Recent findings from our group, obtained on experimental in vivo and ex vivo models of pancreatitis, reveal that this disease causes a profound dysfunction of key cellular organelles, lysosomes and mitochondria. We found that autophagy, the main cellular degradative, lysosome-driven process, is activated but also impaired in acute pancreatitis because of its’ inefficient progression/resolution (flux) resulting from defective function of lysosomes. One mechanism underlying the lysosomal dysfunction in pancreatitis is abnormal processing (maturation) and activation of cathepsins, major lysosomal hydrolases; another is a decrease in pancreatic levels of key lysosomal membrane proteins LAMP-1 and LAMP-2. Our data indicate that lysosomal dysfunction plays an important initiating role in pancreatitis pathobiology. The impaired autophagy mediates vacuole accumulation in acinar cells; furthermore, the abnormal maturation and activation of cathepsins leads to increase in intra-acinar trypsin, the hallmark of pancreatitis; and LAMP-2 deficiency causes inflammation and acinar cell necrosis. Thus, the autophagic and lysosomal dysfunctions mediate key pathologic responses of pancreatitis. On the other hand, we showed that pancreatitis causes acinar cell mitochondria depolarization, mediated by the permeability transition pore (PTP). Genetic (via deletion of cyclophilin D) inactivation of PTP prevents mitochondrial depolarization and greatly ameliorates the pathologic responses of pancreatitis. Further, our data suggest that mitochondrial damage, by stimulating autophagy, increases the demand for efficient lysosomal degradation and therefore aggravates the pathologic consequences of lysosomal dysfunction. Thus, the combined autophagic, lysosomal and mitochondrial dysfunctions are key to the pathogenesis of pancreatitis.
lysosome; cathepsin; LAMP protein; permeability transition pore; pancreatic mitochondria
Acute pancreatitis is an inflammatory disease of exocrine pancreas that carries considerable morbidity and mortality; its pathophysiology remains poorly understood. During the past decade, new insights have been gained into signaling pathways and molecules that mediate the inflammatory response of pancreatitis and death of acinar cells (the main exocrine pancreas cell type). By contrast, much less is known about the acinar cell organellar damage in pancreatitis and how it contributes to the disease pathogenesis.
This review summarizes recent findings from our group, obtained on experimental in vivo and ex vivo models, which reveal disordering of key cellular organelles, namely, mitochondria, autophagosomes, and lysosomes, in pancreatitis. Our results indicate a critical role for mitochondrial permeabilization in determining the balance between apoptosis and necrosis in pancreatitis, and thus the disease severity. We further investigate how the mitochondrial dysfunction (and hence acinar cell death) is regulated by Ca2+, reactive oxygen species, and Bcl-xL, in relation to specific properties of pancreatic mitochondria. Our results also reveal that autophagy, the principal cellular degradative, lysosome-driven pathway, is impaired in pancreatitis due to inefficient lysosomal function, and that impaired autophagy mediates two key pathological responses of pancreatitis—accumulation of vacuoles in acinar cells and the abnormal, intra-acinar activation of digestive enzymes such as trypsinogen.
Critical Issues and Future Directions
The findings discussed in this review indicate critical roles for mitochondrial and autophagic/lysosomal dysfunctions in the pathogenesis of pancreatitis and delineate directions for detailed investigations into the molecular events that underlie acinar cell organellar damage. Antioxid. Redox Signal. 15, 2699–2710.
Cigarette smoking has been linked to many diseases, including pancreatic cancer and more recently, pancreatitis.
Electronic searches of primarily PubMed from 1990 to August 2011 were conducted and only articles published in English were reviewed. Original articles and reviews were selected based on screening of article abstracts and their relevance to tobacco smoking, its components, nicotine and its metabolites, and their effects particularly on the pancreas.
Smoking may affect the risk of developing chronic pancreatitis or its progression. Smoking may also affect the risk for developing acute pancreatitis. Its effects in pancreatitis appear to be dose dependent and its effects may be alcohol independent but synergize with alcohol.
Specific constituents of cigarette smoke, including nicotine and its metabolites, could mediate effects on the pancreas.
