Down-regulation of steroidogenic response to gonadotropins in human and rat preovulatory granulosa cells involves mitogen-activated protein kinase activation and modulation of DAX-1 and steroidogenic factor-1.
Gonadotropins were recently demonstrated to be able to activate the MAPK cascade, but the physiological significance of this activation is still obscure. In the present work we demonstrate that highly luteinized human granulosa cells obtained from in vitro fertilization patients respond to human LH as well as to forskolin in phosphorylation of extracellular-signal regulated kinases 1 and 2 (ERK1 and -2). Moreover, the potent MAPK inhibitors, PD98059 and UO126, augment progesterone production in these cell cultures concomitantly with specific elevation of intracellular steroidogenic acute regulatory protein (StAR). Intracellular levels of the cytochrome P450 side-chain cleavage enzyme system do not seem to be affected. Similar observations were made with rat preovulatory or preantral granulosa cells stimulated with LH, FSH, or forskolin. Elevation of StAR expression by the MAPK inhibitors involved elevation of StAR mRNA, as demonstrated by RT-PCR in the human cells. Immunocytochemical studies using specific antibodies to StAR demonstrate a higher content of mitochondrial StAR in control as well as in gonadotropin-stimulated cells in the presence of PD98059 compared with cells not treated with PD98059. The cultured cells express the transcription factor steroidogenic factor-1 (SF-1), the phosphorylation of which is known to activate the expression of StAR, as well as dosage-sensitive sex reversal adrenal hypoplasia congenita, critical region on the X chromosome gene-1 (DAX-1), which is known to negate SF-1 activity. Intracellular levels of DAX-1 decreased significantly during 24 h of incubation of cells with or without LH in the presence of PD98059 or UO126 compared with those in cultures incubated in the absence of the MAPK inhibitors. The expression of SF-1 was suppressed by LH, whereas MAPK inhibitor could block this effect and further elevate SF-1 levels. Thus, activation of the MAPK cascade by gonadotropins may serve as a novel mechanism to down-regulate steroidogenesis via attenuation of StAR expression. Moreover, modulation of DAX-1 and SF-1 intracellular levels in these cells suggests that these transcription factors could be involved in MAPK suppression of StAR expression.
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 71600, Israel.
Kimihisa
K
Tajima
Ada
A
Dantes
Zhong
Z
Yao
Ksenia
K
Sorokina
Fumikazu
F
Kotsuji
Rony
R
Seger
Abraham
A
Amsterdam
D015124:8-Bromo Cyclic Adenosine Monophosphate; D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D051717:Fushi Tarazu Transcription Factors; D015870:Gene Expression; D006062:Gonadotropins / Q000494:pharmacology*; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism; D018398:Homeodomain Proteins; D006801:Humans; D007986:Luteinizing Hormone / Q000494:pharmacology; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010060:Ovulation; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid / Q000032:analysis / Q000378:metabolism*; D012097:Repressor Proteins; D054339:Steroidogenic Factor 1; D013256:Steroids / Q000096:biosynthesis*; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism*; D000098682:Steroidogenic Acute Regulatory Protein
D006063:Chorionic Gonadotropin; D057137:DAX-1 Orphan Nuclear Receptor; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D051717:Fushi Tarazu Transcription Factors; D006062:Gonadotropins; D018398:Homeodomain Proteins; C539965:NR0B1 protein, human; C515057:NR5A1 protein, human; C539966:Nr0b1 protein, mouse; C539967:Nr0b1 protein, rat; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D018168:Receptors, Retinoic Acid; D012097:Repressor Proteins; D054339:Steroidogenic Factor 1; D013256:Steroids; D014157:Transcription Factors; D000098682:Steroidogenic Acute Regulatory Protein; C515058:steroidogenic factor 1, mouse; C515060:steroidogenic factor 1, rat; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one