Ost is unique among anion membrane transport proteins in molecular structure, requiring for activity two proteins of highly different structural nature. In Xenopus
oocytes, Ost mediates transport of estrone 3-sulfate, dehydroepiandrosterone 3-sulfate, taurocholate, digoxin, and prostaglandin E2
. For some years, the existence of an activity functioning in mammals as the primary ileal basolateral transporter responsible for intestinal reabsorption of bile acids was postulated, but not identified (Ballatori 2005
). Recent evidence is that Ost functions in this capacity in rodents and humans (Seward et al., 2003
; Dawson et al., 2005
; Lee et al., 2006
). It has been speculated that this transporter is likely to be essential for absorption of dietary fats and vitamins, as well as bile flow and cholesterol homeostasis (Ballatori, 2005
). Experiments in mammalian cell cultures have demonstrated that Ost mediates apical to basolateral vectorial transport of taurocholate and is physiologically regulated (Ballatori, 2005
; Boyer et al., 2006
Although Ost was initially identified in the little skate, very little additional work has been carried out in this animal, and no work with Ost has been reported with any other nonmammalian organisms. The skate is an elasmobranch, of the class Chondrichthyes, first appearing about 400 million years ago. Skates and rays (Superorder Batoidea) diverged from other elasmobranchs about 200 million years ago. Despite this primitive origin, the liver of the little skate exhibits many of the biochemical properties and architecture of the liver of later appearing vertebrates, including mammals. Liver functions include metabolism and clearance of endogenous anions such as bile salts, conjugated steroids, and xenobiotic lipophilic organic toxins.
Recently we derived the SAE cell line, the first from a cartilaginous fish (Parton et al., 2007
), and subsequently applied the same approach to develop LEE-1. The successful derivation of cell lines from cartilaginous fish is based on the concept that culture medium formulation should be based on the physiology of the cells and a minimum of heterologous components such as FBS, rather than an adaptation process of cells to a standard medium formulation (eg., basal nutrient medium supplemented only with 10% FBS). Interestingly, the cells did not require urea in the medium, although elasmobranchs maintain a high blood urea concentration as an osmoregulator. Skate embryo cells at early stages may not require or produce high concentrations of circulating urea because they may be protected by the egg casing from a full sea water environment.
We have established that LEE-1 expresses Ost-beta at both the mRNA and protein level and confirmed that the mRNA in the cell line is a single species of the same size as that found in skate liver. Our Northern blots identified an OST-beta band of about 900 nt, somewhat smaller than the band of approximately 1 kb seen in human and rodent liver, intestine and kidney. Thus the LEE-1 cell line provides a reliable comparative model for the study of Ost in vitro
. Immunolocalization in LEE-1 cells detected Ost-beta both in the plasma membrane and intracellularly, with particularly strong localization in perinuclear areas that may be endoplasmic reticulum (see ). Similarly, Ost-beta has been found in areas other than the plasma membrane in immunolocalization studies with mammalian cells (Dawson et al. 2005
; Ballatori et al. 2005
; Li et al. 2007
). These results suggest other functions for Ost-beta or that intercellular pools of Ost-beta are available for recruitment upon the appropriate signal. For instance, Ost-beta has been suggested to function as a chaperone, and may act in a manner similar to RAMPs, which are crucial for endoplasmic reticulum-to-Golgi translocation and plasma membrane trafficking of some receptors in mammals (Dawson et al., 2005 Bouschet et al., 2005
; Parameswaran and Spielman, 2006
; Hay et al., 2006
, Li et al., 2007
Ost-beta previously has been reported to be expressed in most tissues of skates, mice and humans, although expression levels vary among the tissues (Wang et al., 2001
; Ballatori et al., 2005
; Dawson et al., 2005
) The present results demonstrate ubiquitous expression in the skate tissues examined. Skate Ost beta expression previously has been reported in liver, kidney, intestine and heart, (Wang et al., 2001
). Expression in the mouse has been reported to be high in kidney and intestine, and highest levels in humans are found in the testis, colon, liver, small intestine, kidney, ovary and adrenal gland, with lower levels in a number of other tissues of both mouse and human, including brain, spleen, and heart. (Ballatori et al., 2005
; Dawson et al., 2005
; Li et al., 2007
Primary sequence comparisons among L. erinacea. S. acanthias, G. gallus, D. Rerio and H. sapiens showed almost complete identity between the two cartilaginous fish, while considerable deviation from this pattern existed for the teleost D. rerio (zebrafish). The deviation is primarily the result of insertions of long amino acid regions, and is responsible for the greater length of the deduced protein, which is almost twice that of human Ost-beta. Often primary sequence comparisons among teleosts and other vertebrates are poorly related, possibly because of a recent tetraploidization event and subsequent radiation among the bony fishes. G. gallus also showed an increased length of primary structure; about 50% greater than that of the human Ost-beta.
In all cases, conserved regions were obvious in the transmembrane area, including those of shark, zebrafish and chicken; extending this information to that of these other vertebrates. A stretch of approximately 20 amino acids toward the carboxy-terminal end of the protein on the intracellular side of the membrane (Li et al., 2007
) also was found to be conserved, including in chicken and zebrafish. The function of this conserved area is unknown. The role of Ost in nonmammalian and non-elasmobranch species remains to be elucidated. Evidence that the protein exists in a reasonably conserved form in both chicken and bony fish suggests transport functions that may be similar to that seen in the skate (eg., liver transport functions) or may show other critical activities similar to that seen in mammals (eg., intestinal transport functions). The tissue distributions of Ost in chicken and zebrafish are not known.
An animo acid motif of asn-arg-X-arg-lys was conserved in all species listed in , except human. Masking of the core arg-X-arg portion of this sequence is involved in successful transport from the endoplasmic reticulum (Margeta-Mitrovic et al., 2001). Neither of the N-linked extracellular glycosylation sites predicted for the skate protein (Wang et al.; 2001
) were uniformly conserved among the five species compared. Ost-beta from L. erinacea
shared 23% amino acid identity with H. sapiens
, 14% with G. gallus
and 11% with D. rerio
. The latter, in general, showed least amino acid identity with the other species, and we have observed a similar pattern at the noncoding nucleotide level for this species (Forest et al., 2007
Routinely, a lipid mixture was added as a supplement to the LEE-1 cells, and we found that this mixture induced Ost, indicating that the protein is under positive regulation in the usual cell culture medium. Among the individual components of this mixture, arachidonic acid was the most effective at inducing Ost-beta expression. It is interesting that Ost can transport some eicosanoids, and the principal eicosanoids (prostaglandins and thromboxanes) in humans are derived from arachidonic acid. The farnesoid X receptor (FXR) has been shown to positively regulate Ost, and is likely a critical component in signaling mechanisms controlling bile acid homeostasis (Landrier et al., 2005; Boyer et al., 2006
; Lee et al., 2006
; Cai et al., 2007
). Our PCR evidence indicates that FXR mRNA is expressed in LEE-1 cells (not shown). Arachidonic acid has been reported to function as an FXR ligand to positively regulate FXR-mediated expression of the bile salt export pump (Zhao et al., 2004
), and thus, this may be the mechanism by which the molecule stimulates expression of Ost-beta as well. However, additional studies are needed to test this possibility.