Differential regulation of STARD1, STARD4 and STARD6 in the human ovary.

Cells actively engaged in de novo steroidogenesis rely on an expansive intracellular network to efficiently transport cholesterol. The final link in the transport chain is STARD1, which transfers cholesterol to the enzyme complex that initiates steroidogenesis. However, the regulation of ovarian STARD1 is not fully characterized, and even less is known about the upstream cytosolic cholesterol transporters STARD4 and STARD6. Here, we identified both STARD4 and STARD6 mRNAs in the human ovary but only detected STARD4 protein since the primary STARD6 transcript turned out to be a splice variant. Corpora lutea contained the highest levels of STARD4 and STARD1 mRNA and STARD1 protein, while STARD4 protein was uniformly distributed across ovarian tissues. Cyclic AMP analog (8Br-cAMP) and phorbol ester (PMA) individually increased STARD1 and STARD4 mRNA along with STARD1 protein and its phosphoform in cultured primary human luteinized granulosa cells (hGCs). STARD6 transcripts and STARD4 protein were unresponsive to these stimuli. Combining lower doses of PMA and 8Br-cAMP blunted the 8Br-cAMP stimulation of STARD1 protein. Increasing cholesterol levels by blocking its conversion to steroid with aminoglutethimide or by adding LDL reduced the STARD4 mRNA response to stimuli. Sterol depletion reduced the STARD1 mRNA and protein response to PMA. These data support a possible role for STARD4, but not STARD6, in supplying cholesterol for steroidogenesis in the ovary. We demonstrate for the first time how cAMP, PMA and sterol pathways separately and in combination differentially regulate STARD4, STARD6 and STARD1 mRNA levels, as well as STARD1 and STARD4 protein in human primary ovarian cells.

STARD6 is expressed in steroidogenic cells of the ovary and can enhance de novo steroidogenesis.

STARD6 is a member of the StAR-related lipid transfer (START) domain family of proteins whose function thus far remains obscure. While it recently was shown to facilitate steroidogenesis in a cell-free setting, it has not been localized to steroidogenic cells of normal reproductive tissues. In a recent microarray study, we detected STARD6 mRNA in cultured porcine ovarian granulosa cells which are steroidogenic. In the present study, we examined regulation of STARD6 mRNA in porcine granulosa cultures, and found that it was not regulated by cyclic AMP, but it was reduced by combined knockdown of the transcription factors GATA4 and GATA6. We detected both STARD6 mRNA and protein in fresh granulosa cells and whole antral follicles and different stage corpora lutea of pig. The highest levels were discovered in the mid-luteal phase corpus luteum. Immunolocalization within ovarian tissues indicated robust STARD6 immunoreactivity in steroidogenic cells of the corpus luteum. Relatively lesser amounts of STARD6 signal were found in granulosa cells, theca cells, and oocytes. To test the ability of STARD6 to facilitate de novo steroidogenesis, non-steroidogenic COS-1 cells were co-transfected with components of the P450 cholesterol side-chain cleavage system, enabling them to make pregnenolone, and STARD6. STARD6 increased pregnenolone production by two- to three-fold over the empty vector control. In summary, STARD6 is found in the pig ovary, exhibits the strongest expression in highly steroidogenic luteal cells, and significantly enhances pregnenolone production in transfected COS cells independent of cyclic AMP treatment. Collectively, these findings indicate that STARD6 may contribute to steroidogenesis in ovarian cells, but also suggests other cellular functions that require cholesterol trafficking.