for intestinal inflammation, which is known to predispose patients to colon cancer, we have demonstrated that IL-1-induced COX-2 expression requires PKC activation in human colonic myofibroblasts138, 139 (Figure 3C). IL-1-induced COX-2 expression is independent of classical and novel PKC activation and only requires aPKC (aPKCζ) activation. PKCζ activation generates reactive oxygen intermediates in myofibroblasts, which potentiates COX-2 transcription. These data, coupled with those in the colonic epithelial cells, suggest that the regulation of the COX-2 promoter is cell and stimulus type specific because cPKCBII played no role in IL-1-induced COX-2 expression in the myofibroblasts.

Our understanding of the role of PKC in gastrointestinal carcinogenesis is still rudimentary. Because of cell and stimulation differences, the same PKC isozymes may not play the same role in each experimental model. Furthermore, the cross talk between the isozymes is far from understood. For example, although cPKCβII over expression results in hyperproliferation, cPKCα and PKCδ are known inhibitors of cell proliferation.104, 106 Over expression of another aPKC, aPKCι, affects invasiveness of intestinal epithelial cells, thus increasing their metastatic potential.140 aPKCι activity is downstream of cPKCβII activation.141 Furthermore, cPKCα, although inhibiting proliferation, also contributes to the migratory activity of human colon carcinoma cells.142 Thus, PKCs play a role in carcinogenesis via mechanisms both dependent and independent of their effects on cell cycle regulation.

Back to Article Outline

Conclusions 

PKC activation mediates various signaling pathways critical for the formation, regulation, and maintenance of the gastrointestinal tract. The list of PKC-mediated gastrointestinal functions is growing every day, and the complexity and number of pathways that they regulate seems to be growing even faster. Ablation or inhibition of a single isozyme has been useful to demonstrate the importance of a specific PKC in a particular process. aPKCλ and PKC-3 are crucial in development, and nPKCϵ is often seen as a key regulator of transport and secretion. However, it is also becoming clear that PKCs can simultaneously activate opposing pathways to maintain a controlled response as seen in inflammation. Furthermore, interpretation of data is often limited because of the nonspecific effects of the inhibitors currently available. Thus, care must be taken in interpreting studies performed using pharmacologic inhibitors that often block an entire subfamily of isozymes. Although the use of nonpharmacologic approaches (ie, PKC knockout mice, gene ablation, or constitutively active mutants) ameliorates some of these issues, they cannot account for redundancy in pathways or adaptations to the mutation. Although powerful to examine the effect of dysregulating one family (pharmacologic) or one specific PKC (gene ablation), both methodologies are limited in their ability to address how PKCs work in harmony or in opposition to one another. Despite these technical limitations, the data demonstrate that PKCs are key regulators in both normal gastrointestinal function and disease. Their importance in the regulation of gastrointestinal development, secretion, absorption, motility, inflammation, and oncogenesis necessitate increasing our understanding of their regulation and impact on cellular response. Elucidating the regulatory mechanisms by which these kinases act as both affectors and effectors will open a new area of therapeutics in combating gastrointestinal disease.

Back to Article Outline

References 

  1. Johnson GL , Lapadat R . Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases . Science . 2002;298:1911–1912
  2. Dempsey EC, Newton AC, Mochly-Rosen D, Fields AP, Reyland ME, Insel PA, et al. Protein kinase C isozymes and the regulation of diverse cell responses . Am J Physiol Lung Cell Mol Physiol . 2000;279:L429–L438
