F. Müller-Pillasch

Enhancement of transforming growth factor β1 expression in the rat pancreas during regeneration from caerulein-induced pancreatitis

Abstract Synthesis of extracellular matrix components is enhanced in the rat pancreas during regeneration from caerulein‐induced pancreatitis. To study the involvement of transforming growth factor β1 (TGFβ1), one of the most potent modulators of the extracellular matrix, in the process of pancreatic regeneration we examined the expression of this gene on the transcript and protein level. Pancreatic RNA was extracted from rats killed 0h, 12h, 24h, 2, 3 and 7 days after induction of caerulein pancreatitis. Transcript levels for TGFβ1 were measured by slot–blot analysis and mRNA in situ hybridization. Total amount of TGFβ1‐protein was measured using a radioligand binding assay. TGFβ1 protein was increased twofold after 24h and 48h and returned to control values 7 days after induction of pancreatitis, TGFβ1‐mRNA reached maximal values (3‐fold over controls) after 2 days. The largest amount of TGFβ1‐mRNA was found in pancreatic acinar cells and in stromal cells. In summary, expression of TGFβ1 in acinar and stromal cells of the rat pancreas is enhanced during regeneration from caerulein‐induced pancreatitis, which may indicate an involvement of TGFβ1 in the regulation of extracellular matrix regeneration in the rat pancreas after caerulein‐induced pancreatitis.

Identification of genes with specific expression in pancreatic cancer by cDNA representational difference analysis

cDNA representational difference analysis (cDNA‐RDA) is a polymerase‐chain‐reaction‐coupled subtractive and kinetic enrichment procedure for the isolation of differentially expressed genes. In this study, the technique was used to isolate novel genes specifically expressed in pancreatic cancer. cDNA‐RDA was done on cDNA reverse transcribed from a poly(A)+ mRNA pool made from 10 cancer tissues (tester) by using as a driver a cDNA from a poly(A)+ mRNA pool made from a combination of 10 tissues of chronic pancreatitis and 10 healthy pancreatic tissues. The use of chronic pancreatitis in addition to healthy pancreas mRNA in the driver preparation eliminated the influence of stromal tissue components present as contamination in the cancer‐specific preparations. Such cDNA‐RDA led to the isolation of 16 distinct, cancer‐specific gene fragments. These were confirmed to be overexpressed in pancreatic cancer tissues by Northern blot analysis. Sequence analysis revealed homologies to five genes previously implicated in the carcinogenesis of the pancreas or other tissues. Eleven fragments had no significant homology to any known gene and thus represent novel candidate disease genes. The experiments demonstrate that cDNA‐RDA is a reproducible and highly efficient method for the identification of novel genes with cancer‐specific expression. Genes Chromosom. Cancer 19:97–103, 1997.

Role of Extracellular Matrix in Pancreatic Diseases

Accessible online at: http://BioMedNet.com/karger The extracellular matrix (ECM) has been traditionally thought of as the structurally stable material that provides support for cells and tissues. However, a number of discoveries over the past decades have changed this view. First, it has been shown that four major classes of macromolecules – the collagens, proteoglycans, structural glycoproteins, and elastin – collectively comprise the ECM of animal cells [1–6]. Furthermore, with the exception of elastin, each class of matrix macromolecules has been found to contain families of related proteins with each member being a unique gene product. Second, individual members of each class and family of ECM molecules were found to exhibit a degree of tissue-specific distribution implicating the matrix in development and tissue function [7, 8]. Third, specific cell-surface receptors for ECM components were identified, which provided a rational basis for linking the ECM with the cell [9–11]. From these discoveries it is now evident that the extracellular matrix is composed of a number of different macromolecules whose structural integrity and functional composition are important in maintaining normal tissue architecture, in development and in tissue-specific function [2, 4, 6, 8, 12]. Finally, it has been recognized that dysfunctional matrix components and abnormalities in ECM biosynthesis and catabolism are of importance in both inherited and acquired diseases and in normal wound healing [2, 3, 5, 6]. In particular pancreatic diseases as, e.g., pancreatic cancer and chronic pancreatitis are characterized by profound alterations of ECM formation and composition. The following review summarizes some of the major aspects that have emerged in the recent years concerning composition, formation and regulation of the ECM in human pancreatic diseases and in experimental models of pancreatic fibrosis.

Cloning of a new Kunitz-type protease inhibitor with a putative transmembrane domain overexpressed in pancreatic cancer

In a previous large scale screen for differentially expressed genes in pancreatic cancer, we identified a gene highly overexpressed in cancer encoding a novel putative transmembrane protein with two Kunitz-type serine protease inhibitor domains. The identified gene named kop (Kunitz domain containing protein overexpressed in pancreatic cancer) was assigned to chromosome 19 in the region 19q13.1. Kop was detected at high levels in pancreatic cancer cell lines and was overexpressed in pancreatic cancer tissues as compared to both, normal pancreas and chronic pancreatitis tissues. Being a member of the Kunitz-type serine protease inhibitor family, this new gene may participate in tumour cell invasion and metastasis and in the development of the marked desmoplastic reaction typical for human pancreatic cancer tissues. In this context, the fact that kop has a putative transmembrane domain may have functional implications of particular interest.

