Introduction of a gonadotropin receptor expression plasmid into immortalized granulosa cells leads to reconstitution of hormone-dependent steroidogenesis.

Suh, Byung Sun; Sprengel, Rolf; Keren-Tal, Iris; Himmelhoch, S.; Amsterdam, Abraham

doi:10.1083/jcb.119.2.439

Cited by 49 publications

(27 citation statements)

References 46 publications

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“…Cell Lines-rLHR-4 cell line was established by cotransfection of rat preovulatory granulosa cells with mutated p53 (Val 135 -p53), Ha-ras genes and plasmid expressing the rat LH/CG receptor (17). The rFSHR-17 cell line was established by immortalization of preovulatory rat granulosa cells via cotransfection of primary cells with SV40 DNA and an HA-ras gene.…”

Section: Methodsmentioning

confidence: 99%

“…This notion is supported by our preliminary findings that in primary rat granulosa cells obtained from preovulatory follicles and containing p450scc, PD98059 by itself increased progesterone production. On the other hand, MEK inhibitors do synergize with gonadotropin/cAMP stimulation of stereoidogenesis because of the de novo synthesis of the cytochrome p450scc, which is stimulated by gonadotropin/ cAMP in the granulosa cell lines (17,31).…”

Section: -Erk)mentioning

confidence: 99%

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The ERK Signaling Cascade Inhibits Gonadotropin-stimulated Steroidogenesis

Seger¹,

Hanoch²,

Rosenberg

et al. 2001

Journal of Biological Chemistry

212

110

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The response of granulosa cells to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH-and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the steroidogenic acute regulatory protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.Gonadotropic hormones, follicle-stimulating hormone (FSH) 1 and luteinizing hormone (LH), which are released from the pituitary, play a crucial role in controlling reproductive function in males and females. The pleotropic effects of gonadotropins are manifested in various cells of the reproductive system including LH and FSH in ovarian granulosa cells, LH in theca interna cells, FSH in testicular Sertoli cells, and LH in Leydig cells (1-3). One of the main effects of both LH and FSH on the ovary is the stimulation of the production of estradiol and progesterone, which play important roles in ovarian function and control of the reproductive cycle (reviewed in Ref. 4). The mechanisms involved in the regulation of progesterone production by ovarian granulosa cells have been characterized in detail. Gonadotropins exert their stimulatory activity via interaction with specific seven-transmembrane receptors, the LH receptor and FSH receptor. Upon binding of the gonadotropins, both receptors stimulate the G s protein, which activates the membrane-associated adenylyl cyclase, causing an elevation of intracellular cAMP (5). This cyclic nucleotide serves as a second messenger for the up-regulation of the steroidogenic acute regulatory protein (StAR) and the cytochrome P450 (P450scc) enzyme system (reviewed in Refs. 6 and 7).Activation of alternative signaling pathways by the gonadotropin receptors was described in the last decade, including calcium ion mobilization, activation of the phosphoinositol pathway, and stimulation of chloride ion influx (reviewed in Ref. 8). However, these gonadotropin-induced signaling processes were not previously implicated in the modulation of s...

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Section: Methodsmentioning

confidence: 99%

Section: -Erk)mentioning

confidence: 99%

The ERK Signaling Cascade Inhibits Gonadotropin-stimulated Steroidogenesis

Seger¹,

Hanoch²,

Rosenberg

et al. 2001

Journal of Biological Chemistry

212

110

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“…Another possibility is that the cells lost the receptors due to transformation (35). At present we are conducting experiments transfecting these cells with either human LH/CG or human FSH receptors (41,42).…”

Section: Discussionmentioning

confidence: 99%

Induction of Ad4BP/SF-1, Steroidogenic Acute Regulatory Protein, and Cytochrome P450scc Enzyme System Expression in Newly Established Human Granulosa Cell Lines

