Arachidonic acid-induced expression of the organic solute and steroid transporter-beta (Ost-beta) in a cartilaginous fish cell line

Abstract

The organic solute and steroid transporter (OST/Ost) is a unique membrane transport protein heterodimer composed of subunits designated alpha and beta, that transports conjugated steroids and prostaglandin E(2) across the plasma membrane. Ost was first identified in the liver of the cartilaginous fish Leucoraja erinacea, the little skate, and subsequently was found in many other species, including humans and rodents. The present study describes the isolation of a new cell line, LEE-1, derived from an early embryo of L. erinacea, and characterizes the expression of Ost in these cells. The mRNA size and amino acid sequence of Ost-beta in LEE-1 were identical to that previously reported for Ost-beta from skate liver, and the primary structure was identical to that of the spiny dogfish shark (Squalus acanthias) with the exception of a single amino acid. Ost-beta was found both on the plasma membrane and intracellularly in LEE-1 cells, consistent with its localization in other cell types. Interestingly, arachidonic acid, the precursor to eicosanoids, strongly induced Ost-beta expression in LEE-1 cells and a lipid mixture containing arachidonic acid also induced Ost-alpha. Overall, the present study describes the isolation of a novel marine cell line, and shows that this cell line expresses relatively high levels of Ost when cultured in the presence of arachidonic acid. Although the function of this transport protein in embryo-derived cells is unknown, it may play a role in the disposition of eicosanoids or steroid-derived molecules.

Figure 1

Reverse-transcription PCR of mRNA from skate liver and the LEE-1 skate embryo cell line. Primer sequences are listed in Table 1. (1), skate liver primers, 1-Forward/1-Reverse primers; (2), skate liver, primers 2-Forward/2-Reverse; (3), skate liver, primers 3-Forward/3-Reverse; (4) control (no sample) (5), molecular weight markers; (6), LEE-1 cell line, primers 1-Forward/1-Reverse; (7), LEE-1 cell line, primers 2-Forward/2-Reverse; (8), LEE-1 cell line, primers 3-Forward/3-Reverse. PCR was carried out as described in Materials and Methods.

Figure 2

Northern blot of Ost-beta mRNA in skate liver and LEE-1 cell line. (A), LEE-1 cell line; (B), skate liver. Procedures were as described in Materials and Methods.

Figure 3

Immunohistochemical detection of Ost-beta in LEE-1 cells. (1), cells treated with anti-skate Ost beta antiserum ; (2), phase photomicrograph of the same field; (3), cells treated with preimmune serum ; (4), phase photomicrograph of the same field. Immunolocalization of Ost-beta was carried out as described in Materials and Methods.

Figure 4

Quantitative PCR analysis of Ost-beta in skate tissue. Primer set used was F1/R1 as shown in Table 1. Procedures are given in Materials and Methods.

Figure 5

Sequence alignment of primary structure of Ost-beta. Bold shadings indicate amino acid identity to the skate sequence; gray shadings indicate identity among amino acids of the other species.

Figure 6

Expression of Ost in LEE-1 cells in vitro requires the chemically defined lipid mixture (CDL). Cells were cultured for 4 days in the full culture medium with all supplements, or under a series of conditions in which each of seven medium supplements listed below were individually omitted from the medium. Total RNA was isolated from each sample and abundance of Ost-beta mRNA estimated by RT-PCR with elongation factor 1-alpha as control. (1), all medium components present; (2), fetal bovine serum omitted; (3), insulin omitted; (4), supplementary glutamine omitted; (5), transferrin omitted; (6), CDL omitted; (7), fibroblast growth factor omitted; (8), epidermal growth factor omitted.

Figure 7

Induction of Ost-beta in LEE-1 cells by arachidonic acid. Cultures in unsupplemented, basal nutrient media were incubated in the presence or absence of CDL components individually for 4 days and Ost-beta expression visualized in the antibody-linked assay as described in Materials and Methods. Incubation of cells in the presence of arachidonic acid strongly induced Ost-beta. (A), incubation with CDL: (B), phase photomicrograph of the same field: (C), incubation without supplemental lipid: (D), phase photomicrograph of the same field: (E), incubation with arachidonic acid (0.1 uM): (F), phase photomicrograph of the same field.

Figure 8

Induction of Ost-alpha in LEE-1 cells by chemically defined lipids (CDL). Cultures in unsupplemented, basal nutrient media were incubated in the presence or absence of CDL for 4 days and Ost-alpha expression visualized by in situ hybridization as described in Materials and Methods. Incubation of cells in the presence of CDL induced Ost-alpha. (A), cells incubated with CDL, assayed with antisense OST-alpha RNA: (B), cells incubated with CDL, assayed with sense OST-alpha RNA (negative control): (C), phase photomicrograph of the same field as (A): (D), phase photomicrograph of the same field as (B): (E), cells incubated without CDL, assayed with antisense Ost-alpha RNA: (F), cells incubated without CDL, assayed with sense Ost-alpha RNA (negative control); (G), phase photomicrograph of the same field as (E): (H), phase photomicrograph of the same field as (F).