Pancreatic Stellate Cells Increase the Invasion of Human Pancreatic Cancer Cells through the Stromal Cell-Derived Factor-1/CXCR4 Axis

Aim: Both pancreatic stellate cells (PSCs) and the stromal cell-derived factor-1(SDF-1)/CXCR4 receptor ligand system have important roles in pancreatic cancer progression. This study set out to detect if PSCs express SDF-1 and promote the invasion of pancreatic cancer through the SDF-1/CXCR4 receptor ligand axis. Methods: RT-PCR was performed to detect the expression of SDF-1 and CXCR4 in PSCs, pancreatic cancer lines and cancer tissue samples. ELISA was used to investigate the concentration of SDF-1 in PSC supernatants. An MTT assay was applied to detect the proliferation of pancreatic cancer cells. A transwell chamber migration assay was employed to detect the migration of AsPC-1 cells. An in vitro invasion assay was used to detect the invasion of AsPC-1 cells. Results: CXCR4 expression was detected in PSCs; AsPC-1, SW1990 and BxPC-3 cancer cells; and cancer tissues. SDF-1 was detected in PSCs and cancer tissues, but not in AsPC-1, SW1990 and BxPC-3 cells. SDF-1α protein was found in PSC supernatants. PSC-conditioned media can promote the proliferation, migration and invasion of pancreatic cancer cells. SDF-1 neutralizing antibody or AMD3100 can significantly inhibit these promotive effects. Conclusion: PSCs can secrete SDF-1 and increase the invasion of pancreatic cancer cells through the SDF-1/CXCR4 axis.     

Isolation of Quiescent Human Pancreatic Stellate Cells: A Promising in vitro Tool for Studies of Human Pancreatic Stellate Cell Biology

Background: Pancreatic stellate cells (PSCs) play a critical role in pancreatic fibrosis. To date, human PSC biology has been studied using cancer- or inflammation-associated (preactivated) PSCs, but an in vitro model of quiescent normal human PSCs (NhPSCs) has been lacking. Aims: To (i) isolate and characterize quiescent NhPSCs, and (ii) evaluate the response of culture-activated NhPSCs to cytokines and LPS. Methods: Quiescent NhPSCs were isolated from normal pancreatic tissue using density gradient centrifugation. PSC markers, glial fibrillary acidic protein (GFAP), desmin, α-smooth muscle actin (αSMA) and the lipopolysaccharide (LPS) receptors TLR4 and CD14 were identified by immunoblotting and immunocytochemistry. The effect of plateletderived growth factor (PDGF), transforming growth factor β (TGFβ) and LPS on NhPSC activation was also assessed. Results: Freshly isolated NhPSCs displayed a polygonal appearance with refringent cytoplasmic lipid droplets. Culture-activated NhPSCs expressed GFAP, desmin, αSMA, TLR4 and CD14, and were responsive to PDGF, TGFβ and LPS. Conclusion: Isolated NhPSCs expressed the same markers as rat PSCs and human cancer-associated PSCs and responded to PDGF and TGFβ similarly to rat PSCs. NhPSC preparations provide a useful in vitro tool to study the biology of PSCs in their physiological, non-activated state.     

Connective tissue growth factor (CCN2) in rat pancreatic stellate cell function: integrin alpha5beta1 as a novel CCN2 receptor

