Matrix metalloproteinase 7 controls pancreatic acinar cell transdifferentiation by activating the Notch signaling pathway - PubMed (original) (raw)

Matrix metalloproteinase 7 controls pancreatic acinar cell transdifferentiation by activating the Notch signaling pathway

Eric T Sawey et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Acinar-to-ductal metaplasia in the pancreas is associated with an increased risk for tumorigenesis. Molecular dissection of this process in vitro has shown that primary acinar cells, in response to EGF receptor ligands, can transdifferentiate into duct-like epithelia, passing through a nestin-positive intermediate, in a Notch pathway-dependent manner. Here, we show that in vitro acinar transdifferentiation depends on matrix metalloproteinase 7 (MMP-7), a proteinase expressed in most metaplastic epithelia in vivo. MMP-7 was found to be required for Notch activation, which leads to dedifferentiation of acinar cells to the nestin-positive transitional cell. Besides being necessary for acinar transdifferentiation, it was found that MMP-7 activity was sufficient to induce the process, indicating that molecular signals capable of initiating MMP-7 expression also have the potential to induce formation of metaplastic epithelia in the pancreas.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

MMP-7 is expressed in primary acinar cells treated with TGF-α. Pancreatic explants from wild-type mice embedded in collagen were treated with TGF-α. MMP-7 expression (green) was confirmed by immunofluorescence 1 (A), 2 (B), and 3 days (C) after culture, respectively. Staining was evident in the majority of acini on day 2. (Scale bar, 50 μm.) Cells were costained for amylase (red) and DAPI (blue). Data are representative of five independent experiments.

Fig. 2.

Fig. 2.

MMP-7 is required for acinar-to-ductal transdifferentiation. Primary acinar cells from wild-type (A–C) and MMP-7−/− (D–I) mice were embedded in collagen and treated with TGF-α. Coimmunofluorescence for the acinar cell marker amylase (red) and the duct cell marker cytokeratin-19 (green) was performed on cells fixed on day 0 (A, D, and G), day 3 (B, E, and H), and day 5 (C, F, and I). TGF-α treatment of wild-type cultures induced acinar-to-ductal transdifferentiation by day 5 (C). TGF-α treatment of MMP-7−/− acinar cells showed virtually no conversion to ductal structures (D–F). Addition of 200 ng/ml rMMP-7 with TGF-α to MMP-7−/− acinar cells (G–I) restored transdifferentiation. (Scale bar, 100 μm.) DAPI is shown in blue. Data are representative of >10 independent experiments.

Fig. 3.

Fig. 3.

Notch and MMP-7 are required for transition to the nestin-positive intermediate. Nestin immunofluorescence (green) was detectable on day 3 in wild-type cultures treated with DMSO (A), but not in cultures treated with 20 μM the γ-secretase inhibitor, WPE III-31C (GSI) (B). Infection with an adenovirus encoding a constitutively active V5-tagged Notch-1 intracellular domain (Ad-N1ICD-V5) bypassed the GSI, allowing nestin expression (C). Day 3 MMP-7−/− cultures did not show nestin immunoreactivity (D). (Scale bar, 50 μm.) Shown are amylase in red and DAPI in blue. Data are representative of at least three independent experiments.

Fig. 4.

Fig. 4.

Constitutively active Notch bypasses the requirement for MMP-7 in acinar transdifferentiation. Primary acinar cells from wild-type (A–F) or MMP-7−/− mice (G–L) were infected either with Ad-GFP, an adenovirus encoding GFP (A–C and G–I) or Ad-N1ICD-V5 (D–F and J–L) and embedded in collagen. After 3 days in culture, transdifferentiation in N1ICD cultures was evident (D and J) and was coincident with successful infection, confirmed by V5 immunofluorescence (E and K). Transdifferentiation was confirmed by immunofluorescence for CK-19 (F and L). (Scale bar, 50 μm.) DAPI is shown in blue. Data are representative of three independent experiments.

Fig. 5.

Fig. 5.

MMP-7 activity is sufficient to induce Notch-dependent acinar-to-ductal transdifferentiation. (A and B) Primary acinar cells from wild-type mice were treated with 200 ng/ml rMMP-7 along with DMSO (A) or 20 μM WPE III-31C (GSI) (B). Coimmunofluorescence for amylase (red) and CK-19 (green) was performed on day 5. (C and D) Primary acinar cells infected with an adenovirus encoding a dominant-negative RBP-Jκ also blocked transdifferentiation induced by coinfection with Ad-N1ICD-V5 (amylase in red, V5 in green) (C) or by rMMP-7 (amylase in red, CK-19 in green) (D). (Scale bar, 50 μm.) DAPI is shown in blue. Data are representative of at least three independent experiments.

Fig. 6.

Fig. 6.

MMP-7 activity induces γ-secretase cleavage of Notch-1. (A–C) Immunoblots for the γ-secretase-cleaved form of Notch-1 (Cleaved N1 Val-1744). (A) COS-7 cells expressing Notch-1 were treated with 100 ng/ml pro-MMP-7, 50 ng/ml active rMMP-7 with DMSO, or 20 μM WPE III-31C. (B) COS-7 cells expressing Notch-1 were treated with 200 ng/ml rMMP-7 for 0, 2, 4, 6, and 18 h. (C) COS-7 cells expressing Notch-1 were treated with varied rMMP-7 concentrations for 4 h. (D) COS-7 cells expressing Notch-1 with C-terminal V5 were treated with 200 ng/ml for 18 h. Data are representative of five independent experiments.

Fig. 7.

Fig. 7.

MMP-7 activity leads to nuclear translocation of N1ICD and expression of hes-1. (A and B) COS-7 cells expressing full-length Notch-1 with a C-terminal V5 tag were treated for 4 h with MMP-7. Shown is immunofluorescence for Cleaved N1 Val-1744 antibody (green) and V5 antibody (red). (Scale bar, 20 μm.) Cells were treated for 4 h with medium alone (A) or with rMMP-7 (B). Arrows indicate cells with nuclear cleaved Notch-1. DAPI is shown in blue. (C) Quantification of Notch-1 nuclear translocation. Percentage represents cells with nuclear Notch-1 divided by total cells expressing Notch-1. (D) RT-PCR for hes1 from COS-7 cells expressing Notch-1 treated with MMP-7 or EDTA. Numbers represent fold expression relative to medium alone. Data are representative of five independent experiments.

Fig. 8.

Fig. 8.

MMP-7 is required for Notch activation in acinar explants. (A–C) Acinar explants immunostained for Cleaved N1 Val-1744 (green) and amylase (red) on day 3. Wild-type acinar cells exhibited Notch cleavage (A), whereas MMP-7−/− cells did not (B). Inclusion of 200 ng/ml rMMP-7 rescued Notch cleavage in MMP-7−/− acinar cells (C). (Scale bar, 50 μm.) DAPI is shown in blue. (D) RT-PCR for hes1 and hey1 showed increased Notch activation in wild-type acinar cells and MMP-7−/− cells treated with rMMP-7, relative to MMP-7−/− cells. Data are representative of five independent experiments.

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