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Autocrine stimulation of human pancreatic duct-like development by soluble isoforms of epimorphin in vitro.

Lehnert L, Lerch MM, Hirai Y, Kruse ML, Schmiegel W, Kalthoff H - J. Cell Biol. (2001)

Bottom Line: A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells.Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5-6-d-old hollow spheres.A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology, Department of General and Thoracic Surgery, Christian-Albrechts-University, Kiel, Germany.

ABSTRACT
Epimorphin was recently described as a mesenchymal factor modulating morphogenesis of murine mammary ducts, skin, liver, and lung in vitro. In this study epimorphin was analyzed in a human, pancreatic adenocarcinoma cell line (A818-6) which develops single layer epithelial hollow spheres resembling normal pancreatic ductal structures in vitro. Soluble 34- and 31-kD isoforms of epimorphin were found in the culture supernatant of A818-6 cells. In lysates of A818-6 cells we detected the 34-and 31-kD isoforms and the dimers, and in lysates of fibroblasts the 150-kD tetramers of epimorphin additionally. A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells. Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5-6-d-old hollow spheres. A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1. In this novel system for human duct-like differentiation of pancreatic epithelial cells, we provide evidence for an autocrine and paracrine function of epimorphin as a major mediator for morphogenesis.

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Detection of epimorphin in protein lysates of pancreatic carcinoma cell lines and fibroblasts. (A) Western blot analyses of epimorphin in protein lysates of A818-6 monolayer cells (lane 1), hollow sphere cells (lane 2), and Kif-5 fibroblasts (lane 3). Monolayer cells expressed the 34- and 31-kD isoforms, whereas hollow sphere cells lacked the 31-kD isoform. Fibroblasts expressed larger amounts of the 31-kD isoforms of epimorphin than of the 34-kD isoforms (lane 3). (B) The 150-kD tetrameric complex, 70-kD dimer, and monomers of epimorphin detected in the lysate of Kif-5 fibroblasts. (C) Western blot analysis of epimorphin in protein lysates of the pancreatic carcinoma cell lines Capan-1 (lane 2), Capan-2 (lane 3), A818-4 (lane 4), Panc-1 (lane 5), BxPC-3 (lane 6), and A818-6 hollow sphere cells (lane 1) as control; it was also taken as negative control by omitting the primary antibody (lane 7). All cell lines which were capable of lumen formation (A818-6, Capan-2, BxPC-3) expressed the 34-kD isoforms of epimorphin occasionally together with the 31-kD isoform. Cell lines which did not develop lumenal structures (Capan-1, A818-4, Panc-1) expressed no epimorphin or only the 31-kD isoform. Epimorphin isoforms are indicated by arrows.
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Figure 9: Detection of epimorphin in protein lysates of pancreatic carcinoma cell lines and fibroblasts. (A) Western blot analyses of epimorphin in protein lysates of A818-6 monolayer cells (lane 1), hollow sphere cells (lane 2), and Kif-5 fibroblasts (lane 3). Monolayer cells expressed the 34- and 31-kD isoforms, whereas hollow sphere cells lacked the 31-kD isoform. Fibroblasts expressed larger amounts of the 31-kD isoforms of epimorphin than of the 34-kD isoforms (lane 3). (B) The 150-kD tetrameric complex, 70-kD dimer, and monomers of epimorphin detected in the lysate of Kif-5 fibroblasts. (C) Western blot analysis of epimorphin in protein lysates of the pancreatic carcinoma cell lines Capan-1 (lane 2), Capan-2 (lane 3), A818-4 (lane 4), Panc-1 (lane 5), BxPC-3 (lane 6), and A818-6 hollow sphere cells (lane 1) as control; it was also taken as negative control by omitting the primary antibody (lane 7). All cell lines which were capable of lumen formation (A818-6, Capan-2, BxPC-3) expressed the 34-kD isoforms of epimorphin occasionally together with the 31-kD isoform. Cell lines which did not develop lumenal structures (Capan-1, A818-4, Panc-1) expressed no epimorphin or only the 31-kD isoform. Epimorphin isoforms are indicated by arrows.

Mentions: All forms of epimorphin described previously by others were detected with an affinity-purified antibody against epimorphin, as assessed by Western blot analysis (Fig. 9A and Fig. B). The 150-kD tetramer of epimorphin was only detected in the lysates of fibroblasts (Fig. 9 B), whereas it was not detectable by Western blot analysis in lysates of A818-6 monolayer cells and hollow sphere cells. No difference in the expression pattern of 70-kD dimers was observed upon comparison of fibroblasts, A818-6 monolayer cells, and A818-6 hollow sphere cells. Differences were observed in the expression pattern of monomeric 34- and 31-kD isoforms of epimorphin. All monomeric isoforms were expressed in A818-6 monolayer cells whereas 3D hollow spheres exclusively expressed the larger 34-kD isoforms. Fibroblasts expressed larger amounts of the smaller, 31-kD isoform, than of the 34-kD isoforms (Fig. 9 A). Subsequently, we compared cell lines that did not develop lumenal structures with cell lines which grew with a lumenal phenotype under our cell culture conditions. All cell lines capable of lumen formation expressed the larger 34-kD isoforms of epimorphin occasionally together with the 31-kD isoform. Cell lines not capable of lumen formation expressed only the smaller 31-kD isoform or failed to express any (Fig. 9 C). The cell lines Capan-2 and BxPC-3 that expressed the 34-kD isoforms responded upon fibroblast coculture with stimulation of the development of lumenal structures. Cell lines expressing only the 31-kD isoform or none (Capan-1, A818-4, Panc-1) showed no induction of lumen formation in fibroblast coculture (data not shown).


