Molecular mechanism for repression of 17alpha-hydroxylase expression and androstenedione production in granulosa cells.
BACKGROUND
According to the traditional two-cell two-gonadotropin model of follicular steroidogenesis, androgen production arises exclusively from theca cells. The granulosa cells, in turn, utilize androstenedione and testosterone, which are aromatized into estrone and estradiol, respectively. Differential expression of the activator protein-1 (AP-1) transcription factor, c-fos, has been postulated to result in distinct patterns of steroidogenesis in the theca and granulosa cell compartments. We hypothesize that c-fos functions to inhibit the production of 17alpha-hydroxylase 17,20 lyase (CYP17) in granulosa cells, thereby suppressing androgen synthesis.


OBJECTIVE
Our objective was to define the role of c-fos in the regulation of CYP17 production in granulosa cells.


METHODS
Human luteinized granulosa (HGL5) cells were utilized for all experiments. The following techniques were used: mRNA extraction, steroid quantification, small interfering RNA silencing, microarray analysis, and immunohistochemistry.


RESULTS
Immunohistochemistry studies demonstrated significant staining of c-fos in the granulosa cell layer, but absent staining for CYP17. Conversely, the theca cell layer did not stain for c-fos, but staining was evident for CYP17. Treatment of HGL5 cells with the MAPK kinase inhibitor PD98059 resulted in an 11-fold increase in CYP17 mRNA levels. In c-fos gene silenced cells, CYP17 mRNA levels increased 8-fold. Androstenedione production was increased 13-fold after treatment with PD98059.


CONCLUSIONS
These results suggest that the AP-1 transcription factor, c-fos, may be one of the factors responsible for CYP17 repression and hence suppression of androstenedione production in granulosa cells. This may provide an explanation for the lack of CYP17 in granulosa cells.
Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9032, USA.
Satin S
SS
Patel
Victor E
VE
Beshay
Julian C
JC
Escobar
Takashi
T
Suzuki
Bruce R
BR
Carr
D000328:Adult; D000735:Androstenedione / Q000096:biosynthesis* / Q000235:genetics; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004794:Enzyme Repression; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics / Q000502:physiology; D006107:Granulosa Cells / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D010053:Ovary / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000037:antagonists & inhibitors / Q000502:physiology; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013254:Steroid 17-alpha-Hydroxylase / Q000096:biosynthesis* / Q000235:genetics; D013256:Steroids / Q000378:metabolism
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005419:Flavonoids; D016760:Proto-Oncogene Proteins c-fos; D013256:Steroids; D000735:Androstenedione; D012313:RNA; D013254:Steroid 17-alpha-Hydroxylase; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one