Acute and chronic pancreatitis; Zymogen activation; Cellular injury; Nicotine; NNK; Tobacco smoking
Alcohol abuse is the leading etiologic factor of pancreatitis, although many heavy drinkers do not develop pancreatic damage. Alcohol promotes pancreatitis through a combination of remote (e.g., increased gut permeability to bacterial products such as lipopolysaccharide) and more proximal effects (e.g., altered pancreatic cholinergic inputs), including oxidative damage at the level of the pancreatic acinar cell. Recent evidence indicates that alcohol exposure to rodents disturbs proteostasis in the exocrine pancreas, an effect counterbalanced by homeostatic processes that include both the unfolded protein response (UPR) and autophagy. A corollary to this notion is that pancreatitis results when adaptive responses are insufficiently robust to alleviate the cellular stress caused by alcohol.
acinar cell; alcohol abuse; pancreas; ERAD; UPR; XBP1
Alcohol abuse is one of the most common causes of pancreatitis. The risk of developing alcohol-induced pancreatitis is related to the amount and duration of drinking. However, only a small portion of heavy drinkers develop disease, indicating that other factors (genetic, environmental or dietary) contribute to disease initiation. Epidemiologic studies suggest roles for cigarette smoking and dietary factors in the development of alcoholic pancreatitis. The mechanisms underlying alcoholic pancreatitis are starting to be understood. Studies from animal models are revealing that alcohol sensitizes the pancreas to key pathobiologic processes that are involved in pancreatitis. Current studies are focused on the mechanisms responsible for the sensitizing effect of alcohol; recent findings reveal disordering of key cellular organelles including endoplasmic reticulum, mitochondria, and lysosomes. As our understanding of alcohol’s effects continue to advance to the level of molecular mechanisms, insights into potential therapeutic strategies will emerge providing opportunities for clinical benefit.
Alcohol abuse is a common cause of both acute and chronic pancreatitis. There is a wide spectrum of pancreatic manifestations in heavy drinkers from no apparent disease in most individuals to acute inflammatory and necrotizing pancreatitis in a minority of individuals with some progressing to chronic pancreatitis characterized by replacement of the gland by fibrosis and chronic inflammation. Both smoking and African-American ethnicity are associated with increased risk of alcoholic pancreatitis. In this review we describe how our recent studies demonstrate that ethanol feeding in rodents causes oxidative stress in the endoplasmic reticulum (ER) of the digestive enzyme synthesizing acinar cell of the exocrine pancreas. This ER stress is attenuated by a robust unfolded protein response (UPR) involving X-box binding protein-1 (XBP1) in the acinar cell. When the UPR activation is prevented by genetic reduction in XBP1, ethanol feeding causes significant pathological responses in the pancreas. These results suggest that the reason most individuals who drink alcohol heavily do not get significant pancreatic disease is because the pancreas mounts an adaptive UPR to attenuate the ER stress that ethanol causes. We hypothesize that disease in the pancreas results when the UPR is insufficiently robust to alleviate the ER stress caused by alcohol abuse.
Pancreas; Pancreatitis; Unfolded protein response; Alcohol; Smoking
Inflammation and acinar cell necrosis are two major pathological responses of acute pancreatitis, a serious disorder with no current therapies directed to its molecular pathogenesis. Serine/threonine protein kinase D family, which includes PKD/PKD1, PKD2, and PKD3, has been increasingly implicated in the regulation of multiple physiological and pathophysiological effects. We recently reported that PKD/PKD1, the predominant PKD isoform expressed in rat pancreatic acinar cells, mediates early events of pancreatitis including NF-κB activation and inappropriate intracellular digestive enzyme activation. In current studies, we investigated the role and mechanisms of PKD/PKD1 in the regulation of necrosis in pancreatic acinar cells by using two novel small molecule PKD inhibitors CID755673 and CRT0066101 and molecular approaches in in vitro and in vivo experimental models of acute pancreatitis. Our results demonstrated that both CID755673 and CRT0066101 are PKD-specific inhibitors and that PKD/PKD1 inhibition by either the chemical inhibitors or specific PKD/PKD1 siRNAs attenuated necrosis while promoting apoptosis induced by pathological doses of cholecystokinin-octapeptide (CCK) in pancreatic acinar cells. Conversely, up-regulation of PKD expression in pancreatic acinar cells increased necrosis and decreased apoptosis. We further showed that PKD/PKD1 regulated several key cell death signals including inhibitors of apoptotic proteins, caspases, receptor-interacting protein kinase 1 to promote necrosis. PKD/PKD1 inhibition by CID755673 significantly ameliorated necrosis and severity of pancreatitis in an in vivo experimental model of acute pancreatitis. Thus, our studies indicate that PKD/PKD1 is a key mediator of necrosis in acute pancreatitis and that PKD/PKD1 may represent a potential therapeutic target in acute pancreatitis.