The Influence of Transforming Growth Factor β1 on the Expression of Genes Coding for Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases During Regeneration from Cerulein-Induced Pancreatitis

Enhanced synthesis and deposition of extracellular matrix (ECM) components is a characteristic feature during regeneration from acute cerulcin-induced pancreatitis in rats. Transforming growth factor β1 (TGFβ1) has been suggested to be an important modulator of the ECM by interfering with a number of essential processes such as the synthesis of ECM components. To study the involvement of the ECM degrading proteases (matrix metalloproteinases; MMPs) and their specific inhibitors in the process of pancreatic regeneration, we examined the expression of these genes on the transcript level and the activation of the corresponding enzymes by use of zymographies. Pancreatic RNA and protein were extracted from rats sacrificed 1, 2, 3, 5, and 7 days after induction of cerulein pancreatitis. To investigate the influence of TGFβ on gene expression of ECM proteases and their specific inhibitors, we blocked the activity of TGFβ1 during regeneration from acute pancreatitis by use of neutralizing antibodies against TGFβ1. Steady levels of 72-kD type IV collagenase (MMP-2), stromelysin (MMP-3), and tissue inhibitor of metalloproteinase 2 (TIMP-2) mRN A were significantly increased 2 days after induction of pancreatitis. MMP-9 and MMP-3 enzyme activity was elevated 12 h after induction of pancreatitis, whereas MMP-2 activity increased 12 h later. Inhibition of TGFβ1 by neutralizing antibodies only reduced the amount of stromelysin transcripts throughout pancreatic regeneration. In summary, ECM degrading proteases, in particular stromelysin, appear to be involved in ECM remodeling during pancreatic regeneration. TGFβ1 may be responsible for regulation of stromelysin transcription.

Supramaximal Secretagogue Stimulation Enhances Heat Shock Protein Expression in the Rat Pancreas

Heat shock or stress proteins (HSPs) are synthesized by various cell types in response to different metabolic insults (e.g., hyperthermia). Although the function of HSPs is not fully understood, they are believed to be an evolutionary conserved intracellular defense mechanism. In an attempt to characterize the autoprotective potential of pancreatic acinar cells, we investigated the regulation of HSPs of the 70-kD family and the small HSP ubiquitin in vitro and in vivo during supramaximal cerulein stimulation. Infusion of the secretagogue cerule-in induces a mild edematous form of pancreatitis in vivo and is characterized by a marked disturbance of the intracellular transport and segregation of enzymes. Synthesis of HSP70 mRN A is upregulated in isolated pancreatic lobules by either cerulein (100 nM) or hyperthermia (42°C for 60 min). In contrast, expression of ubiquitin mRNA was not altered by either secretagogue treatment or hyperthermia. This heat shock-like response of pancreatic acinar cells could be reproduced in vivo: Pancreatitis was induced in male Wistar rats by intravenous infusion of supramaximal doses of cerulein (10 μg/kg/h). Analysis of mRNA expression revealed a significant upregulation of HSP70 RNA during supramaximal secretagogue stimulation. mRNA levels encoding for ubiquitin remained unchanged. Western blot analysis demonstrated that the transcriptional upregulation of HSP70 in vivo was reflected on the protein level. This study demonstrates that the marked intracellular disturbance observed in secretagogue-induced pancreatitis is associated with enhanced expression and synthesis of a major stress protein. Given the autoprotective potential of HSPs, this upregulation may indicate a self-defense mechanism of pancreatic acinar cells in experimental pancreatitis.

Extracellular Matrix in Pancreatic Diseases

It has been suggested that extracellular matrix (ECM) components play an important role in the strong desmoplastic reaction observed in pancreatic cancer, which is characterised by remarkable proliferation of interstitial connective tissue [1]. Quantitative analysis of collagen subtypes I, III and V in human pancreatic cancer, tumour-associated chronic pancreatitis (TACP) and alcoholic chronic pancreatitis (AICP) showed that mean collagen content was elevated in both pancreatic cancer and TACP tissue and to a lesser extent in AICP. The compositional proportions of type I, III and V appear to be similar in all tissues examined compared to normal pancreatic tissues [2]. In grade I and II tumours of the pancreas, collagen type I and fibronectin form small septa between tumorous structures whereas in grade III tumours, fibronectin and collagen type I were present as unoriented thick fibrous bundles. In large areas of the tumour, connective tissue was the main constituent of the ECM [3]. Laminin, a major component of basement membranes, was found only in well-differentiated pancreatic tumour tissue whereas, with progressive dedifferentiation, basal laminin was absent or discontinuously expressed [3, 4]. Similar expression patterns of basal laminar components were observed in colorectal carcinoma [5] and breast carcinoma [6]. In vitro cultivation of pancreatic carcinoma cell lines on laminin substrates resulted in an arrest of proliferation, while normal proliferation was found on collagen type I and fibronectin substrates [3].