TGFβ1 autocrine growth control in isolated pancreatic fibroblastoid cells/stellate cells in vitro

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Abstract

TGFβ1 is a multifunctional factor, controlling cellular growth and extracellular matrix production. Deletion of the TGFβ1 gene in mice results in multiple inflammatory reactions. Targeted overexpression of TGFβ1 in pancreatic islet cells leads to fibrosis of the exocrine pancreas in transgenic mice. In pancreatic fibrosis interstitial fibroblasts are primary candidates for production and deposition of extracellular matrix. Still, little is known about regulation of these cells during development of pancreatic disease. We established primary cell lines of pancreatic fibroblastoid/stellate cells (PFC) from rat pancreas. Investigation of rPFCs in vitro shows TGFβ1 expression by RT-PCR analysis. Mature TGFβ1 was detected in culture supernatants by immunoassay. Rat PFCs in culture possess both receptors TGFβ receptor type I, and type II, necessary for TGFβ1 signal transduction. Inhibition of TGFβ1 activity by means of neutralizing antibodies interferes with an autocrine loop and results in a 2-fold stimulation of cell growth. So far, pancreatic fibroblastoid/stellate cells in vitro were known as a target of TGFβ1 action, but not as a source of TGFβ1. Our data indicate TGFβ1 activity in rat pancreas extends beyond regulation of matrix production, but appears to be important in growth control of pancreatic fibroblastoid cells.

Introduction

Pancreatic fibrosis is seen in chronic pancreatitis and pancreatic cancer. Fibrosis is characterized by replacement of functional tissue by connective tissue consisting mostly of densely packed extracellular matrix (ECM). Thus fibrosis appears to result from dysregulation of cells producing extracellular matrix. One of the factors assumed responsible for regulation of fibroblast cell function and matrix deposition is transforming growth factor-β (TGFβ) [1], [2], [3], [4]. In pancreas TGFβ has been functionally investigated in experimental pancreatitis in rodents [5], [6], [7]. Investigation of TGFβ expression in human pancreatitis was carried out mostly by immunohistochemistry [8], [9], [10]. Generally, expression of TGFβ1 in pancreatic tissue is predominantly described in ductal and acinar cells. These findings implicate paracrine mechanisms for regulation of cells responsible for extracellular matrix (ECM) production, with parenchymal cells controlling fibroblasts.

We have established long-term culture of pancreatic fibroblastoid/stellate cells from rat pancreas. Calling these cells pancreatic fibroblastoid cells/stellate cells (PFC) pays respect to isolation and long-term culture [11]. While other investigators have analysed the effect of exogenously added TGFβ on pancreatic stellate cells regarding production of extracellular matrix [12], [13], [14], [15], [16], we were mainly interested in the question of whether these cells themselves express TGFβ. We describe here the production of biologically active TGFβ1 by fibroblastoid/stellate cells from rat pancreas in vitro. Furthermore we provide evidence for an autocrine loop for TGFβ1 activity derived from rPFCs. Inhibition of autocrine TGFβ1 activity by neutralising antibodies results in growth stimulation in vitro, depending on cell density.

Section snippets

Materials and methods

All chemicals were of highest analytical purity purchased from Sigma (Deisenhofen, Germany), Biomol (Hamburg, Germany) or Merck (Darmstadt, Germany). All tissue culture reagents were from Life Technologies (Karlsruhe, Germany), unless otherwise stated. Secondary antibodies were fluorescein (FITC)-coupled donkey anti-goat antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Alkaline phosphatase-coupled anti-rabbit secondary antibodies were from New England Biolabs (Beverly, MA).

Results

We have investigated TGFβ production and activity in rat pancreatic fibroblastoid/stellate cells (rPFCs) grown on collagen matrix. The cells were positive for vimentin, a marker for mesenchymal origin. Furthermore, these cells express α-smooth muscle actin (αSMA) and desmin as described before [11].

Discussion

TGFβ is a multifunctional growth/differentiation factor which has been investigated extensively in pancreas, and was mainly found in parenchymal cells by in situ hybridisation and immunohistochemistry [5], [8], [9], [10]. A major target of TGFβ1 activity are cells producing extracellular matrix [3], [4]. Detection of immunoreactivity for TGFβ1 or its mRNA suggests parenchymal expression and paracrine activity. We were most interested, whether isolated rat pancreatic fibroblastoid cells/stellate

Acknowledgements

Parts of the work described here are results from the doctoral thesis of PBH and CT.

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