BACKGROUND & AIMS: Pancreatic stellate cells (PSCs) are proposed to play a key role in the development of pancreatic fibrosis. The aim of this study was to evaluate the production by rat activated PSCs of the fibrogenic protein, connective tissue growth factor (CCN2), and to determine the effects of CCN2 on PSC function. METHODS: CCN2 production was evaluated by immunoprecipitation and promoter activity assays. Expression of integrin alpha5beta1 was examined by immunoprecipitation and Western blot. Binding between CCN2 and integrin alpha5beta1 was determined in cell-free systems. CCN2 was assessed for its stimulation of PSC adhesion, migration, proliferation, DNA synthesis, and collagen I synthesis. RESULTS: CCN2 was produced by activated PSCs, and its levels were enhanced by transforming growth factor beta1 treatment. CCN2 promoter activity was stimulated by transforming growth factor beta1, platelet-derived growth factor, alcohol, or acetaldehyde. CCN2 stimulated integrin alpha5beta1-dependent adhesion, migration, and collagen I synthesis in PSCs. Integrin alpha5beta1 production by PSCs was verified by immunoprecipitation, while direct binding between integrin alpha5beta1 and CCN2 was confirmed in cell-free binding assays. Cell surface heparan sulfate proteoglycans functioned as a partner of integrin alpha5beta1 in regulating adhesion of PSCs to CCN2. PSC proliferation and DNA synthesis were enhanced by CCN2. CONCLUSIONS: PSCs synthesize CCN2 during activation and after stimulation by profibrogenic molecules. CCN2 regulates PSC function via cell surface integrin alpha5beta1 and heparan sulfate proteoglycan receptors. These data support a role for CCN2 in PSC-mediated fibrogenesis and highlight CCN2 and its receptors as potential novel therapeutic targets.     

ERAP2 is a novel target involved in autophagy and activation of pancreatic stellate cells via UPR signaling pathway

Background/objectivesPancreatic ductal adenocarcinoma (PDAC) is characterized by excessive desmoplasia and autophagy-dependent tumorigenic growth. Pancreatic stellate cells (PSCs) as a predominant stromal cell type play a critical role in PDAC biology. We have previously reported that autophagy facilitates PSC activation, however, the mechanism remains unknown. We investigated the mechanism of autophagy in PSC activation.MethodsWe compared gene expression profiles between patient-derived PSCs from pancreatic cancer and chronic pancreatitis using a microarray. The stromal expression of target gene in specimen of PDAC patients (n = 63) was analyzed. The effect of target gene on autophagy and activation of PSCs was investigated by small interfering RNAs transfection, and the relationship between autophagy and ER stress was investigated. We analyzed the growth and fibrosis of xenografted tumor by orthotopic models.ResultsIn analysis of gene expression microarray, endoplasmic reticulum aminopeptidase 2 (ERAP2) upregulated in cancer-associated PSCs was identified as the target gene. High stromal ERAP2 expression is associated with a poor prognosis of PDAC patients. Knockdown of ERAP2 inhibited unfolded protein response mediated autophagy, and led to inactivation of PSCs, thereby attenuating tumor-stromal interactions by inhibiting production of IL-6 and fibronectin. In vivo, the promoting effect of PSCs on xenografted tumor growth and fibrosis was inhibited by ERAP2 knockdown.ConclusionsOur findings demonstrate a novel mechanism of PSCs activation regulated by autophagy. ERAP2 as a promising therapeutic target may provide a novel strategy for the treatment of PDAC.     

Pancreatic stellate cells respond to inflammatory cytokines: potential role in chronic pancreatitis

BACKGROUND: It is now generally accepted that chronic pancreatic injury and fibrosis may result from repeated episodes of acute pancreatic necroinflammation (the necrosis-fibrosis sequence). Recent studies suggest that pancreatic stellate cells (PSCs), when activated, may play an important role in the development of pancreatic fibrosis. Factors that may influence PSC activation during pancreatic necroinflammation include cytokines known to be important in the pathogenesis of acute pancreatitis, such as tumour necrosis factor alpha (TNF-alpha), and the interleukins 1, 6, and 10 (IL-1, IL-6, and IL-10). AIM: To determine the effects of these cytokines on PSC activation, as assessed by cell proliferation, alpha smooth muscle actin (alpha-SMA) expression, and collagen synthesis. METHODS: Cultured rat PSCs were incubated with cytokines for 24 hours. Cell proliferation was assessed by measuring (3)H thymidine incorporation into cellular DNA, alpha-SMA expression by western blotting, and collagen synthesis by incorporation of (14)C proline into collagenase sensitive protein. mRNA levels for procollagen alpha(1)(1) in PSCs were determined by northern and dot blotting methods. RESULTS: Expression of alpha-SMA by PSCs was increased on exposure to each of the cytokines used in the study. Stellate cell proliferation was stimulated by TNF-alpha but inhibited by IL-6, while IL-1 and IL-10 had no effect on PSC proliferation. Collagen synthesis by PSCs was stimulated by TNF-alpha and IL-10, inhibited in response to IL-6, and unaltered by IL-1. Changes in collagen protein synthesis in response to TNF-alpha, IL-10, and IL-6 were not regulated at the mRNA level in the cells. CONCLUSION: This study has demonstrated that PSCs have the capacity to respond to cytokines known to be upregulated during acute pancreatitis. Persistent activation of PSCs by cytokines during acute pancreatitis may be a factor involved in the progression from acute pancreatitis to chronic pancreatic injury and fibrosis.     