  3. Martelli AM , Faenza I , Billi AM , Fala F , Cocco L , Manzoli L . Nuclear protein kinase C isoforms (key players in multiple cell functions?) . Histol Histopathol . 2003;18:1301–1312
  4. Martelli AM , Sang N , Borgatti P , Capitani S , Neri LM . Multiple biological responses activated by nuclear protein kinase C . J Cell Biochem . 1999;74:499–521
  5. Musashi M , Ota S , Shiroshita N . The role of protein kinase C isoforms in cell proliferation and apoptosis . Int J Hematol . 2000;72:12–19
  6. Newton AC . Protein kinase C (structure, function, and regulation) . J Biol Chem . 1995;270:28495–28498
  7. Toker A . Signaling through protein kinase C . Front Biosci . 1998;3:D1134–D1147
  8. Newton AC . Regulation of protein kinase C . Curr Opin Cell Biol . 1997;9:161–167
  9. Newton AC . Regulation of the ABC kinases by phosphorylation (protein kinase C as a paradigm) . Biochem J . 2003;370:361–371
  10. Borner C , Filipuzzi I , Wartmann M , Eppenberger U , Fabbro D . Biosynthesis and posttranslational modifications of protein kinase C in human breast cancer cells . J Biol Chem . 1989;264:13902–13909
  11. Tsutakawa SE , Medzihradszky KF , Flint AJ , Burlingame AL , Koshland DE . Determination of in vivo phosphorylation sites in protein kinase C . J Biol Chem . 1995;270:26807–26812
  12. Chou MM, Hou W, Johnson J, Graham LK, Lee MH, Chen CS, et al. Regulation of protein kinase C zeta by PI 3-kinase and PDK-1 . Curr Biol . 1998;8:1069–1077
  13. Dutil EM , Toker A , Newton AC . Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1) . Curr Biol . 1998;8:1366–1375
  14. Le Good JA , Ziegler WH , Parekh DB , Alessi DR , Cohen P , Parker PJ . Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1 . Science . 1998;281:2042–2045
  15. Srinivasan N , Bax B , Blundell TL , Parker PJ . Structural aspects of the functional modules in human protein kinase-C α deduced from comparative analyses . Proteins . 1996;26:217–235
  16. Orr JW , Newton AC . Intrapeptide regulation of protein kinase C . J Biol Chem . 1994;269:8383–8387
  17. Poole DP , Hunne B , Robbins HL , Furness JB . Protein kinase C isoforms in the enteric nervous system . Histochem Cell Biol . 2003;120:51–61
  18. Sossin WS , Schwartz JH . Ca(2+)-independent protein kinase Cs contain an amino-terminal domain similar to the C2 consensus sequence . Trends Biochem Sci . 1993;18:207–208
  19. Li C , Fultz ME , Wright GL . PKC-α shows variable patterns of translocation in response to different stimulatory agents . Acta Physiol Scand . 2002;174:237–246
  20. Battistella-Patterson AS , Fultz ME , Li C , Geng W , Norton M , Wright GL . PKCα translocation is microtubule-dependent in passaged smooth muscle cells . Acta Physiol Scand . 2000;170:87–97
  21. Haller H , Maasch C , Lindschau C , Brachmann M , Buchner K , Luft FC . Intracellular targeting and protein kinase C in vascular smooth muscle cells (specific effects of different membrane-bound receptors) . Acta Physiol Scand . 1998;164:599–609
  22. Haller H , Lindschau C , Maasch C , Olthoff H , Kurscheid D , Luft FC . Integrin-induced protein kinase Cα and Cϵ translocation to focal adhesions mediates vascular smooth muscle cell spreading . Circ Res . 1998;82:157–165
  23. Chang BY , Chiang M , Cartwright CA . The interaction of Src and RACK1 is enhanced by activation of protein kinase C and tyrosine phosphorylation of RACK1 . J Biol Chem . 2001;276:20346–20356
  24. Chang BY , Harte RA , Cartwright CA . RACK1 (a novel substrate for the Src protein-tyrosine kinase) . Oncogene . 2002;21:7619–7629
  25. Yang C , Kazanietz MG . Divergence and complexities in DAG signaling (looking beyond PKC) . Trends Pharmacol Sci . 2003;24:602–608
