Characterization of a 95 kDa high affinity human high density lipoprotein-binding protein.

A new human 95 kDa high density lipoprotein (HDL)-binding protein (HBP) corresponding to a high affinity HDL-binding site with K(d) = 1.67 microg/mL and a capacity of 13.4 ng/mg was identified in human fetal hepatocytes. The HDL binding with the 95 kDa HBP plateaus at 2.5-5 microg/mL under reducing and nonreducing conditions. The association of HDL(3) with the 95 kDa HBP plateaued in 15-30 min while dissociation was complete in 30 min. HDL(3), apoA-I, and apoA-II were recognized by the 95 kDa HBP while low density lipoproteins (LDL) and tetranitromethane-modified HDL were not. The 95 kDa HBP predominantly resides on the surface of cells since trypsin treatment of HepG2 cells eliminated nearly 70% of HDL binding. All studied human cells and cell lines (HepG2, Caco-2, HeLa, fibroblasts, SKOV-3, PA-I) demonstrated the presence of the 95 kDa protein. Both RT-PCR and Western blotting for HB-2/ALCAM were negative in human fetal hepatocytes while Gp96/GRP94 was clearly differentiated from the 95 kDa HBP by two-dimensional electrophoretic mobility. Moreover, deglycosylation of HepG2 membrane preparations did not affect either HDL binding to the 95 kDa HBP or its size, while in contrast it affected the molecular weights of HB-2/ALCAM and SR-BI/CLA-1. We conclude that the 95 kDa HBP is a new HDL receptor candidate widely expressed in human cells and cell lines.

Apolipoprotein specificity for lipid efflux by the human ABCAI transporter.

ABCAI, a member of the ATP binding cassette family, mediates the efflux of excess cellular lipid to HDL and is defective in Tangier disease. The apolipoprotein acceptor specificity for lipid efflux by ABCAI was examined in stably transfected Hela cells, expressing a human ABCAI-GFP fusion protein. ApoA-I and all of the other exchangeable apolipoproteins tested (apoA-II, apoA-IV, apoC-I, apoC-II, apoC-III, apoE) showed greater than a threefold increase in cholesterol and phospholipid efflux from ABCAI-GFP transfected cells compared to control cells. Expression of ABCAI in Hela cells also resulted in a marked increase in specific binding of both apoA-I (Kd = 0.60 microg/mL) and apoA-II (Kd = 0.58 microg/mL) to a common binding site. In summary, ABCAI-mediated cellular binding of apolipoproteins and lipid efflux is not specific for only apoA-I but can also occur with other apolipoproteins that contain multiple amphipathic helical domains.

Heat shock protein 60 is a high-affinity high-density lipoprotein binding protein.

A new 55-kDa HDL/apolipoprotein binding protein was demonstrated in plasma membrane preparations of the human cell lines and primary cultured hepatocytes. Analysis of specific binding by ligand immunoblots of HDL, apoA-I, and apoA-II to a partially purified 55-kDa PA-I plasma membrane preparation demonstrated a K(d,HDL) = 50 nM (10 microg/ml), K(d,apoA-II) = 20 nM (0.4 microg/ml), and K(d, apoA-I) = 330 nM (10 microg/ml). Following preparative SDS-PAGE electrophoresis of a plasma membrane preparation isolated from human PA-I cells, fractions with apoA-II binding activity were collected, concentrated, and subjected to two-dimensional electrophoresis. Internal microprotein sequencing of the 55-kDa protein band revealed the binding protein as being heat shock protein 60 (hsp60). The hsp60 monoclonal antibody LK-1 blocked apoA-II binding to the 55-kDa HBP preparation. In summary, these results provide a potential mechanism to explain the known association between immunity developed against hsp60 and the development of atherosclerosis.

Evidence for direct action of testosterone on rat liver cells: in vivo and in vitro induction of unusual estrogen-binding protein.