pancreatic acinar cells; CCK; CID755673; CRT0066101; apoptosis; necrosis
AIM: To determine the effect of ellagic acid on apop-tosis and proliferation in pancreatic cancer cells and to determine the mechanism of the pro-survival effects of ellagic acid.
METHODS: The effect of ellagic acid on apoptosis was assessed by measuring Phosphatidylserine externalization, caspase activity, mitochondrial membrane potential and DNA fragmentation; and proliferation by measuring DNA thymidine incorporation. Mitochondrial membrane potential was measured in permeabilized cells, and in isolated mitochondria. Nuclear factor κB (NF-κB) activity was measured by electromobility shift assay (EMSA).
RESULTS: We show that ellagic acid, a polyphenolic compound in fruits and berries, at concentrations 10 to 50 mmol/L stimulates apoptosis in human pancreatic adenocarcinoma cells. Further, ellagic acid decreases proliferation by up to 20-fold at 50 mmol/L. Ellagic acid stimulates the mitochondrial pathway of apoptosis associated with mitochondrial depolarization, cytochrome C release, and the downstream caspase activation. Ellagic acid does not directly affect mitochondria. Ellagic acid dose-dependently decreased NF-κB binding activity. Furthermore, inhibition of NF-κB activity using IkB wild type plasmid prevented the effect of ellagic acid on apoptosis.
CONCLUSION: Our data indicate that ellagic acid stimulates apoptosis through inhibition of the prosu-rvival transcription factor NF-κB.
Ellagic acid; Nuclear factor-κB; Apoptosis; Pancreatic cancer
BACKGROUND & AIMS
Endoplasmic reticulum (ER) stress responses (collectively known the unfolded protein response, UPR) have important roles in several human disorders, but their contribution to alcoholic pancreatitis is not known. We investigated the role of X box-binding protein 1 (XBP1), an UPR regulator, in prevention of alcohol-induced ER stress in the exocrine pancreas.
Wild-type and Xbp1+/− mice were fed control or ethanol diets for 4 weeks. Pancreatic tissue samples were then examined by light and electron microscopy to determine pancreatic alterations; UPR regulators were analyzed biochemically.
In wild-type mice, ethanol activated a UPR, increasing pancreatic levels of XBP1 and XBP1 targets such as protein disulfide isomerase (PDI). In these mice, pancreatic damage was minor. In ethanol-fed Xbp1+/− mice, XBP1 and PDI levels were significantly lower than in ethanol-fed, wild-type mice. The combination of XBP1 deficiency and ethanol feeding reduced expression of regulators of ER function and the upregulation of pro-apoptotic signals. Moreover, ethanol feeding induced oxidation of PDI, which might compromise PDI-mediated disulfide bond formation during ER protein folding. In ethanol-fed Xbp1+/− mice, ER stress was associated with disorganized and dilated ER, loss of zymogen granules, accumulation of autophagic vacuoles, and increased acinar cell death.
