Andre Menke

Identification, culture, and characterization of pancreatic stellate cells in rats and humans☆☆☆

BACKGROUND & AIMS Until now, the basic matrix-producing cell type responsible for pancreas fibrosis has not been identified. In this report, retinoid-containing pancreatic stellate cells (PSCs) in rat and human pancreas are described, and morphological and biochemical similarities to hepatic stellate cells are shown. METHODS Electron and immunofluorescence microscopy (collagen types I and III, fibronectin, laminin, alpha-actin, and desmin) was performed using pancreatic tissue and cultured PSCs. Extracellular matrix synthesis was shown using quantitative immunoassay and Northern blot analysis. RESULTS PSCs are located in interlobular areas and in interacinar regions. Early primary cultured PSCs contain retinol and fatty acid retinyl-esters. Addition of retinol to passaged cells resulted in retinol uptake and esterification. During primary culture, the cells changed from a quiescent fat-storing phenotype to a highly synthetic myofibroblast-like cell expressing iso-alpha-smooth muscle actin (>90%) and desmin (20%-40%) and showing strong positive staining with antibodies to collagen types I and III, fibronectin, and laminin. As determined on protein and messenger RNA level, serum growth factors stimulated the synthesis of collagen type I and fibronectin. CONCLUSIONS The identification of PSCs, particularly in fibrotic areas, and the similarities of these cells to hepatic stellate cells suggest that PSCs participate in the development of pancreas fibrosis.

Role of MT‐MMPs and MMP‐2 in pancreatic cancer progression

Activation of matrix metalloproteinase‐2 (MMP‐2) by the membrane‐type matrix metalloproteinases (MT‐MMPs) has been associated with tumor progression. In the present study, we examined the role of MMP‐2 and its activators MT1‐MMP, MT2‐MMP and MT3‐MMP in pancreatic tumor cell invasion and the development of the desmoplastic reaction characteristic of pancreatic cancer tissues. Northern blot analyses revealed that transcript levels of MT1‐MMP and MT2‐MMP, but not MT3‐MMP, were enhanced in pancreatic cancer tissues (n = 18) compared with both chronic pancreatitis (n = 9) and healthy pancreas (n = 9). A good correlation was found between MT1‐MMP and both MMP‐2 expression (p < 0.01) and activity in pancreatic cancer tissues. In addition, expression and activation of MMP‐2 were strongly associated with the extent of the desmoplastic reaction in pancreatic cancer tissues. Invasion assays showed a good correlation between MMP‐2 expression and activity and the invasive potential of pancreatic cancer cell lines. In cell lines with high levels of MMP‐2 expression and activity, the MMP inhibitor Batimastat led to significant reduction of the number of invading cells. Our results suggest that MT1‐MMP is involved in the progression of pancreatic cancer via activation of MMP‐2. MMP‐2 itself plays an important role in tumor cell invasion and appears to be associated with the development of the characteristic desmoplastic reaction in pancreatic cancer. Int. J. Cancer 85:14–20, 2000.

Platelet-Derived Growth Factors Stimulate Proliferation and Extracellular Matrix Synthesis of Pancreatic Stellate Cells: Implications in Pathogenesis of Pancreas Fibrosis

At present, the cell-cell interactions and molecular mechanisms of pancreas fibrogenesis are largely unknown. The purpose of this study was to investigate paracrine stimulatory loops between platelets and pancreatic stellate cells (PSC). Human PSC were obtained by outgrowth from fibrotic human pancreas. Native platelet lysate (nPL) and transiently acidified platelet lysate (aPL) were added to cultured PSC (passage 4 to 7) in the absence of serum. The synthesis of collagen types I and III and c-fibronectin (cFN) was demonstrated on protein (immunofluorescence and quantitative immunoassay) and mRNA (Northern blot) level. Using sections of human pancreas with acute pancreatitis, platelet aggregates in capillaries were demonstrated by transmission electron microscopy. nPL, and to an even greater extent aPL, significantly increased the synthesis of collagen types I and III and of c-FN (120 μl/ml aPL increased collagen type I concentration in PSC supernatants by 1.99 ± 0.17 times and c-FN of 2.49 ± 0.28 times, mean ± sd, n = 3). nPL and aPL also significantly stimulated cell proliferation (increased bromodeoxyuridine (BrdU) incorporation by 6.4 ± 0.78 times and 10 ± 0.29 times, respectively). By preincubating aPL with transforming growth factor β (TGFβ)- and platelet-derived growth factor (PDGF)-neutralizing antibodies and the TGFβ-latency associated peptide, respectively, TGFβ1 was identified as the main mediator stimulating matrix synthesis and PDGF as the responsible mitogen. Our data demonstrate that platelets contain fibrogenic mediators that stimulate proliferation (PDGF) and matrix synthesis (TGFβ1) of cultured PSC. We suggest that platelets and PSC cooperate in the development of pancreas fibrosis.