Vitamin A inhibits pancreatic stellate cell activation: implications for treatment of pancreatic fibrosis

BACKGROUND AND AIMS: Activated pancreatic stellate cells (PSCs) are implicated in the production of alcohol induced pancreatic fibrosis. PSC activation is invariably associated with loss of cytoplasmic vitamin A (retinol) stores. Furthermore, retinol and ethanol are known to be metabolised by similar pathways. Our group and others have demonstrated that ethanol induced PSC activation is mediated by the mitogen activated protein kinase (MAPK) pathway but the specific role of retinol and its metabolites all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-RA) in PSC quiescence/activation, or its influence on ethanol induced PSC activation is not known. Therefore, the aims of this study were to (i) examine the effects of retinol, ATRA, and 9-RA on PSC activation; (ii) determine whether retinol, ATRA, and 9-RA influence MAPK signalling in PSCs; and (iii) assess the effect of retinol supplementation on PSCs activated by ethanol. METHODS: Cultured rat PSCs were incubated with retinol, ATRA, or 9-RA for varying time periods and assessed for: (i) proliferation; (ii) expression of alpha smooth muscle actin (alpha-SMA), collagen I, fibronectin, and laminin; and (iii) activation of MAPKs (extracellular regulated kinases 1 and 2, p38 kinase, and c-Jun N terminal kinase). The effect of retinol on PSCs treated with ethanol was also examined by incubating cells with ethanol in the presence or absence of retinol for five days, followed by assessment of alpha-SMA, collagen I, fibronectin, and laminin expression. RESULTS: Retinol, ATRA, and 9-RA significantly inhibited: (i) cell proliferation, (ii) expression of alpha-SMA, collagen I, fibronectin, and laminin, and (iii) activation of all three classes of MAPKs. Furthermore, retinol prevented ethanol induced PSC activation, as indicated by inhibition of the ethanol induced increase in alpha-SMA, collagen I, fibronectin, and laminin expression. CONCLUSIONS: Retinol and its metabolites ATRA and 9-RA induce quiescence in culture activated PSCs associated with a significant decrease in the activation of all three classes of MAPKs in PSCs. Ethanol induced PSC activation is prevented by retinol supplementation.     

New high-throughput screening detects compounds that suppress pancreatic stellate cell activation and attenuate pancreatic cancer growth

Background/objectivesPancreatic stellate cells (PSCs) are involved in abundant desmoplasia, which promotes cancer cell aggressiveness and resistance to anti-cancer drugs. Therefore, PSCs are suggested to be a promising therapeutic target by attenuating PSC activation to inhibit tumor-stromal interactions with pancreatic cancer cells. Here, we developed a screen to identify compounds that reduce the activity of PSCs and investigated the effect of candidates on pancreatic cancer.MethodsLipid droplet accumulation in PSCs was used to observe differences in PSC activity and a new high-throughput screening platform that quantified lipid droplets in PSCs was established. A library of 3398 Food and Drug Administration-approved drugs was screened by this platform. Validation assays were performed in vitro and in vivo.ResultsThirty-two compounds were finally selected as candidate compounds by screening. These compounds decreased α-smooth muscle actin expression and inhibited autophagic flux in PSCs in vitro. Among the candidates, three drugs selected for validation assays inhibited the proliferation and migration of PSCs and invasion of cancer cells by disrupting tumor-stromal interactions. Production of extracellular matrix molecules was also decreased significantly by this treatment. In vivo testing in xenograft models showed that dopamine antagonist zuclopenthixol suppressed tumor growth; this suppression was significantly increased when combined with gemcitabine.ConclusionsA new screening platform that focused on the morphological features of PSCs was developed. Candidate drugs from this screening suppressed PSC activation and tumor growth. This screening system may be useful to discover new compounds that attenuate PSC activation.     

