Secretion polarity of interferon-b in epithelial cell lines Kiyo Nakanishi, a Yoshihiko Watanabe, b Masato Maruyama, c Fumiyoshi Yamashita, a Yoshinobu Takakura, c and Mitsuru Hashida a, * a Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan b Department of Molecular Microbiology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan c Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan Received 2 January 2002, and in revised form 2 April 2002 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 inves- tigated. Here, we have studied the secretion polarity of IFN-b expressed via gene transfection in mouse epithelial Pam-T cells on a bicameral culture system. In transient expression, IFN-b was predominantly secreted from the cell membrane side on which the transfectionwascarriedout.Meanwhile,thesecretionofconstitutiveIFN-b fromstabletransformantswasapparentlyunpolarized. Interestingly, the transformants displayed a polarized secretion of transiently expressed IFN-b in a transfection-side-dependent manner, their stable IFN-b secretion remaining unpolarized. These results suggest that epithelial cells have at least dual protein sorting–secretionpathways,transientandstable,forthesamesecretoryproteins,suchasIFNs. Ó 2002ElsevierScience(USA).All rights reserved. Keywords: Epithelial cells; Interferon-b; Secretion polarity; Secretory protein; Transfection Epithelial cells cover various tissues and organs in animal bodies [1,2] and form barriers via cell adherent systems,includingthetightjunction,whichseparatecell surface membranes into two domains, apical and baso- lateral.Thetwodomainsaredistinctfromeachotherin terms of function and composition, or are polarized. Epithelial cells thereby play a fundamental role in the vectorial transport and secretion of proteins in tissues and organs [3–6]. A number of studies on protein se- cretion in cultured epithelial cells have demonstrated various modes of secretion: apical, basolateral, or non- polar secretion. The mode of secretion is primarily de- terminedbysortingsignalsonthepeptide.Forinstance, N-glycans are thought to act as a signal for apical sorting of secretory proteins [7,8], although this is still controversial [9–11]. Thus, epithelial cells, when transferred with a gene encoding secretory protein, will secrete the gene product from the apical and/or basolateral membrane, depending on the nature of the protein.Therefore,epithelialcellsareconsideredtobea potential target for local delivery of genes for secre- tory proteins such as cytokines, including interferon (IFN), 1 which have therapeutic activities. Little is known, however, about the secretion polarity of the gene products. IFNs exhibit a wide range of biological activities, includingantiviraleffects,inhibitionofcellproliferation, regulation of cell differentiation, and modulation of the immune system [12,13], and they have been used clini- cally as antiviral and antitumor reagents. In addition, IFN gene transfer to tumors is being attempted for therapeutic purposes [14,15]. Hence, IFN genes are a promising candidate for gene transfer into epithelial cells for therapeutic purposes. The secretion polarity of Archives of Biochemistry and Biophysics 402 (2002) 201–207 www.academicpress.com ABB * Corresponding author. Fax: +81-75-753-4575. E-mail address: hashidam@pharm.kyoto-u.ac.jp (M. Hashida). 1 Abbreviations used: IFN,interferon;FBS,fetalbovineserum;PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; TER, transepithelial electrical resistance; MDCK, Madin–Darby canine kidney. 0003-9861/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0003-9861(02)00093-0