Role of Cadherin-mediated Cell-Cell Adhesion in Pancreatic Exocrine-to-Endocrine Transdifferentiation

Kohtaro Minami,Hirotoshi Okano,Akinori Okumachi,Susumu Seino

doi:10.1074/jbc.m710034200

Kohtaro Minami, Hirotoshi Okano + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m710034200

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Abstract

Although pancreatic exocrine acinar cells have the potential to transdifferentiate into pancreatic endocrine cells, the mechanisms are poorly understood. Here we report that intracellular signaling pathways, including those involving MAPK and phosphatidylinositol 3 (PI3)-kinase, are activated by enzymatic dissociation of pancreatic acinar cells and that spherical cell clusters are formed by cadherin-mediated cell-cell adhesion during transdifferentiation. Inhibition of PI3-kinase by LY294002 prevents spheroid formation by degrading E-cadherin and beta-catenin, blocking transdifferentiation into insulin-secreting cells. In addition, neutralizing antibody against E-cadherin suppresses the induction of genes characteristic of pancreatic beta-cells. We also show that loss of cadherin-mediated cell-cell adhesion induces and maintains a dedifferentiated state in isolated pancreatic acinar cells. Thus, disruption and remodeling of cadherin-mediated cell-cell adhesion is critical in pancreatic exocrine-to-endocrine transdifferentiation, in which the PI3-kinase pathway plays an essential role.

Highlights

The phenotypic plasticity of pancreatic cells has been shown in many studies [4, 11, 12]
We found that formation of spherical three-dimensional structures by cadherin-mediated cell-cell adhesion plays a critical role in the induction of ␤-cell-specific gene expression in acinar-derived cells and that PI3kinase activity is involved in both the formation of spheroids and the completion of transdifferentiation into insulin-secreting cells
Intracellular Signals Essential for Transdifferentiation of Pancreatic Acinar Cells—We have previously shown that enzymatic dissociation of pancreas causes tyrosine phosphorylation of the EGF receptor in pancreatic acinar cells, leading to activation of its downstream intracellular signals and enabling the isolated acinar cells to transdifferentiate into insulin-secreting cells even without the addition of EGF [14]

Summary

EXPERIMENTAL PROCEDURES

From Upstate Biotechnology) were cloned into pENTR vector (Invitrogen). Adenoviruses were generated by using ViraPower. Isolation and Culture of Pancreatic Acinar Cells—Pancreatic adenoviral expression system (Invitrogen) according to the manacinar cells were isolated from streptozotocin-injected 8-week- ufacturer’s instructions. Cells were infected with these adenoviold male C57Bl/6 mice as described [14, 17]. Secondary antibodies crine acinar cell-enriched fraction recovered as a pellet was used were Alexa Fluor 488- or 546-conjugated IgGs (Molecular stained with sterilized dithizone solution (0.1 mg/ml) to con- Probes) (1:400). Subcellular fractionation of the cells was performed by using a ProteoExtract subcellular extraction kit (Calbiochem) according to the manufacturer’s instructions. The cells were preincubated in the buffer with 3 mM glucose for 30 min at 37 °C. The solutions were collected to measure insulin concentration by enzyme-linked immunosorbent assay (Shibayagi). Total protein was extracted in 1 N NaOH, and the concentration was determined using Coomassie Brilliant BlueG250 reagent (Bio-Rad Laboratories)

RESULTS

Pancreatic acinar cells are known

DISCUSSION