Secretion polarity of interferon-β in epithelial cell lines

doi:10.1016/S0003-9861(02)00093-0

Archives of Biochemistry and Biophysics

Volume 402, Issue 2, 15 June 2002, Pages 201-207

https://doi.org/10.1016/S0003-9861(02)00093-0 Get rights and content

Abstract

Epithelial cells are an attractive target for local gene delivery in gene therapy for which cytokine genes such as interferon (IFN) genes are promising. However, how the secretion of the gene products is regulated in epithelial cells has been insufficiently investigated. Here, we have studied the secretion polarity of IFN-β expressed via gene transfection in mouse epithelial Pam-T cells on a bicameral culture system. In transient expression, IFN-β was predominantly secreted from the cell membrane side on which the transfection was carried out. Meanwhile, the secretion of constitutive IFN-β from stable transformants was apparently unpolarized. Interestingly, the transformants displayed a polarized secretion of transiently expressed IFN-β in a transfection-side-dependent manner, their stable IFN-β secretion remaining unpolarized. These results suggest that epithelial cells have at least dual protein sorting–secretion pathways, transient and stable, for the same secretory proteins, such as IFNs.

Section snippets

Materials and methods

Cell lines. Mouse epithelial squamous cell carcinoma Pam-T cells [16], [17] were cultured in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 100 U/ml penicillin, $100 μg$ /ml streptomycin (GIBCO–Invitrogen, Carlsbad, CA), and 10% fetal bovine serum (FBS). Mouse L and human FL cells were maintained in Eagle's minimum essential medium (Nissui Pharmaceutical) supplemented with 100 U/ml penicillin, $100 μg$ /ml streptomycin, and 6% FBS and used for the bioassay of mouse and human

Formation of impermeable Pam-T cell monolayers

Pam-T cells seeded on Transwell filters grow to form tight monolayers which do not allow the solute in the medium to cross from one side compartment to the other through the sheet of cells. The process of the monolayer formation was monitored by measuring the TER and observing penetration through the monolayer of trypan blue added into the upper medium as a permeability marker, as shown in Fig. 1A. The TERs increased gradually and reached a plateau after 7–8 days of culture in a

Discussion

In this study, we examined the secretion polarity of IFN-β exogenously expressed in mouse epithelial Pam-T cells using the bicameral culture system. According to a widely accepted model for protein sorting in epithelial cells, glycoproteins are trafficked in the cytoplasm via the endoplasmic reticulum–Golgi vehicle systems, being bridled into the systems by the leader sequence, regulated by other signal sequences for sorting direction, and thereby secreted toward the apical or the basolateral

Acknowledgements

This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

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Secretion polarity of interferon-β in epithelial cell lines

Abstract

Section snippets

Materials and methods

Formation of impermeable Pam-T cell monolayers

Discussion

Acknowledgements

Cell

Curr. Opin. Cell Biol.

Exp. Cell Res.

FEBS Lett.

FEBS Lett.

Biochim. Biophys. Acta

Cancer Lett.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Prog. Nucleic Acid Res. Mol. Biol.

Hum. Gene Ther.

Hum. Gene Ther.