Hosokawa¹

1998

Endocrinology

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We have established immortalized human granulosa cells by triple transfection of primary cells obtained from in vitro fertilization patients with SV40 DNA, Ha-ras oncogene, and a temperature sensitive (ts) mutant of the tumor suppressor gene p53 (p53val135). Forty-one clones were isolated, and their steroidogenic responses were analyzed. While all the cell lines proliferate rapidly and show only traces of progesterone production, upon stimulation with 50 M of forskolin (FK), which elevates intracellular cAMP, they become steroidogenic as evidenced by progesterone production. The steroidogenic response of the cell lines was stable even after 20 generations and several cycles of freezing and thawing. A highly responsive cell line (HO-23) was further examined for characteristics of the steroidogenic response. Cells stimulated with FK and 8-Br-cAMP produced high levels of pregnenolone, progesterone, and 20␣-hydroxy-4-pregnen-3-one (20␣-OH-progesterone) comparable with amounts produced by highly differentiated primary human granulosa-luteal cells. Hydrocortisone and dexamethasone highly augment the cAMP-stimulated progesterone production, whereas testosterone and PRL enhanced cAMPinduced progesterone synthesis only moderately. Estradiol, insulinlike growth factor I, and insulin showed no significant effect on cAMPinduced steroidogenesis. The phorbol ester TPA, and basic fibroblast growth factor, dramatically suppress cAMP-induced production of progesterone, whereas bovine corneal endothelial cell ECM (BCE/ ECM) enhanced cAMP-induced progesterone and antagonized basic fibroblast growth factor suppression of cAMP-induced steroidogenesis. Steroidogenic factor 1 (Ad4BP/SF-1) was expressed in control cells, and its expression was augmented by FK, whereas the steroidogenic acute regulatory protein showed low expression in the nonstimulated cells but was clearly elevated upon cAMP stimulation and was slightly decreased by TPA in cAMP-stimulated cells. Expression of the electron carrier adrenodoxin (ADX), which is a part of the cytochrome P450scc enzyme system, was very low in nonstimulated cells but was dramatically elevated in FK-and 8-Br-cAMP-stimulated cells, whereas no reduction of ADX was evident in cells costimulated with FK and TPA. Immunocytochemical studies revealed a weak staining of ADX in mitochrondria of nonstimulated cells and intensive staining in highly clustered mitochondria of FK-or 8-Br-cAMP-stimulated cells. Only moderate reduction in ADX staining was evident in cells costimulated with FK and TPA. These unique cell lines can provide a useful model for the investigation of induced steroidogenesis in human granulosa cells. (Endocrinology 139: 4679 -4687, 1998)

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“…LH-15 cells (a gift from Prof. A. Amsterdam, Weizmann Institute of Science, Israel; Suh et al 1992) were cultured in hormone-free DMEM/Ham F12 1:1 (DMEM-F12) supplemented with penicillin and streptomycin (100 IU/ml and 100 mg/ml respectively; Biological Industries, Beit-Ha'emek, Israel) and 10% Charcoal-stripped fetal bovine serum (CS-FBS; Invitrogen; culture medium). Cells were serum-starved for 16 h (0 or 0.1% CS-FBS) prior to and during the various stimulations.…”

Section: Cell Culturementioning

confidence: 99%

Pigment epithelium-derived factor regulation by human chorionic gonadotropin in granulosa cells

Bar‐Joseph¹,

Ben‐Ami²,

Ron-El³

et al. 2016

Reproduction

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Human chorionic gonadotropin (hCG) is a known trigger of ovarian hyperstimulation syndrome (OHSS), a potentially life-threatening complication of assisted reproduction. Administration of hCG results in the release of vascular endothelial growth factor (VEGF) from the ovary. We have previously shown that expression of pigment epithelium-derived factor (PEDF) in granulosa cell line is regulated by hCG, reciprocally to VEGF, and that the PEDF-VEGF balance is impaired in OHSS. Our aim was to explore the signaling network by which hCG downregulates the expression of PEDF mRNA and protein in granulosa cells. We applied specific chemical inhibitors and stimuli to human primary granulosa cells and rat granulosa cell line. We found that PKA and protein kinase C, as well as EGFR, ERK1/2 and PI3K, participate in the signaling network. The finding that hCG-induced PEDF downregulation and VEGF upregulation are mediated by similar signaling cascades emphasizes the delicate regulation of ovarian angiogenesis.

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Introduction of a gonadotropin receptor expression plasmid into immortalized granulosa cells leads to reconstitution of hormone-dependent steroidogenesis.

Cited by 49 publications

References 46 publications

The ERK Signaling Cascade Inhibits Gonadotropin-stimulated Steroidogenesis

The ERK Signaling Cascade Inhibits Gonadotropin-stimulated Steroidogenesis

Induction of Ad4BP/SF-1, Steroidogenic Acute Regulatory Protein, and Cytochrome P450scc Enzyme System Expression in Newly Established Human Granulosa Cell Lines

Pigment epithelium-derived factor regulation by human chorionic gonadotropin in granulosa cells

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