  26. Chang Q , Tepperman BL . The role of protein kinase C isozymes in TNF-α-induced cytotoxicity to a rat intestinal epithelial cell line . Am J Physiol Gastrointest Liver Physiol . 2001;280:G572–G583
  27. Schechtman D , Craske ML , Kheifets V , Meyer T , Schechtman J , Mochly-Rosen D . A critical intramolecular interaction for protein kinase C(epsilon) translocation . J Biol Chem . 2004;279:15831–15840
  28. Patterson RL , van Rossum DB , Barrow RK , Snyder SH . RACK1 binds to inositol 1,4,5-trisphosphate receptors and mediates Ca2+ release . PNAS . 2004;101:2328–2332
  29. Jans DA , Xiao CY , Lam MH . Nuclear targeting signal recognition (a key control point in nuclear transport?) . Bioessays . 2000;22:532–544
  30. Horne-Badovinac S, Lin D, Waldron S, Schwarz M, Mbamalu G, Pawson T, et al. Positional cloning of heart and soul reveals multiple roles for PKC lambda in zebra fish organogenesis . Curr Biol . 2001;11:1492–1502
  31. Leung B , Hermann GJ , Priess JR . Organogenesis of the Caenorhabditis elegans intestine . Dev Biol . 1999;216:114–134
  32. Peterson RT , Mably JD , Chen JN , Fishman MC . Convergence of distinct pathways to heart patterning revealed by the small molecule concentramide and the mutation heart-and-soul . Curr Biol . 2001;11:1481–1491
  33. Ward KW , Rogers EH , Hunter ES . Dysmorphogenic effects of a specific protein kinase C inhibitor during neurulation . Reprod Toxicol . 1998;12:525–534
  34. Yoo J, Nichols A, Mammen J, Calvo I, Song JC, Worrell RT, et al. Bryostatin-1 enhances barrier function in T84 epithelia through PKC-dependent regulation of tight junction proteins . Am J Physiol Cell Physiol . 2003;285:C300–C309
  35. Matter K , Balda MS . Signalling to and from tight junctions . Nat Rev Mol Cell Biol . 2003;4:225–236
  36. Chen ML , Pothoulakis C , LaMont JT . Protein kinase C signaling regulates ZO-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile toxin A . J Biol Chem . 2002;277:4247–4254
  37. Tabuse Y , Izumi Y , Piano F , Kemphues KJ , Miwa J , Ohno S . Atypical protein kinase C cooperates with PAR-3 to establish embryonic polarity in Caenorhabditis elegans . Development . 1998;125:3607–3614
  38. Wu SL , Staudinger J , Olson EN , Rubin CS . Structure, expression, and properties of an atypical protein kinase C (PKC3) from Caenorhabditis elegans. PKC3 is required for the normal progression of embryogenesis and viability of the organism . J Biol Chem . 1998;273:1130–1143
  39. Hiramatsu Y, Sekiguchi N, Hayashi M, Isshiki K, Yokota T, King GL, et al. Diacylglycerol production and protein kinase C activity are increased in a mouse model of diabetic embryopathy . Diabetes . 2002;51:2804–2810
  40. Berkes J , Viswanathan VK , Savkovic SD , Hecht G . Intestinal epithelial responses to enteric pathogens (effects on the tight junction barrier, ion transport, and inflammation) . Gut . 2003;52:439–451
  41. Savkovic SD , Koutsouris A , Hecht G . PKC ζ participates in activation of inflammatory response induced by enteropathogenic E coli . Am J Physiol Cell Physiol . 2003;285:C512–C521
  42. Crane JK , Oh JS . Activation of host cell protein kinase C by enteropathogenic Escherichia coli . Infect Immun . 1997;65:3277–3285
  43. Terres AM, Pajares JM, Hopkins AM, Murphy A, Moran A, Baird AW, et al. Helicobacter pylori disrupts epithelial barrier function in a process inhibited by protein kinase C activators . Infect Immun . 1998;66:2943–2950
  44. Wooten MW . Function for NF-κB in neuronal survival (regulation by atypical protein kinase C) . J Neurosci Res . 1999;58:607–611
  45. Webb PR , Wang KQ , Scheel-Toellner D , Pongracz J , Salmon M , Lord JM . Regulation of neutrophil apoptosis (a role for protein kinase C and phosphatidylinositol-3-kinase) . Apoptosis . 2000;5:451–458
  46. Brown JF , Chang Q , Soper BD , Tepperman BL . Protein kinase C mediates experimental colitis in the rat . Am J Physiol . 1999;276:G583–G590