Collagen type I induces disruption of E-cadherin-mediated cell-cell contacts and promotes proliferation of pancreatic carcinoma cells

Pancreatic cancer is characterized by its invasiveness, early metastasis, and the production of large amounts of extracellular matrix (ECM). We analyzed the influence of type I collagen and fibronectin on the regulation of cellular adhesion in pancreatic cancer cell lines to characterize the role of ECM proteins in the development of pancreatic cancer. We show that collagen type I is able to initiate a disruption of the E-cadherin adhesion complex in pancreatic carcinoma cells. This is due to the increased tyrosine phosphorylation of the complex protein beta-catenin, which correlates with collagen type I-dependent activation of the focal adhesion kinase and its association with the E-cadherin complex. The activation and recruitment of focal adhesion kinase to the E-cadherin complex depends on the interaction of type I collagen with beta1-containing integrins and an integrin-mediated activation of the cellular kinase Src. The disassembly of the E-cadherin adhesion complex correlates with the nuclear translocation of beta-catenin, which leads to an increasing expression of the beta-catenin-Lef/Tcf target genes, cyclin D1 and c-myc. In addition to that, cells grown on collagen type I show enhanced cell proliferation. We show that components of the ECM, produced by the tumor, contribute to invasiveness and metastasis by reducing E-cadherin-mediated cell-cell adhesion and enhance proliferation in pancreatic tumor cells.

TGFβ-induced downregulation of E-cadherin-based cell-cell adhesion depends on PI3-kinase and PTEN

Transforming growth factor beta (TGFβ) has profound growth-suppressive effects on normal epithelial cells, but supports metastasis formation in many tumour types. In most epithelial tumour cells TGFβ1 treatment results in epithelial dedifferentiation with reduced cell aggregation and enhanced cellular migration. Here we show that the epithelial dedifferentiation, accompanied by dissociation of the E-cadherin adhesion complex, induced by TGFβ1 depended on phosphatidylinositol 3-kinase (PI3-kinase) and the phosphatase PTEN as analysed in PANC-1 and Smad4-deficient BxPC-3 pancreatic carcinoma cells. TGFβ1 treatment enhanced tyrosine phosphorylation of α- and β-catenin, which resulted in dissociation of the E-cadherin/catenin complex from the actin cytoskeleton and reduced cell-cell adhesion. The PI3-kinase and PTEN were found associated with the E-cadherin/catenin complex via β-catenin. TGFβ1 treatment reduced the amount of PTEN bound to β-catenin and markedly increased the tyrosine phosphorylation of β-catenin. By contrast, forced expression of PTEN clearly reduced the TGFβ1-induced phosphorylation of β-catenin. The TGFβ1-induced β-catenin phosphorylation was also dependent on PI3-kinase and Ras activity. The described effects of TGFβ1 were independent of Smad4, which is homozygous deleted in BxPC-3 cells. Collectively, these data show that the TGFβ1-induced destabilisation of E-cadherin-mediated cell-cell adhesion involves phosphorylation of β-catenin, which is regulated by E-cadherin adhesion complex-associated PI3-kinase and PTEN.

The Role of Metastasis-Associated Protein 1 in Prostate Cancer Progression

Distinguishing aggressive prostate cancer from indolent disease represents an important clinical challenge, as current therapy requires over treating men with prostate cancer to prevent the progression of a few cases. Expression of the metastasis-associated protein 1 (MTA1) has previously been found to be associated with progression to the metastatic state in various cancers. Analyzing DNA microarray data, we found MTA1 to be selectively overexpressed in metastatic prostate cancer compared with clinically localized prostate cancer and benign prostate tissue. These results were validated by demonstrating overexpression of MTA1 in metastatic prostate cancer by immunoblot analysis. MTA1 protein expression was evaluated by immunohistochemistry in a broad spectrum of prostate tumors with tissue microarrays containing 1940 tissue cores from 300 cases. Metastatic prostate cancer demonstrated significantly higher mean MTA1 protein expression intensity (score = 3.4/4) and percentage of tissue cores staining positive for MTA1 (83%) compared with clinically localized prostate cancer (score = 2.8/4, 63% positive cores) or benign prostate tissue (score = 1.5/4, 25% positive cores) with a mean difference of 0.54 and 1.84, respectively (P < 0.00001 for both). Paradoxically, for localized disease, higher MTA1 protein expression was associated with lower rates of prostate specific antigen recurrence after radical prostatectomy for localized disease. In summary, this study identified an association of MTA1 expression and prostate cancer progression.

Wound‐healing defect of CD18−/− mice due to a decrease in TGF‐β1 and myofibroblast differentiation

We studied the mechanisms underlying the severely impaired wound healing associated with human leukocyte‐adhesion deficiency syndrome‐1 (LAD1) using a murine disease model. In CD18−/− mice, healing of full‐thickness wounds was severely delayed during granulation‐tissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers α‐smooth muscle actin and ED‐A fibronectin were substantially reduced in wounds of CD18−/− mice, suggesting an impaired myofibroblast differentiation. TGF‐β signalling was clearly involved since TGF‐β1 and TGF‐β receptor type‐II protein levels were decreased, while TGF‐β1 injections into wound margins fully re‐established wound closure. Since, in CD18−/− mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18−/− neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF‐β1. Indeed, in neutrophil–macrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF‐β1 release by macrophages due to defective adhesion to, and subsequent impaired phagocytic clearance of, neutrophils. Our data demonstrates that the paracrine secretion of growth factors is essential for cellular differentiation in wound healing.