Chronic ethanol feeding causes oxidative ER stress, which activates a UPR and increases XBP1 levels and activity. A defective UPR, due to XBP1 deficiency, results in ER dysfunction and acinar cell pathology.
alcohol disease; transcription factors; ER protein folding; organelle
The mechanisms initiating pancreatitis in patients with chronic alcohol abuse are poorly understood. Although alcohol feeding has been previously suggested to alter cholinergic pathways, the effects of these cholinergic alterations in promoting pancreatitis have not been characterized. For the present study, we determined the role of the cholinergic system in ethanol-induced sensitizing effects on cerulein pancreatitis.
Rats were pair fed control and ethanol-containing Lieber-DeCarli diets for 6 weeks followed by parenteral administration of four hourly intraperitoneal injections of the cholecystokinin analogue, cerulein at 0.5 μg/Kg. This dose of cerulein was selected because it caused pancreatic injury in ethanol-fed but not in control-fed rats. Pancreatitis was preceded by treatment with the muscarinic receptor antagonist atropine or by bilateral subdiaphragmatic vagotomy. Measurement of pancreatic pathology included serum lipase activity, pancreatic trypsin and caspase-3 activities, and markers of pancreatic necrosis, apoptosis and autophagy. In addition, we measured the effects of ethanol feeding on pancreatic acetylcholinesterase activity and pancreatic levels of the muscarinic acetylcholine receptors m1 and m3. Finally, we examined the synergistic effects of ethanol and carbachol on inducing acinar cell damage.
We found that atropine blocked almost completely pancreatic pathology caused by cerulein administration in ethanol fed rats, while vagotomy was less effective. Ethanol feeding did not alter expression levels of cholinergic muscarinic receptors in the pancreas but significantly decreased pancreatic acetylcholinesterase activity, suggesting that acetylcholine levels and cholinergic input within the pancreas can be higher in ethanol fed rats. We further found that ethanol treatment of pancreatic acinar cells augmented pancreatic injury responses caused by the cholinergic agonist, carbachol.
These results demonstrate key roles for the cholinergic system in the mechanisms of alcoholic pancreatitis.
Pancreas; alcohol feeding; cerulein; cholinergic pathways; acetylcholinesterase
The exocrine pancreas has the greatest protein synthetic capacity of any mammalian organ and is challenged with the synthesis, processing and transporting a large load of digestive enzymes. Based on recent findings we present a hypothesis proposing that mutations in the digestive enzymes and environmental risks impacting the pancreas (i.e., alcohol abuse, smoking, metabolic disorders, and drugs) cause endoplasmic reticulum (ER) stress. We review recent findings showing that in normal pancreas the ER stress resulting from alcohol abuse leads to an adaptive unfolded protein response (UPR) allowing for maintenance of protein synthesis, processing, and transport. However, when key pathways necessary for the adaptive UPR are altered, the exocrine cell of the pancreas is unable to maintain these processes and cellular pathology results. These findings may explain why some individuals with alcohol abuse disorders develop organ injury and disease while most do not. Further, the findings allow us to hypothesize that the UPR in the exocrine pancreas adapts the protein synthetic machinery of the ER stress resulting from mutational and environmental stressors. When the ability of the UPR to adapt to the stressors is exceeded, pathologic pathways and disease develop.
UPR; pancreas; gastrointestinal; research; exocrine; pancreatic function
Isoforms of protein kinase C (PKC) have been shown to modulate some cellular responses such as pathological secretion and generation of inflammatory mediators during acute pancreatitis (AP). We propose that PKC also participates in premature zymogen activation within the pancreatic acinar cell, a key event in the initiation of AP. This hypothesis was examined in in vivo and cellular models of caerulein-induced AP using PKC activators and inhibitors. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA, 200 nM), a known activator of PKC, enhanced zymogen activation at both 0.1 nM and 100 nM caerulein, concentrations which mimic physiological and supraphysiological effects of the hormone cholecystokinin, respectively, in preparations of pancreatic acinar cells. Isoform-specific PKC inhibitors for PKC-δ and PKC-ε reduced supraphysiological caerulein-induced zymogen activation. Using a cell-free reconstitution system, we showed that inhibition of PKC-δ and -ε, reduced zymogen activation in both zymogen granule-enriched and microsomal fractions. In dispersed acinar cells, 100 nM caerulein stimulation caused PKC-δ and -ε isoform translocation to microsomal membranes using cell fractionation and immunoblot analysis. PKC translocation was confirmed with in vivo studies and immunofluorescence microscopy in pancreatic tissues from rats treated with or without 100 nM caerulein. PKC-ε redistributed from an apical to a supranuclear region following caerulein administration. The signal for PKC-ε overlapped with granule membrane protein, GRAMP-92, an endosomal/lysosomal marker, in a supranuclear region where zymogen activation takes place. These results indicate that PKC-δ and -ε isoforms translocate to specific acinar cell compartments and modulate zymogen activation.