  47. Trushin SA , Pennington KN , Algeciras-Schimnich A , Paya CV . Protein kinase C and calcineurin synergize to activate IκB kinase and NF-κB in T lymphocytes . J Biol Chem . 1999;274:22923–22931
  48. Touqui L , Alaoui-El-Azher M . Mammalian secreted phospholipases A2 and their pathophysiological significance in inflammatory diseases . Curr Mol Med . 2001;1:739–754
  49. Gukovskaya AS , Gukovsky I , Jung Y , Mouria M , Pandol SJ . Cholecystokinin induces caspase activation and mitochondrial dysfunction in pancreatic acinar cells. Roles in cell injury processes of pancreatitis . J Biol Chem . 2002;277:22595–22604
  50. Pongracz J , Webb P , Wang K , Deacon E , Lunn OJ , Lord JM . Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein kinase C-δ . J Biol Chem . 1999;274:37329–37334
  51. Kim YS, Ko J, Kim IS, Jang SW, Sung HJ, Lee HJ, et al. PKCδ-dependent cleavage and nuclear translocation of annexin A1 by phorbol 12-myristate 13-acetate . Eur J Biochem . 2003;270:4089–4094
  52. Sakanoue Y , Hatada T , Horai T , Shoji Y , Kusunoki M , Utsunomiya J . Protein kinase C activity of colonic mucosa in ulcerative colitis . Scand J Gastroenterol . 1992;27:275–280
  53. Jones BA , Rao YP , Stravitz RT , Gores GJ . Bile salt-induced apoptosis of hepatocytes involves activation of protein kinase C . Am J Physiol . 1997;272:G1109–G1115
  54. Tepperman BL , Chang Q , Soper BD . Protein kinase C mediates lipopolysaccharide- and phorbol-induced nitric-oxide synthase activity and cellular injury in the rat colon . J Pharmacol Exp Ther . 2000;295:1249–1257
  55. Carpenter L , Cordery D , Biden TJ . Protein kinase Cδ activation by interleukin-1β stabilizes inducible nitric-oxide synthase mRNA in pancreatic β-cells . J Biol Chem . 2001;276:5368–5374
  56. Chang Q , Soper BD , Yacyshyn BR , Tepperman BL . Alterations in protein kinase C isoforms in experimentally-induced colitis in the rat . Inflamm Res . 2000;49:27–35
  57. Banan A , Fields JZ , Talmage DA , Zhang L , Keshavarzian A . PKC-ζ is required in EGF protection of microtubules and intestinal barrier integrity against oxidant injury . Am J Physiol Gastrointest Liver Physiol . 2002;282:G794–G808
  58. Banan A , Fields JZ , Talmage DA , Zhang Y , Keshavarzian A . PKC-β1 mediates EGF protection of microtubules and barrier of intestinal monolayers against oxidants . Am J Physiol Gastrointest Liver Physiol . 2001;281:G833–G847
  59. Banan A , Fields JZ , Zhang LJ , Shaikh M , Farhadi A , Keshavarzian A . Zeta isoform of protein kinase C prevents oxidant-induced nuclear factor-κB activation and I-κBα degradation (a fundamental mechanism for epidermal growth factor protection of the microtubule cytoskeleton and intestinal barrier integrity) . J Pharmacol Exp Ther . 2003;307:53–66
  60. Banan A , Fields JZ , Zhang Y , Keshavarzian A . Key role of PKC and Ca2+ in EGF protection of microtubules and intestinal barrier against oxidants . Am J Physiol Gastrointest Liver Physiol . 2001;280:G828–G843
  61. Banan A , Zhang L , Fields JZ , Farhadi A , Talmage DA , Keshavarzian A . PKC-ζ prevents oxidant-induced iNOS upregulation and protects the microtubules and gut barrier integrity . Am J Physiol Gastrointest Liver Physiol . 2002;283:G909–G922
  62. Keshavarzian A, Banan A, Farhadi A, Komanduri S, Mutlu E, Zhang Y, et al. Increases in free radicals and cytoskeletal protein oxidation and nitration in the colon of patients with inflammatory bowel disease . Gut . 2003;52:720–728
  63. Gukovskaya AS, Hosseini S, Satoh A, Cheng JH, Nam KJ, Gukovsky I, et al. Ethanol differentially regulates NF-κB activation in pancreatic acinar cells through calcium and protein kinase C pathways . Am J Physiol Gastrointest Liver Physiol . 2004;286:G204–G213
  64. Williams JA . Intracellular signaling mechanisms activated by cholecystokinin-regulating synthesis and secretion of digestive enzymes in pancreatic acinar cells . Annu Rev Physiol . 2001;63:77–97