We demonstrate that on the rat liver, testosterone (T) induced differentiated functions and enhanced unusual estrogen-binding protein (UEBP) content through mechanisms dependent on cell activation by androgens, the presence of growth hormone (GH) and the hormonal status of the animal. To determine whether liver cells are a target for androgens, we measured T effects on UEBP in gonadectomized adult male and female rats in vivo and in vitro. In ovariectomized rats, T increased 8- to 9-fold UEBP levels that remained constant during 10 days. Also in vitro, using hepatocytes from ovariectomized rats, T alone increased UEBP levels 3-fold in a dose-response pattern. Combining a fixed low dose of GH with different concentrations of T increased UEBP 2-fold above T alone. Whereas GH alone had no effects in ovariectomized rats, hepatocytes were responsive to GH, in a dose dependent pattern that was abolished when T was used together with GH. On the other hand, T alone had no effect in hypophysectomized-ovariectomized animals. The latter group was rendered T responsive after the simultaneous injection of GH with T that increased UEBP content 6.6-fold in vivo. Castrated males revealed a marked responsiveness to T and GH in vivo and in vitro, when added separately or in combination. The results obtained suggest a complex regulatory system and we conclude that T acts directly on rat liver as: (1) an inducer of sex differentiation; and (2) a regulator of UEBP production in males. In addition, liver regeneration studies in castrated-hypophysectomized males revealed the UEBP phenotype in daughter cells in the absence of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo and in vitro estimations of the direct effect of estrogen on rat hepatocytes tested by the changes in the unusual estrogen-binding protein content.

The direct effect of estradiol (E2) on the hepatocytes of mature male rats has been examined by measuring the changes in the unusual estrogen-binding protein (UEBP) content and parallel measuring the level of liver estrogen receptors (ER). The content of UEBP (NUEBP) and ER (NER) in the liver were determined using the quantitative methods for differential specific determination of the E2-binding sites of these proteins. It has been shown that the administration of E2 in vivo induced a considerable decrease in hepatic NUEBP not only in intact males, but also in hypophysectomized males during the initial period after the operation (when the content of hepatic ER was still high) and produced no effect in hypophysectomized males during the later period (when liver ER were depleted). Repeated administration of human growth hormone (hGH) (twice a day) resulted in a considerable increase in NER in hypophysectomized males and restored the sensitivity to the subsequent inhibitory effect of E2 on UEBP. We also used rat hepatocytes after a 4-day primary culturing. These cells had a stable morpho-functional status, high ER level, and sex-differentiated UEBP content. Culturing of mature male rat hepatocytes in the medium containing E2 at concentrations close to physiological levels (10(-10)-10(-7) M) decreased NUEBP in a dose-dependent manner. Hexestrol (10(-7) M) but not cholesterol (10(-5) M) also exhibited a direct effect on NUEBP in cultured rat hepatocytes. The effect of E2 was reversible: statistically significant increase in NUEBP was observed 3 days after 10(-9) M E2 had been removed from the culturing medium. It was concluded that hepatocytes may be a primary target for E2 under physiological conditions and that GH may modulate the direct effect of E2 at the hepatic level by modifying the content of liver ER.

The unusual estrogen-binding protein (UEBP) of rat liver: the role of sex steroids and hypophysis in its regulation.

The number of estradiol (E2) binding sites of rat liver unusual estrogen-binding protein (NUEBP) was measured, using a novel modification of the quantitative method of specific UEBP determination. In liver cytosol of mature male and female rats, NUEBP amounted to 6.83 +/- 0.49 and less than 0.05 pmol/mg protein, respectively. Neonatal administration of testosterone-propionate (TP) and TP injections at later periods of ontogenesis increased NUEBP in female rat liver in a similar fashion. The elevated NUEBP was found in the liver of mature ovariectomized females 30 days after cessation of TP injections. Hypophysectomy (but not adrenalectomy or thyroidectomy) prevented TP induction of elevated NUEBP in pubertal females. E2 injections reversibly decreased NUEBP in the liver of all animals under study except of hypophysectomized males. A stimulating regulatory effect of TP on NUEBP in male rat liver was observed only in the case of endogenous androgen deficiency and low NUEBP. TP prevented the E2-dependent decrease of NUEBP upon their simultaneous injections and increased the E2-reduced NUEBP when injected after E2. Hypophysectomy led to a decrease of NUEBP in pubertal males but only slightly affected that in castrated animals. After TP injections to hypophysectomized males, NUEBP returned to a level next to the initial one. It was concluded that estrogen-androgen regulation of the UEBP level led to the maintenance of sex differences in the UEBP content.