translocation; PKC activator phorbol ester; PKC inhibitor
To define the role of protein kinase C δ (PKC δ) in acinar cell responses to the hormone cholecystokinin-8 (CCK) using isoform-specific inhibitors and a previously unreported genetic deletion model.
Pancreatic acinar cells were isolated from 1) rat, and pre-treated with a PKC-δ-specific inhibitor or 2) PKC δ deficient and wild type mice. Isolated cells were stimulated with CCK (0.001-100 nM) and cell responses measured.
The PKC δ inhibitor did not affect stimulated amylase secretion from rat pancreatic acinar cells. CCK stimulation induced a typical biphasic dose response curve for amylase secretion in acinar cells isolated from both PKC δ -/- and wild type mice, with maximal stimulation at 10 pM CCK. CCK (100 nM) induced zymogen and NFkB activation in both PKC δ -/- and wild type mice, although it was up to 50% less in PKC δ -/-.
In contrast to previous studies, this study has used specific and complimentary approaches to examine PKC δ mediated acinar cell responses. We could not confirm that it mediates amylase release, but corroborated its role in the early stages of acute pancreatitis.
Physiological stimulation of pancreatic acinar cells by cholecystokinin and acetylcholine activate a spatial-temporal pattern of cytosolic [Ca+2] changes that are regulated by a coordinated response of inositol 1,4,5-trisphosphate receptors (IP3Rs), ryanodine receptors (RyRs) and calcium-induced calcium release (CICR). For the present study, we designed experiments to determine the potential role of Bcl-2 proteins in these patterns of cytosolic [Ca+2] responses. We used small molecule inhibitors that disrupt the interactions between prosurvival Bcl-2 proteins (i.e. Bcl-2 and Bcl-xl) and proapoptotic Bcl-2 proteins (i.e. Bax) and fluorescence microfluorimetry techniques to measure both cytosolic [Ca+2] and endoplasmic reticulum [Ca+2]. We found that the inhibitors of Bcl-2 protein interactions caused a slow and complete release of intracellular agonist-sensitive stores of calcium. The release was attenuated by inhibitors of IP3Rs and RyRs and substantially reduced by strong [Ca2+] buffering. Inhibition of IP3Rs and RyRs also dramatically reduced activation of apoptosis by BH3I-2′. CICR induced by different doses of BH3I-2′ in Bcl-2 overexpressing cells was markedly decreased compared with control. The results suggest that Bcl-2 proteins regulate calcium release from the intracellular stores and suggest that the spatial-temporal patterns of agonist-stimulated cytosolic [Ca+2] changes are regulated by differential cellular distribution of interacting pairs of prosurvival and proapoptotic Bcl-2 proteins.
Pancreas; Pancreatic acinar cell; Acetylcholine; Transport; Signal transduction; Cell death
Background & Aims
Ethanol abuse can lead to hepatic steatosis and evolve into cirrhosis and hepatocellular carcinoma. Pigment epithelium-derived factor (PEDF) is a multifunctional secreted glycoprotein that is expressed by hepatocytes. Proteomic, experimental, and clinical studies implicate PEDF’s role in lipid regulation. Because matrix metalloproteinase (MMP)-2/9 activity regulates PEDF levels, we investigated whether PEDF degradation by MMPs has a permissive role in ethanol-induced hepatic steatosis.
PEDF levels were examined in liver biopsy specimens from patients with ethanol-induced steatosis. Hepatic PEDF levels and MMP activity were assessed in 2 animal models of ethanol feeding (rats on an alcohol-containing liquid diet and mice given intragastric infusion of ethanol). The consequences of PEDF depletion in the liver were examined in PEDF-null mice.