  65. Satoh A, Gukovskaya AS, Nieto JM, Cheng JH, Gukovsky I, Reeve JR, et al. PKC-δ and -ϵ regulate NF-κB activation induced by cholecystokinin and TNF-α in pancreatic acinar cells . Am J Physiol Gastrointest Liver Physiol . 2004;287:G582–G591
  66. Song JC , Hanson CM , Tsai V , Farokhzad OC , Lotz M , Matthews JB . Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms . Am J Physiol Cell Physiol . 2001;281:C649–C661
  67. Saksena S, Gill RK, Syed IA, Tyagi S, Alrefai WA, Ramaswamy K, et al. Inhibition of apical Cl−/OH− exchange activity in Caco-2 cells by phorbol esters is mediated by PKCϵ . Am J Physiol Cell Physiol . 2002;283:C1492–C1500
  68. Song JC , Hrnjez BJ , Farokhzad OC , Matthews JB . PKC-ϵ regulates basolateral endocytosis in human T84 intestinal epithelia (role of F-actin and MARCKS) . Am J Physiol . 1999;277:C1239–C1249
  69. Song JC , Rangachari PK , Matthews JB . Opposing effects of PKCα and PKCϵ on basolateral membrane dynamics in intestinal epithelia . Am J Physiol Cell Physiol . 2002;283:C1548–C1556
  70. Powell DW . Intestinal water and electrolyte transport . In:  Johnson LR editors. Physiology of the gastrointestinal tract . 2nd ed.. New York: Raven; 1987;p. 1267–1305
  71. Benedetti A , Strazzabosco M , Ng OC , Boyer JL . Regulation of activity and apical targeting of the Cl−/HCO3− exchanger in rat hepatocytes . Proc Natl Acad Sci U S A . 1994;91:792–796
  72. Chew CS , Zhou CJ , Parente JA . Ca2+-independent protein kinase C isoforms may modulate parietal cell HCl secretion . Am J Physiol . 1997;272:G246–G256
  73. Umar S , Sellin JH , Morris AP . Murine colonic mucosa hyperproliferation. II. PKC-β activation and cPKC-mediated cellular CFTR over expression . Am J Physiol Gastrointest Liver Physiol . 2000;278:G765–G774
  74. Anderson NG , Hanson PJ . Involvement of calcium-sensitive phospholipid-dependent protein kinase in control of acid secretion by isolated rat parietal cells . Biochem J . 1985;232:609–611
  75. Beil W , Mannschedel W , Sewing KF . Protein kinase C and parietal cell function . Biochem Biophys Res Commun . 1987;149:720–728
  76. Brown MR , Chew CS . Multiple effects of phorbol ester on secretory activity in rabbit gastric glands and parietal cells . Can J Physiol Pharmacol . 1987;65:1840–1847
  77. McKenna JP , Hanson PJ . Inhibition by Ro 31-8220 of acid secretory activity induced by carbachol indicates a stimulatory role for protein kinase C in the action of muscarinic agonists on isolated rat parietal cells . Biochem Pharmacol . 1993;46:583–588
  78. Tuo BG , Chow JY , Barrett KE , Isenberg JI . Protein kinase C potentiates cAMP-stimulated mouse duodenal mucosal bicarbonate secretion in vitro . Am J Physiol Gastrointest Liver Physiol . 2004;286:G814–G821
  79. Alrefai WA, Scaglione-Sewell B, Tyagi S, Wartman L, Brasitus TA, Ramaswamy K, et al. Differential regulation of the expression of Na(+)/H(+) exchanger isoform NHE3 by PKC-α in Caco-2 cells . Am J Physiol Cell Physiol . 2001;281:C1551–C1558
  80. Cavet ME , Akhter S , Murtazina R , Sanchez de Medina F , Tse CM , Donowitz M . Half-lives of plasma membrane Na(+)/H(+) exchangers NHE1-3 (plasma membrane NHE2 has a rapid rate of degradation) . Am J Physiol Cell Physiol . 2001;281:C2039–C2048
  81. Kandasamy RA , Yu FH , Harris R , Boucher A , Hanrahan JW , Orlowski J . Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways . J Biol Chem . 1995;270:29209–29216
  82. Lee-Kwon W , Kawano K , Choi JW , Kim JH , Donowitz M . Lysophosphatidic acid stimulates brush border Na+/H+ exchanger 3 (NHE3) activity by increasing its exocytosis by an NHE3 kinase A regulatory protein-dependent mechanism . J Biol Chem . 2003;278:16494–16501
  83. Mendez CF, Leibiger IB, Leibiger B, Hoy M, Gromada J, Berggren PO, et al. Rapid association of protein kinase C-ϵ with insulin granules is essential for insulin exocytosis . J Biol Chem . 2003;278:44753–44757
