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