Autocrine stimulation of human pancreatic duct-like development by soluble isoforms of epimorphin in vitro.

Lehnert L, Lerch MM, Hirai Y, Kruse ML, Schmiegel W, Kalthoff H - J. Cell Biol. (2001)

Detection of epimorphin in protein lysates of pancreatic carcinoma cell lines and fibroblasts. (A) Western blot analyses of epimorphin in protein lysates of A818-6 monolayer cells (lane 1), hollow sphere cells (lane 2), and Kif-5 fibroblasts (lane 3). Monolayer cells expressed the 34- and 31-kD isoforms, whereas hollow sphere cells lacked the 31-kD isoform. Fibroblasts expressed larger amounts of the 31-kD isoforms of epimorphin than of the 34-kD isoforms (lane 3). (B) The 150-kD tetrameric complex, 70-kD dimer, and monomers of epimorphin detected in the lysate of Kif-5 fibroblasts. (C) Western blot analysis of epimorphin in protein lysates of the pancreatic carcinoma cell lines Capan-1 (lane 2), Capan-2 (lane 3), A818-4 (lane 4), Panc-1 (lane 5), BxPC-3 (lane 6), and A818-6 hollow sphere cells (lane 1) as control; it was also taken as negative control by omitting the primary antibody (lane 7). All cell lines which were capable of lumen formation (A818-6, Capan-2, BxPC-3) expressed the 34-kD isoforms of epimorphin occasionally together with the 31-kD isoform. Cell lines which did not develop lumenal structures (Capan-1, A818-4, Panc-1) expressed no epimorphin or only the 31-kD isoform. Epimorphin isoforms are indicated by arrows.
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Related In: Results  -  Collection

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Figure 9: Detection of epimorphin in protein lysates of pancreatic carcinoma cell lines and fibroblasts. (A) Western blot analyses of epimorphin in protein lysates of A818-6 monolayer cells (lane 1), hollow sphere cells (lane 2), and Kif-5 fibroblasts (lane 3). Monolayer cells expressed the 34- and 31-kD isoforms, whereas hollow sphere cells lacked the 31-kD isoform. Fibroblasts expressed larger amounts of the 31-kD isoforms of epimorphin than of the 34-kD isoforms (lane 3). (B) The 150-kD tetrameric complex, 70-kD dimer, and monomers of epimorphin detected in the lysate of Kif-5 fibroblasts. (C) Western blot analysis of epimorphin in protein lysates of the pancreatic carcinoma cell lines Capan-1 (lane 2), Capan-2 (lane 3), A818-4 (lane 4), Panc-1 (lane 5), BxPC-3 (lane 6), and A818-6 hollow sphere cells (lane 1) as control; it was also taken as negative control by omitting the primary antibody (lane 7). All cell lines which were capable of lumen formation (A818-6, Capan-2, BxPC-3) expressed the 34-kD isoforms of epimorphin occasionally together with the 31-kD isoform. Cell lines which did not develop lumenal structures (Capan-1, A818-4, Panc-1) expressed no epimorphin or only the 31-kD isoform. Epimorphin isoforms are indicated by arrows.
Mentions: All forms of epimorphin described previously by others were detected with an affinity-purified antibody against epimorphin, as assessed by Western blot analysis (Fig. 9A and Fig. B). The 150-kD tetramer of epimorphin was only detected in the lysates of fibroblasts (Fig. 9 B), whereas it was not detectable by Western blot analysis in lysates of A818-6 monolayer cells and hollow sphere cells. No difference in the expression pattern of 70-kD dimers was observed upon comparison of fibroblasts, A818-6 monolayer cells, and A818-6 hollow sphere cells. Differences were observed in the expression pattern of monomeric 34- and 31-kD isoforms of epimorphin. All monomeric isoforms were expressed in A818-6 monolayer cells whereas 3D hollow spheres exclusively expressed the larger 34-kD isoforms. Fibroblasts expressed larger amounts of the smaller, 31-kD isoform, than of the 34-kD isoforms (Fig. 9 A). Subsequently, we compared cell lines that did not develop lumenal structures with cell lines which grew with a lumenal phenotype under our cell culture conditions. All cell lines capable of lumen formation expressed the larger 34-kD isoforms of epimorphin occasionally together with the 31-kD isoform. Cell lines not capable of lumen formation expressed only the smaller 31-kD isoform or failed to express any (Fig. 9 C). The cell lines Capan-2 and BxPC-3 that expressed the 34-kD isoforms responded upon fibroblast coculture with stimulation of the development of lumenal structures. Cell lines expressing only the 31-kD isoform or none (Capan-1, A818-4, Panc-1) showed no induction of lumen formation in fibroblast coculture (data not shown).

Bottom Line: A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells.Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5-6-d-old hollow spheres.A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology, Department of General and Thoracic Surgery, Christian-Albrechts-University, Kiel, Germany.

ABSTRACT
Epimorphin was recently described as a mesenchymal factor modulating morphogenesis of murine mammary ducts, skin, liver, and lung in vitro. In this study epimorphin was analyzed in a human, pancreatic adenocarcinoma cell line (A818-6) which develops single layer epithelial hollow spheres resembling normal pancreatic ductal structures in vitro. Soluble 34- and 31-kD isoforms of epimorphin were found in the culture supernatant of A818-6 cells. In lysates of A818-6 cells we detected the 34-and 31-kD isoforms and the dimers, and in lysates of fibroblasts the 150-kD tetramers of epimorphin additionally. A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells. Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5-6-d-old hollow spheres. A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1. In this novel system for human duct-like differentiation of pancreatic epithelial cells, we provide evidence for an autocrine and paracrine function of epimorphin as a major mediator for morphogenesis.

Show MeSH
Related in: MedlinePlus