  84. Hong DH , Petrovics G , Anderson WB , Forstner J , Forstner G . Induction of mucin gene expression in human colonic cell lines by PMA is dependent on PKC-ϵ . Am J Physiol . 1999;277:G1041–G1047
  85. Nagashio Y , Hirohata Y , Akiyama T , Otsuki M . Dibutyltin dichloride modifies amylase release from isolated rat pancreatic acini . Pancreas . 2002;25:57–62
  86. Nozu F , Tsunoda Y , Ibitayo AI , Bitar KN , Owyang C . Involvement of RhoA and its interaction with protein kinase C and Src in CCK-stimulated pancreatic acini . Am J Physiol . 1999;276:G915–G923
  87. Guyton AC . Secretory functions of the alimentary tract . Philadelphia: W.B. Saunders; 1991;
  88. Evers BM . Endocrine gene neurotensin (molecular mechanisms and a model of intestinal differentiation) . World J Surg . 2002;26:799–805
  89. Li J , Hellmich MR , Greeley GH , Townsend CM , Evers BM . Phorbol ester-mediated neurotensin secretion is dependent on the PKC-α and -δ isoforms . Am J Physiol Gastrointest Liver Physiol . 2002;283:G1197–G1206
  90. Murthy KS , Zhou H , Grider JR , Brautigan DL , Eto M , Makhlouf GM . Differential signalling by muscarinic receptors in smooth muscle (m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway) . Biochem J . 2003;374:145–155
  91. Sarna SK . Neuronal locus and cellular signaling for stimulation of ileal giant migrating and phasic contractions . Am J Physiol Gastrointest Liver Physiol . 2003;284:G789–G797
  92. Murthy KS , Grider JR , Kuemmerle JF , Makhlouf GM . Sustained muscle contraction induced by agonists, growth factors, and Ca(2+) mediated by distinct PKC isozymes . Am J Physiol Gastrointest Liver Physiol . 2000;279:G201–G210
  93. Sohn UD, Cao W, Tang DC, Stull JT, Haeberle JR, Wang CL, et al. Myosin light chain kinase- and PKC-dependent contraction of LES and esophageal smooth muscle . Am J Physiol Gastrointest Liver Physiol . 2001;281:G467–G478
  94. Murthy KS , Yee YS , Grider JR , Makhlouf GM . Phorbol-stimulated Ca(2+) mobilization and contraction in dispersed intestinal smooth muscle cells . J Pharmacol Exp Ther . 2000;294:991–996
  95. Cao W , Sohn UD , Bitar KN , Behar J , Biancani P , Harnett KM . MAPK mediates PKC-dependent contraction of cat esophageal and lower esophageal sphincter circular smooth muscle . Am J Physiol Gastrointest Liver Physiol . 2003;285:G86–G95
  96. Shi XZ , Sarna SK . Impairment of Ca(2+) mobilization in circular muscle cells of the inflamed colon . Am J Physiol Gastrointest Liver Physiol . 2000;278:G234–G242
  97. Ali I , Sarna SK . Selective modulation of PKC isozymes by inflammation in canine colonic circular muscle cells . Gastroenterology . 2002;122:483–494
  98. Martinez-Palomo A . Ultrastructural modifications of intercellular junctions between tumor cells . In Vitro . 1970;6:15–20
  99. Black JD . Protein kinase C isozymes in colon carcinogenesis (guilt by omission) . Gastroenterology . 2001;120:1868–1872
  100. Jiang YH , Aukema HM , Davidson LA , Lupton JR , Chapkin RS . Localization of protein kinase C isozymes in rat colon . Cell Growth Differ . 1995;6:1381–1386
  101. Kahl-Rainer P , Sedivy R , Marian B . Protein kinase C tissue localization in human colonic tumors suggests a role for adenoma growth control . Gastroenterology . 1996;110:1753–1759
  102. Verstovsek G , Byrd A , Frey MR , Petrelli NJ , Black JD . Colonocyte differentiation is associated with increased expression and altered distribution of protein kinase C isozymes . Gastroenterology . 1998;115:75–85
  103. Black JD . Protein kinase C-mediated regulation of the cell cycle . Front Biosci . 2000;5:D406–D423
  104. Frey MR , Saxon ML , Zhao X , Rollins A , Evans SS , Black JD . Protein kinase C isozyme-mediated cell cycle arrest involves induction of p21(waf1/cip1) and p27(kip1) and hypophosphorylation of the retinoblastoma protein in intestinal epithelial cells . J Biol Chem . 1997;272:9424–9435