Uncoupling protein 2 deifciency reduces proliferative capacity of murine pancreatic stellate cells

BACKGROUND: Uncoupling protein 2 (UCP2) has been sug-gested to inhibit mitochondrial production of reactive oxygen species (ROS) by decreasing the mitochondrial membrane potential. Experimental acute pancreatitis is associated with increased UCP2 expression, whereas UCP2 deifciency retards regeneration of aged mice from acute pancreatitis. Here, we have addressed biological and molecular functions of UCP2 in pancreatic stellate cells (PSCs), which are involved in pancre-atic wound repair and ifbrogenesis.
METHODS: PSCs were isolated from 12 months old (aged) UCP2-/- mice and animals of the wild-type (WT) strain C57BL/6. Proliferation and cell death were assessed by em-ploying trypan blue staining and a 5-bromo-2’-deoxyuridine incorporation assay. Intracellular fat droplets were visualized by oil red O staining. Levels of mRNA were determined by RT-PCR, while protein expression was analyzed by immuno-blotting and immunolfuorescence analysis. Intracellular ROS levels were measured with 2’, 7’-dichlorolfuorescin diacetate. Expression of senescence-associatedβ-galactosidase (SAβ-Gal) was used as a surrogate marker of cellular senescence.
RESULTS: PSCs derived from UCP2-/- mice proliferated at a lower rate than cells from WT mice. In agreement with this observation, the UCP2 inhibitor genipin displayed dose-dependent inhibitory effects on WT PSC growth. Interestingly, ROS levels in PSCs did not differ between the two strains, and PSCs derived from UCP2-/- mice did not senesce faster than those from corresponding WT cells. PSCs from UCP2-/- mice and WT animals were also indistinguishable with respect to the activation-dependent loss of intracellular fat droplets, ex-pression of the activation markerα-smooth muscle actin, type I collagen and the autocrine/paracrine mediators interleu-kin-6 and transforming growth factor-β1.
CONCLUSIONS: A reduced proliferative capacity of PSC from aged UCP2-/- mice may contribute to the retarded regenera-tion after acute pancreatitis. Apart from their slower growth, PSC of UCP2-/- mice displayed no functional abnormalities. The antiifbrotic potential of UCP2 inhibitors deserves further attention.     

Mechanisms of pancreatic fibrosis and applications to the treatment of chronic pancreatitis

Pancreatic stellate cells (PSCs) play a crucial role in pancreatic fibrogenesis in chronic pancreatitis and in the desmoplastic reaction of pancreatic cancer. When PSCs are stimulated by oxidative stress, ethanol and its metabolite acetaldehyde, and cytokines, the phenotype of quiescent fat-storing cells converts to myofibroblastlike activated PSCs, which then produce extracellular matrix, adhesion molecules, and various chemokines in response to cytokines and growth factors. Recent data suggest that PSCs have a phagocytic function. Plateletderived growth factor is a potent stimulator of PSC proliferation. Transforming growth factor β, activin A, and connective tissue growth factor also play a role in PSC-mediated pancreatic fibrogenesis through autocrine and paracrine loops. Following pancreatic damage, pathophysiological processes that occur in the pancreas, including pancreas tissue pressure, hyperglycemia, intracellular reactive oxygen species production, activation of protease-activated receptor 2, induction of cyclooxygenase 2, and bacterial infection play a role in sustaining pancreatic fibrosis through increased PSC proliferation and collagen production by PSCs. Targeting PSCs might be an effective therapeutic approach in chronic pancreatitis. Various substances including vitamin A, vitamin E, polyphenols, peroxisome proliferator-activated receptor γ ligands, and inhibitors of the renin-angiotensin system show great promise of being useful in the treatment of chronic pancreatitis.     

