Abstract
Rat ovarian granulosa rely heavily on lipoprotein-derived cholesterol for steroidogenesis, which is principally supplied by the LDL receptor- and scavenger receptor class B type I (SR-BI)-mediated pathways. In this study, we characterized the hormonal and cholesterol regulation of another member of the LDL receptor superfamily, low density lipoprotein receptor-related protein (LRP), and its role in granulosa cell steroidogenesis. Coincubation of cultured granulosa cells with LDL and N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (Bt2cAMP) greatly increased the mRNA/protein levels of LRP. Bt2cAMP and Bt2cAMP plus human hLDL also enhanced SR-BI mRNA levels. However, there was no change in the expression of receptor-associated protein, a chaperone for LRP, or another lipoprotein receptor, LRP8/apoER2, in response to Bt2cAMP plus hLDL, whereas the mRNA expression of LDL receptor was reduced significantly. The induced LRP was fully functional, mediating increased uptake of its ligand, alpha2-macroglobulin. The level of binding of another LRP ligand, chylomicron remnants, did not increase, although the extent of remnant degradation that could be attributed to the LRP doubled in cells with increased levels of LRP. The addition of lipoprotein-type LRP ligands such as chylomicron remnants and VLDL to the incubation medium significantly increased the progestin production under both basal and stimulated conditions. In summary, our studies demonstrate a role for LRP in lipoprotein-supported ovarian granulosa cell steroidogenesis.
Highlights
Rat ovarian granulosa rely heavily on lipoproteinderived cholesterol for steroidogenesis, which is principally supplied by the LDL receptor- and scavenger receptor class B type I (SR-BI)-mediated pathways
In ovarian granulosa cells that had been cultured in the presence of N 6 (Bt2cAMP) alone, z38% of the degradation was inhibited by the use of the anti-LDL receptor antibody and 85% was inhibited by receptor-associated protein (RAP) either alone or in combination with the antibody (Fig. 8)
This suggests that members of the LDL receptor family mediated most of the specific degradation of chylomicron remnants and that approximately half of this was attributable to the LDL receptor itself
Summary
Materials Na 125I (carrier-free; 643.8 GBq/mg, 17.4 Ci/mg) was purchased from NEN Life Science Products (Boston, MA). Dishes were washed with the medium to remove nonadherent cells and incubated in a binding medium (0.5% BSA and 10 mM HEPES, pH 7.4) containing 125I-labeled a2-macroglobulin (1 mg/ml) or chylomicron remnants (2 mg/ml) in the presence or absence of unlabeled a2-macroglobulin, GST-RAP, or the anti-LDL receptor antibody at 37jC for 4 h. The antisense [32P]complimentary ribonucleic acid probes were synthesized using [a32P]CTP, restriction endonucleaselinearized plasmid (EcoRI for LRP and RAP, XhoI for LDL receptor, HindIII for HMG-CoA reductase, and BamHI for 18S rRNA), and the appropriate T3 or T7 polymerase according to the method described in Stratagene’s in vitro transcription kit Because of their high lability, the riboprobes were always freshly prepared before hybridization. Each sample was measured in triplicate plus a control without reverse transcriptase
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