  105. Saxon ML , Zhao X , Black JD . Activation of protein kinase C isozymes is associated with post-mitotic events in intestinal epithelial cells in situ . J Cell Biol . 1994;126:747–763
  106. Cerda SR, Bissonnette M, Scaglione-Sewell B, Lyons MR, Khare S, Mustafi R, et al. PKC-δ inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases apoptosis in CaCo-2 cells . Gastroenterology . 2001;120:1700–1712
  107. Chiu T , Santiskulvong C , Rozengurt E . ANG II stimulates PKC-dependent ERK activation, DNA synthesis, and cell division in intestinal epithelial cells . Am J Physiol Gastrointest Liver Physiol . 2003;285:G1–G11
  108. Guha S , Lunn JA , Santiskulvong C , Rozengurt E . Neurotensin stimulates protein kinase C-dependent mitogenic signaling in human pancreatic carcinoma cell line PANC-1 . Cancer Res . 2003;63:2379–2387
  109. Baum CL , Wali RK , Sitrin MD , Bolt MJ , Brasitus TA . 1,2-Dimethylhydrazine-induced alterations in protein kinase C activity in the rat preneoplastic colon . Cancer Res . 1990;50:3915–3920
  110. Guillem JG, O’Brian CA, Fitzer CJ, Forde KA, LoGerfo P, Treat M, et al. Altered levels of protein kinase C and Ca2+-dependent protein kinases in human colon carcinomas . Cancer Res . 1987;47:2036–2039
  111. Kahl-Rainer P , Karner-Hanusch J , Weiss W , Marian B . Five of six protein kinase C isoenzymes present in normal mucosa show reduced protein levels during tumor development in the human colon . Carcinogenesis . 1994;15:779–782
  112. Kopp R , Noelke B , Sauter G , Schildberg FW , Paumgartner G , Pfeiffer A . Altered protein kinase C activity in biopsies of human colonic adenomas and carcinomas . Cancer Res . 1991;51:205–210
  113. Gokmen-Polar Y , Murray NR , Velasco MA , Gatalica Z , Fields AP . Elevated protein kinase C βII is an early promotive event in colon carcinogenesis . Cancer Res . 2001;61:1375–1381
  114. Ohba M, Ishino K, Kashiwagi M, Kawabe S, Chida K, Huh NH, et al. Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the η and δ isoforms of protein kinase C . Mol Cell Biol . 1998;18:5199–5207
  115. Wali RK, Frawley BP, Hartmann S, Roy HK, Khare S, Scaglione-Sewell BA, et al. Mechanism of action of chemoprotective ursodeoxycholate in the azoxymethane model of rat colonic carcinogenesis (potential roles of protein kinase C-α, -β II, and -ζ) . Cancer Res . 1995;55:5257–5264
  116. Kinzler KW , Vogelstein B . Lessons from hereditary colorectal cancer . Cell . 1996;87:159–170
  117. Kinzler KW , Vogelstein B . Life (and death) in a malignant tumour . Nature . 1996;379:19–20
  118. Wong NA , Pignatelli M . Beta-catenin—a linchpin in colorectal carcinogenesis? . Am J Pathol . 2002;160:389–401
  119. Pongracz J , Clark P , Neoptolemos JP , Lord JM . Expression of protein kinase C isoenzymes in colorectal cancer tissue and their differential activation by different bile acids . Int J Cancer . 1995;61:35–39
  120. Murray NR , Davidson LA , Chapkin RS , Clay Gustafson W , Schattenberg DG , Fields AP . Overexpression of protein kinase C βII induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis . J Cell Biol . 1999;145:699–711
  121. Yu W, Murray NR, Weems C, Chen L, Guo H, Ethridge R, et al. Role of cyclooxygenase 2 in protein kinase C β II-mediated colon carcinogenesis . J Biol Chem . 2003;278:11167–11174
  122. Chang WC , Chapkin RS , Lupton JR . Predictive value of proliferation, differentiation and apoptosis as intermediate markers for colon tumorigenesis . Carcinogenesis . 1997;18:721–730
  123. Deschner EE , Lytle JS , Wong G , Ruperto JF , Newmark HL . The effect of dietary omega-3 fatty acids (fish oil) on azoxymethanol-induced focal areas of dysplasia and colon tumor incidence . Cancer . 1990;66:2350–2356
  124. Murray NR, Weems C, Chen L, Leon J, Yu W, Davidson LA, et al. Protein kinase C βII and TGFβRII in omega-3 fatty acid-mediated inhibition of colon carcinogenesis . J Cell Biol . 2002;157:915–920