YAP activates pancreatic stellate cells and enhances pancreatic fibrosis

BackgroundPancreatic stellate cells (PSCs) foster the progression of pancreatic adenocarcinoma and chronic pancreatitis (CP) by producing a dense fibrotic stroma. However, the incomplete knowledge of PSCs biology hampers the exploration of antifibrotic therapies. Here, we explored the role of the Hippo pathway in the context of PSCs activation and experimental CP.MethodsCP model was created in rats with the tail vein injection of dibutyltin dichloride (DBTC). The expression of Yes-associated protein (YAP) in CP tissue was assessed. Primary and immortalized rats PSCs were treated with the YAP-inhibitor verteporfin. Furthermore, YAP siRNA was employed. Subsequently, DNA synthesis, cell survival, levels of α-smooth muscle actin (α-SMA) protein, presence of lipid droplets and PSCs gene expression were evaluated. Upstream regulators of YAP signaling were studied by reporter gene assays.ResultsIn DBTC-induced CP, pronounced expression of YAP in areas of tubular structures and periductal fibrosis was observed. Verteporfin diminished DNA replication in PSCs in a dose-dependent fashion. Knockdown of YAP reduced cell proliferation. Primary cultures of PSCs were characterized by a decrease of lipid droplets and increased synthesis of α-SMA protein. Both processes were not affected by verteporfin. At the non-cytotoxic concentration of 100 nmol/L, verteporfin significantly reduced mRNA levels of transforming growth factor-β1 (Tgf-β1) and Ccn family member 1 (Ccn1). YAP signaling was activated by TGF-β1, but repressed by interferon-γ.ConclusionsActivated YAP enhanced PSCs proliferation. The antifibrotic potential of Hippo pathway inhibitors warrants further investigation.     

Glial fibrillary acidic protein promoter targets pancreatic stellate cells

BackgroundPancreatic fibrosis is one of the clinical manifestations of chronic pancreatitis and pancreatic cancer. Pancreatic stellate cells (PSCs) have been recognised as principal effector cells in the development of pancreatic fibrosis. The ability to specifically address PSCs might offer a potential for developing a targeted therapy for pancreatic fibrosis.AimCharacterisation of the 2.2 kb hGFAP (human glial fibrillary acidic protein) promoter for its usefulness to express reporter genes specifically in PSCs in vitro and in vivo.Methods2.2 kb hGFAP-LacZ reporter expressions were examined in four immortalised PSC lines and two non-PSCs, meanwhile, GFAP-LacZ transgenic mice were used to detect LacZ reporter in pancreas tissue. Several kinase inhibitors, vitamin A and its metabolites were applied to study the regulation of 2.2 kb hGFAP promoter in PSCs.ResultsOur results showed that the 2.2 kb hGFAP promoter is capable of regulating the expression of reporter genes exclusively in immortalised and primary PSCs, as well as in PSCs of transgenic GFAP-LacZ mice. When a PSC cell line transfected with the LacZ reporter (SAM-K/LacZ/C1) was treated with different anti-fibrotic agents and kinase inhibitors, the transgenic β-galactosidase activity was found to be regulated by multiple signalling pathways known to be involved in the PSC activation.ConclusionsThis study provides the proof of concept for using the 2.2 kb hGFAP promoter to specifically manipulate PSCs for the development of targeted gene and/or drug therapy in pancreatic fibrosis, and for the screening of anti-fibrotic agents.     

Cell migration: a novel aspect of pancreatic stellate cell biology

BACKGROUND: Pancreatic stellate cells (PSCs), implicated as key mediators of pancreatic fibrogenesis, are found in increased numbers in areas of pancreatic injury. This increase in PSC number may be due to increased local proliferation and/or migration of these cells from adjacent areas. The ability of PSCs to proliferate has been well established but their potential for migration has not been examined. AIMS: Therefore, the aims of this study were to determine whether cultured rat PSCs have the capacity to migrate and, if so, to characterise this migratory capacity with respect to the influence of basement membrane components and the effect of platelet derived growth factor (PDGF, a known stimulant for migration of other cell types). METHODS: Migration of freshly isolated (quiescent) and culture activated (passaged) rat PSCs was assessed across uncoated or Matrigel (a basement membrane-like substance) coated porous membranes (pore size 8 micro m) in the presence or absence of PDGF (10 and 20 ng/ml) in the culture medium. A checkerboard assay was performed to assess whether the effect of PDGF on PSC migration was chemotactic or chemokinetic. RESULTS: Cell migration was observed with both freshly isolated and passaged PSCs. However, compared with passaged (culture activated) cells, migration of freshly isolated cells was delayed, occurring only at or after 48 hours of incubation when the cells displayed an activated phenotype. PSC migration through Matrigel coated membranes was delayed but not prevented by basement membrane components. PSC migration was increased by PDGF and this effect was predominantly chemotactic (that is, in the direction of a positive concentration gradient). CONCLUSIONS: (i) PSCs have the capacity to migrate. (ii) Activation of PSCs appears to be a prerequisite for migration. (iii) PDGF stimulates PSC migration and this effect is predominantly chemotactic. IMPLICATION: Chemotactic factors released during pancreatic injury may stimulate the migration of PSCs through surrounding basement membrane towards affected areas of the gland.     