Activation of Phosphatidylinositol 3-Kinase and Extracellular Signal-Regulated Kinase Is Required for Glial Cell Line-Derived Neurotrophic Factor-Induced Migration and Invasion of Pancreatic Carcinoma Cells

Pancreatic carcinoma cells exhibit a pronounced tendency to invade along and into intra- and extrapancreatic nerves, even at early stages of the disease. The neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) has been shown to promote pancreatic cancer cell invasion. Here, we demonstrate that pancreatic carcinoma cell lines, such as PANC-1, expressed the RET and GDNF family receptor α receptor components for GDNF and that primary pancreatic tumor samples, derived from carcinomas with regional lymph node metastasis, exhibited marked expression of the mRNA encoding the RET51 isoform. Moreover, GDNF was an efficacious and potent chemoattractant for pancreatic carcinoma cells as examined in in vitro and in vivo model systems. Treatment of PANC-1 cells with GDNF resulted in activation of the monomeric GTPases N-Ras, Rac1, and RhoA, in activation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) and in activation of the phosphatidylinositol 3-kinase/Akt pathway. Both inhibition of the Ras-Raf-MEK (mitogen-activated protein/ERK kinase)-ERK cascade by either stable expression of dominant-negative H-Ras(N17) or addition of the MEK1 inhibitor PD98059 as well as inhibition of the phosphatidylinositol 3-kinase pathway by LY294002 prevented GDNF-induced migration and invasion of PANC-1 cells. These results demonstrate that pancreatic tumor cell migration and possibly perineural invasion in response to GDNF is critically controlled by activation of the Ras-Raf-MEK-ERK and the phosphatidylinositol 3-kinase pathway.

Extracellular matrix is reduced by inhibition of transforming growth factor beta1 in pancreatitis in the rat

BACKGROUND & AIMS Regeneration from cerulein-induced pancreatitis is accompanied by a transient synthesis and deposition of extracellular matrix components in the rat pancreas. The pleiotropic transforming growth factor (TGF)-beta1 has been suggested to regulate extracellular matrix remodeling during regeneration from acute pancreatitis. The present study was designed to verify this hypothesis by investigating the effect of TGF-beta1 inhibition. METHODS Experimental acute pancreatitis was induced in rats by supramaximal doses of cerulein. The biological activity of TGF-beta1 was inhibited by injections of neutralizing TGF-beta1 antibody. Changes in the content of extracellular matrix proteins, TGFs, and their messenger RNA concentrations were monitored. RESULTS TGF-beta1 expression in pancreatic cells was suppressed after induction of acute pancreatitis by the application of neutralizing TGF-beta1 antibody. Immunochemical analysis showed a clear reduction of extracellular matrix formation during the regeneration of the pancreas in antibody-treated animals. The hydroxyproline content and the concentration of collagen types I and III, fibronectin on protein, and messenger RNA level were significantly reduced in the pancreas of treated animals. CONCLUSIONS These results provide evidence that TGF-beta1 is involved in the regulation of extracellular matrix remodeling in the rat pancreas during regeneration from acute pancreatitis.

Growth factor-dependent activation of the Ras-Raf-MEK-MAPK pathway in the human pancreatic carcinoma cell line PANC-1 carrying activated K-ras: implications for cell proliferation and cell migration.

Human ductal adenocarcinoma of the pancreas frequently carry activating point mutations in the K-ras protooncogene. We have analysed the activity of the Ras-Raf-MEK-MAPK cascade in the human pancreatic carcinoma cell line PANC-1 carrying an activating K-ras mutation. Serum-starved cells and cells grown in medium with serum did not show constitutively activated c-Raf, MEK-1, or p42 MAPK. Stimulation of cells with epidermal growth factor (EGF) or fetal calf serum (FCS) resulted in activation of N-Ras, but not K-Ras, as well as activation of c-Raf, MEK-1, and p42 MAPK. Preincubation of serum-starved cells with MEK-1 inhibitor PD98059 abolished EGF- and FCS-induced MAPK activation, identifying MEK as the upstream activator of MAPK. PANC-1 cells exhibited marked serum-dependence of anchorage-dependent and -independent cell growth as well as cell migration. EGF, alone or in combination with insulin and transferrin, did not induce cell proliferation of serum-starved PANC-1 cells, indicating that activation of MAPK alone was not sufficient to induce cell proliferation. FCS-induced DNA synthesis was inhibited by 40% by the MEK-1 inhibitor. On the other hand, treatment with either FCS or EGF alone resulted in marked, MEK-dependent increase of directed cell migration. Collectively, our results show that the activating K-ras mutation in PANC-1 cells does not result in constitutively increased Raf-MEK-MAPK signaling. Signal transduction via the Ras-Raf-MEK-MAPK cascade is maintained in these cells and is required for growth factor-induced cell proliferation and directed cell migration.