  125. Reddy BS , Burill C , Rigotty J . Effect of diets high in omega-3 and omega-6 fatty acids on initiation and postinitiation stages of colon carcinogenesis . Cancer Res . 1991;51:487–491
  126. Takahashi M , Minamoto T , Yamashita N , Yazawa K , Sugimura T , Esumi H . Reduction in formation and growth of 1,2-dimethylhydrazine-induced aberrant crypt foci in rat colon by docosahexaenoic acid . Cancer Res . 1993;53:2786–2789
  127. Davidson LA, Brown RE, Chang WC, Morris JS, Wang N, Carroll RJ, et al. Morphodensitometric analysis of protein kinase C β(II) expression in rat colon (modulation by diet and relation to in situ cell proliferation and apoptosis) . Carcinogenesis . 2000;21:1513–1519
  128. Pajari AM , Oikarinen S , Grasten S , Mutanen M . Diets enriched with cereal brans or inulin modulate protein kinase C activity and isozyme expression in rat colonic mucosa . Br J Nutr . 2000;84:635–643
  129. Mutanen M , Pajari AM , Oikarinen SI . Beef induces and rye bran prevents the formation of intestinal polyps in Apc(Min) mice (relation to β-catenin and PKC isozymes) . Carcinogenesis . 2000;21:1167–1173
  130. Du BR . COX-2 in large bowel cancer (a one-sided story) . Gut . 1999;45:636–637
  131. Prescott SM . Is cyclooxygenase-2 the α and the ω in cancer? . J Clin Invest . 2000;105:1511–1513
  132. Sheehan KM, Sheahan K, O’Donoghue DP, MacSweeney F, Conroy RM, Fitzgerald DJ, et al. The relationship between cyclooxygenase-2 expression and colorectal cancer . JAMA . 1999;282:1254–1257
  133. Steinbach G, Lynch PM, Phillips RK, Wallace MH, Hawk E, Gordon GB, et al. The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis . N Engl J Med . 2000;342:1946–1952
  134. Adegboyega PA , Mifflin RC , DiMari JF , Saada JI , Powell DW . Immunohistochemical study of myofibroblasts in normal colonic mucosa, hyperplastic polyps, and adenomatous colorectal polyps . Arch Pathol Lab Med . 2002;126:829–836
  135. Shattuck-Brandt RL , Varilek GW , Radhika A , Yang F , Washington MK , DuBois RN . Cyclooxygenase 2 expression is increased in the stroma of colon carcinomas from IL-10−/− mice . Gastroenterology . 2000;118:337–345
  136. Oshima M, Dinchuk JE, Kargman SL, Oshima H, Hancock B, Kwong E, et al. Suppression of intestinal polyposis in ApcΔ716 knockout mice by inhibition of cyclooxygenase 2 (COX-2) . Cell . 1996;87:803–809
  137. Adegboyega PA , Oolade O , Saada J , Mifflin RC , Di Mari JF , Powell DW . Subepithelial myofibroblasts express COX-2 in colorectal tubular adenomas . Clin Cancer Res . 2004;10:5870–5879
  138. Di Mari JF , Mifflin RC , Adegboyega PA , Saada JI , Powell DW . IL-1α-induced COX-2 expression in human intestinal myofibroblasts is dependent on a PKCζ-ROS pathway . Gastroenterology . 2003;124:1855–1865
  139. Mifflin RC , Saada JI , Di Mari JF , Adegboyega PA , Valentich JD , Powell DW . Regulation of COX-2 expression in human intestinal myofibroblasts (mechanisms of IL-1-mediated induction) . Am J Physiol Cell Physiol . 2002;282:C824–C834
  140. Murray NR, Jamieson L, Yu W, Zhang J, Gokmen-Polar Y, Sier D, et al. Protein kinase Cι is required for Ras transformation and colon carcinogenesis in vivo . J Cell Biol . 2004;164:797–802
  141. Zhang J , Anastasiadis PZ , Liu Y , Aubrey Thompson E , Fields AP . Protein kinase C β II induces cell invasion through a RAS/MEK-, PKC /RAC 1-dependent signaling pathway . J Biol Chem . 2004;279:22118–22123
  142. Masur K , Lang K , Niggemann B , Zanker KS , Entschladen F . High PKC α and low E-cadherin expression contribute to high migratory activity of colon carcinoma cells . Mol Biol Cell . 2001;12:1973–1982

PII: S0016-5085(04)01747-0

doi:10.1053/j.gastro.2004.09.078

Gastroenterology
Volume 128, Issue 7 , Pages 2131-2146, June 2005

Article Tools

* Image Usage & Resolution