Matrix metalloproteinases and diabetic foot ulcers: the ratio of MMP-1 to TIMP-1 is a predictor of wound healing

Aims Matrix metalloproteinases (MMPs) play a major role in wound healing: they can degrade all components of the extracellular matrix. In diabetic foot ulcers there is an excess of MMPs and a decrease of the tissue inhibitors of MMPs (TIMPs). This imbalance is probably one cause of impaired healing. However, little is known about changes in MMPs during wound healing. Methods Sixteen patients with neuropathic diabetic foot ulcers participated. Wound fluid was collected regularly during the 12-week follow-up period, for measurement of MMP-1, MMP-2, MMP-8, MMP-9 and TIMP-1. Results were analysed by the degree of wound healing: good healers (defined by a reduction of at least 82% in initial wound surface at 4 weeks) and poor healers (reduction of less than 82% in wound surface at 4 weeks). Results In good healers, levels of MMP-8 and -9 secreted by inflammatory cells decreased earlier. The initial levels of MMP-1 were similar in good and poor healers (P = 0.1) but rose significantly at week 2 in good healers (P = 0.039). There was a significant correlation between a high ratio of MMP-1/TIMP-1 and good healing (r = 0.65, P = 0.008). Receiver Operator Curve (ROC) analysis showed that an MMP-1/TIMP-1 ratio of 0.39 best predicted wound healing (sensitivity = 71%, specificity = 87.5%). Conclusions A high level of MMP-1 seems essential to wound healing, while an excess of MMP-8 and -9 is deleterious, and could be a target for new topical treatments. The MMP-1/TIMP-1 ratio is a predictor of wound healing in diabetic foot ulcers.     

Effect of prostaglandin E1 and steroid on healing colonic anastomoses

PURPOSE: The effect of prostaglandin E₁ (PGE₁) and corticosteroids alone and in combination were studied in the healing rat colon to determine whether PGE₁ could not only improve healing but reverse the negative effect of steroids on colonic wound healing. METHODS: Colonic anastomoses were performed in 144 male Sprague-Dawley rats divided into four groups. The control group (I) received no further treatment. The steroid group (II) received cortisone acetate (5 mg/kg/day) beginning six days preoperatively and continuing until sacrifice. The PGE₁ group (III) received 2 μg of PGE₁ intra-aortically at surgery and for three days postoperatively. The combination PGE₁/steroid group (IV) received both drugs in the same doses as those in Groups II and III. Animals were sacrificed on postoperative days 6, 10, and 14. Wound healing was evaluated by hydroxyproline content, bursting pressures, and histology. RESULTS: The hydroxyproline assay at day 10 revealed that steroid-treated rats have significantly lower levels than any other group. The PGE₁ group (III) had the highest level of significance in comparison to the steroid group (II) (P=0.001). The addition of PGE₁ to steroid (Group IV) appeared to abolish the negative effect of the steroid as measured by hydroxyproline content on day 10 (P=0.038). When measuring bursting pressures, the PGE₁ group (III) had significantly higher pressures than any other group at day 10. However, no amelioration of the steroid effect on bursting pressures was seen. Histologic evaluation of the anastomosis did not reveal any significant differences among the four groups. CONCLUSIONS: PGE₁ reverses the negative effect of the steroid on hydroxyproline levels at day 10. Furthermore, using bursting pressure as a parameter of wound healing, administration of PGE₁ results in significantly improved anastomotic healing at day 10.     