Liver biopsy samples from patients with ethanol-induced steatosis had reduced PEDF levels, compared with normal liver samples. Ethanol-fed animals had histologic steatosis and increased liver triglyceride content (P < .05), as well as reduced levels of hepatic PEDF and increased MMP-2/9 activity. Ethanol-exposed hepatic lysates degraded PEDF in a MMP-2/9-dependent manner, and liver sections demonstrated abundant MMP-2/9 activity in situ. Addition of recombinant PEDF to PEDF-null hepatocytes, reduced their triglyceride content.
Ethanol exposure leads to marked loss of hepatic PEDF in human livers and in 2 animal models of ethanol feeding. Loss of PEDF contributes to the accumulation of lipids in ethanol-induced hepatic steatosis.
The pathogenic mechanisms underlying acute pancreatitis are not clear. Two key pathologic acinar cell responses of this disease are vacuole accumulation and trypsinogen activation. We show here that both result from defective autophagy, by comparing the autophagic responses in rodent models of acute pancreatitis to physiologic autophagy triggered by fasting. Pancreatitis-induced vacuoles in acinar cells were greater in number and much larger than those induced with fasting. Degradation of long-lived proteins, a measure of autophagic efficiency, was markedly inhibited in in vitro pancreatitis, while it was stimulated by acinar cell starvation. Further, processing of the lysosomal proteases cathepsin L (CatL) and CatB into their fully active, mature forms was reduced in pancreatitis, as were their activities in the lysosome-enriched subcellular fraction. These findings indicate that autophagy is retarded in pancreatitis due to deficient lysosomal degradation caused by impaired cathepsin processing. Trypsinogen activation occurred in pancreatitis but not with fasting and was prevented by inhibiting autophagy. A marker of trypsinogen activation partially localized to autophagic vacuoles, and pharmacologic inhibition of CatL increased the amount of active trypsin in acinar cells. The results suggest that retarded autophagy is associated with an imbalance between CatL, which degrades trypsinogen and trypsin, and CatB, which converts trypsinogen into trypsin, resulting in intra-acinar accumulation of active trypsin in pancreatitis. Thus, deficient lysosomal degradation may be a dominant mechanism for increased intra-acinar trypsin in pancreatitis.
Background & Aims:
The plasminogen (plg) system participates in tissue repair in several
organs, but its role in pancreas repair remains poorly characterized. To
better understand the role of plg in pancreas recovery following injury, we
examined the course of caerulein-induced pancreatitis in plg deficient and
Pancreatitis was induced by caerulein administration (50
μg/kg, 7 ip injections). Mice were sacrificed either at the acute
phase (7 hours after the first caerulein injection) or during recovery (at
2, 4 and 7 days). In pancreatic sections we examined: pancreatic morphology,
trypsin activation, inflammatory cell infiltration, acinar cell death, cell
proliferation, extracellular matrix (ECM) deposition, activation of stellate
cells (PSCs), and components of the plg and metalloproteinase systems.
In plg sufficient mice, pancreatic plg levels and plasmin activity
increased during the acute phase and remained elevated during recovery.
Pancreatitis resolved in plg sufficient mice within 7 days. Pancreas
recovery involved reorganization of the parenchyma structure, removal of
necrotic debris, cell proliferation, transient activation of PSCs and
moderate deposition of ECM proteins. Acute pancreatitis (7-h) was
indistinguishable between plg deficient and sufficient mice. In contrast,
pancreas recovery was impaired in plg deficient mice. Plg deficiency led to
disorganized parenchyma, extensive acinar cell loss, poor removal of
necrotic debris, reduced cell proliferation and fibrosis. Fibrosis was
characterized by deposition of collagens and fibronectin, persistent
activation of PSCs and upregulation of pancreatic TGF-β1.
Plg/plasmin deficiency leads to features similar to those found in
chronic pancreatitis such as parenchymal atrophy and fibrosis.