Regulation of p38 MAPK and glucocorticoid receptor activation by hydrocortisone in mono-and co-cultured pancreatic acinar and stellate cells

Background/objectivesAcute pancreatitis develops as an inflammatory response to pancreatic tissue injury. Postoperative pancreatitis has recently been associated with increased occurrence of complications. Activation of the mitogen-activated protein kinase p38 (p38 MAPK) pathway occurs early in acute pancreatitis and its inhibition has been suggested to alleviate pancreatic inflammation. Glucocorticoids are potent anti-inflammatory steroids whose use in the management of acute pancreatitis remains controversial. Our aim was to examine the effect of crosstalk between pancreatic acinar cells (PACs) and stellate cells (PSCs) on p38 MAPK and glucocorticoid receptor (GR) activation and to assess the impact of hydrocortisone on these events.MethodsThe long-term co-culture setting for mouse PACs and PSCs developed in our laboratory was used. Parallel 4d mono- and co-cultures with or without 10 nM hydrocortisone were performed followed by immunocytochemical analysis of nuclear GR and phospho-p38 MAPK (pp38 MAPK).ResultsHydrocortisone inhibited pp38 MAPK up-regulation evoked by co-culture in PACs and PSCs and increased nuclear translocation of GR in PAC monocultures and in co-cultured PACs and PSCs. In PSC monocultures and co-cultured PACs, ligand-independent expression of nuclear GR was observed. In the former no change in nuclear GR but a significant decrease in total GR as analyzed by Western blot was caused by hydrocortisone.ConclusionsCellular microenvironment plays a significant role on p38 MAPK and GR activation in PACs and PSCs. Hydrocortisone is an effective means to inhibit p38 MAPK activation in PACs and PSCs. Both ligand-dependent and -independent regulatory roles for GR are suggested in the exocrine pancreas.     

Collagenase-1 (MMP-1), matrilysin-1 (MMP-7), and stromelysin-2 (MMP-10) are expressed by migrating enterocytes during intestinal wound healing

Background: Matrix metalloproteinases (MMPs) play a crucial role in wound healing of the skin, airways, and cornea, but data on MMPs in normal intestinal wound healing is limited. The aim of this study was to clarify the role of collagenase-1 (MMP-1), matrilysin-1 (MMP-7), and stromelysin-2 (MMP-10) in intestinal wound repair and to determine the effect of cytokines on the expression of these MMPs in intestinal epithelial cell lines. Methods: Surgical specimens from patients with ischemic colitis (n?=?5) were used as an in vivo model of intestinal re-epithelialization. Fetal ileal explants were used as an ex vivo model. In situ hybridization for MMPs -1, -3, -7, and -10 was performed and immunohistochemical stainings were used to localize MMP-7 and -9 expressing cells. Stainings for cytokeratin and laminin-5 were performed to identify epithelial cells and migrating enterocytes, respectively. Caco-2, HT-29, and WiDr cell lines were treated for 6-48?h with different cytokines (e.g. EGF, KGF, IL-1β, TGF-α, TNF-α, and TGF-β1) and Taqman real-time quantitative RT-PCR was used to investigate their effect on the expression of MMPs-1, -7, and -10. Results: MMP-7, MMP-10, and MMP-1 were expressed by migrating enterocytes bordering intestinal ulcers in 5/5, 3/5, and 3/5 samples, respectively. In the fetal gut model, MMP-1 and MMP-10 were expressed by migrating enterocytes, but matrilysin-1 expression was not detected. Matrilysin-1 was up-regulated by TNF-α and IL-1β, and stromelysin-2 by TNF-α and EGF in Caco-2 and WiDr cell cultures. MMP-1 was up-regulated in Caco-2 cells by TGF-beta, EGF, and IL-1β, but only by EGF in WiDR cells. Conclusions: It is concluded that collagenase-1, stromelysin-2, and matrilysin-1 are involved in intestinal re-epithelialization in vivo and that they are up-regulated by cytokines relevant in wound repair.