Activated stellate cells are considered the principal mediators of chronic alcoholic pancreatitis/fibrosis. However the mechanisms of alcohol action on pancreatic stellate cells (PaSCs) are poorly understood. The aims of this study were to determine the presence and role of the NADPH oxidase system in mediating alcohol effects on PaSCs with specific emphasis on proliferation.
PaSC NADPH oxidase components mRNA and protein were determined by RT-PCR and Western blot. The NADPH oxidase activity was measured by detecting the production of reactive oxygen species using lucigenin-derived chemiluminescence assay. PaSC DNA synthesis, a measure of proliferation, was performed by determining the [3H] thymidine incorporation into DNA.
mRNA for NADPH oxidase components Nox1, gp91phox, Nox4, p22phox, p47phox and p67phox and protein for NADPH oxidase subunits gp91phox, p22phox, p47phox and p67phox are present in PaSCs. Treatment with platelet-derived growth factor (PDGF) significantly increased the NADPH oxidase activity and DNA synthesis in cultured PaSCs. Alcohol treatment markedly augmented both the NADPH oxidase activity and the DNA synthesis caused by PDGF, which was prevented by antioxidant N-acetyl-L-cysteine, ROS scavenger tiron, and the NADPH oxidase inhibitor diphenylene iodium. The effects of PDGF on NADPH oxidase activity and DNA synthesis were prevented in PaSCs isolated from the pancreas of mice with a genetic deficiency of p47phox.
Ethanol causes proliferation of stellate cells by augmenting the activation of the cell's NADPH oxidase system stimulated by PDGF. These results provide new insights into the mechanisms of alcohol-induced fibrosing disorders.
NADPH oxidase; Reactive oxygen species; Proliferation; Platelet-derived growth factor; Ethanol
Pancreatic stellate cells (PaSCs) are myofibroblast-like cells found in the areas of the pancreas that have exocrine function. PaSCs are regulated by autocrine and paracrine stimuli and share many features with their hepatic counterparts, studies of which have helped further our understanding of PaSC biology. Activation of PaSCs induces them to proliferate, to migrate to sites of tissue damage, to contract and possibly phagocytose, and to synthesize ECM components to promote tissue repair. Sustained activation of PaSCs has an increasingly appreciated role in the fibrosis that is associated with chronic pancreatitis and with pancreatic cancer. Therefore, understanding the biology of PaSCs offers potential therapeutic targets for the treatment and prevention of these diseases.
In addition to immune cells, many other cell types are known to produce cytokines. Cultured normal mouse gallbladder epithelial cells, used as a model system for gallbladder epithelium, were examined for their ability to express the mRNA of various cytokines and chemokines in response to bacterial lipopolysaccharide. The synthesis and secretion of the tumor necrosis factor-α (TNF-α) protein by these cells was also measured.
Untreated mouse gallbladder cells expressed mRNA for TNF-α, RANTES, and macrophage inflammatory protein-2 (MIP-2). Upon treatment with lipopolysaccharide, these cells now produced mRNA for Interleukin-1β (IL-1β), IL-6, monocyte chemoattractant protein-1 (MCP-1), and showed increased expression of TNF-α and MIP-2 mRNA. Untreated mouse gallbladder cells did not synthesize TNF-α protein; however, they did synthesize and secrete TNF-α upon treatment with lipopolysaccharide.
Cells were treated with lipopolysaccharides from 3 strains of bacteria. Qualitative and semi-quantitative RT-PCR, using cytokine or chemokine-specific primers, was used to measure mRNA levels of TNFα, IL-1β, IL-6, IL-10, KC, RANTES, MCP-1, and MIP-2. TNF-α protein was measured by immunoassays.
This research demonstrates that gallbladder epithelial cells in response to lipopolysaccharide exposure can alter their cytokine and chemokine RNA expression pattern and can synthesize and secrete TNFα protein. This suggests a mechanism whereby gallbladder epithelial cells in vivo may mediate gallbladder secretory function, inflammation and diseases in an autocrine/paracrine fashion by producing and secreting cytokines and/or chemokines during sepsis.