Extracellular Signal-Regulated Kinases Are Involved in the Acute Activation of Steroidogenesis in Immature Rat Leydig Cells by Human Chorionic Gonadotropin

0013-7227/04/$15.00/0 Printed in U.S.A. Endocrinology 145(10):4629 – 4634 Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2004-0496 Extracellular Signal-Regulated Kinases Are Involved in the Acute Activation of Steroidogenesis in Immature Rat Leydig Cells by Human Chorionic Gonadotropin N. MARTINELLE, M. HOLST, O. SÖDER, AND K. SVECHNIKOV Department of Woman and Child Health, Pediatric Endocrinology Unit, Karolinska Institute and University Hospital, SE-17176 Stockholm, Sweden We studied the involvement of the ERK cascade in human chorionic gonadotropin (hCG)-induced steroidogenesis by primary cultures of immature rat Leydig cells. Our findings indicate that protein kinase A and protein kinase C function as upstream kinases in connection with transduction of the signal from the gonadotropin receptor to the ERK cascade. These MAPKs enhance the stimulatory effects of hCG on the de novo synthesis of the steroidogenic acute regulatory protein and the activity of protein phosphatase 2A, which are associated with increased androgen production by the Leydig cell. Specific inhibition of ERK1/2 by Uo126 suppressed all of these cellular responses to hCG. In contrast, steroidogenesis L H SECRETED BY THE pituitary gland plays a central role in regulation of reproductive function in both male and female mammals. In the case of the testis, LH regulates the expression of receptors on the surface of Leydig cells and maintains the local and peripheral concentrations of androgens required for hormonal and reproductive development (1). Human chorionic gonadotropin (hCG) exhibits pronounced structural similarity to LH and binds to the same receptors. In connection with the signaling cascade of events triggered by gonadotropins and resulting in activation of steroidogenesis, the cAMP-dependent protein kinase [protein kinase A (PKA)] plays a key role (2). PKA activation leads to up-regulation of the expression of the steroidogenic acute regulatory (StAR) protein, the function of which is to translocate cholesterol from the outer to the inner mitochondrial membrane, which constitutes the rate-limiting step in steroid hormone synthesis (3). After this translocation, cholesterol is converted to pregnenolone by the cytochrome P450 sidechain cleavage complex (4). ERKs (1/2), belonging to the family of signaling MAPKs, are involved in the regulation of a number of important biological functions, including cell proliferation, differentiation, and apoptosis as well as carcinogenesis (5). Several recent reports (6, 7) have indicated that ERK1/2 participate Abbreviations: dbcAMP or (Bu)2 cAMP, Dibutyryl cAMP; HBSS, Hanks’ balanced salt solution; hCG, human chorionic gonadotropin; PKA, protein kinase A; PKC, protein kinase C; PP, protein phosphatase; 22R-OHC, 22R-hydroxycholesterol; StAR, steroidogenic acute regulatory. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community. from 22OHC (a cell-permeable form of cholesterol) is not inhibited by Uo126, suggesting that cholesterol delivery to mitochondria is being affected by this compound. We propose that the ERK cascade is an important part of the signal transduction pathway involved in the rapid hormonal responses of Leydig cells to trophic hormones. In hCG-activated Leydig cells, these MAPKs may play a role in controlling the biosynthesis of the steroidogenic acute regulatory protein as well as regulating protein phosphatase 2A activity, thereby governing cholesterol transport across the mitochondrial membrane. (Endocrinology 145: 4629 – 4634, 2004) in the regulation of steroidogenesis in steroid-producing cells, but to our knowledge virtually nothing is known concerning their possible functions with regard to the Leydig cell. In the present investigation, we demonstrate that the steroidogenesis induced in immature rat Leydig cells by hCG is dependent on activation of the ERK cascade. Materials and Methods Materials DMEM-Ham’s nutrient mixture F-12, MEM, Hanks’ balanced salt solution (HBSS) without Ca2⫹ and Mg2⫹, and penicillin-streptomycin were obtained from Life Technologies, Inc./BRL (Paisley, Scotland, UK). BSA (fraction V), Percoll, HEPES, hCG, (Bu)2cAMP (dibutyryl cAMP or dbcAMP), collagenase type I, and 22R-hydroxycholesterol (22R-OHC) (Sigma Chemical Co., St. Louis, MO), Uo126, Calphostin C, and H-89 hydrochloride (Calbiochem, La Jolla, CA), specific phospho-p44/42 MAPK antibodies (mouse monoclonal antibody IgG, affinity purified), and p44/42 MAPK antibodies (rabbit polyclonal IgG, affinity purified) (Cell Signaling Technology, Inc., Beverly, MA), and 35S-methionine (Amersham Pharmacia Biotech, Buckinghamshire, UK) were purchased from the sources indicated. Animals Forty-d-old male Sprague Dawley rats (B&K Laboratories, Sollentuna, Sweden) were used as the source of immature Leydig cells (8). These animals were fed a standard pellet diet and water ad libitum. These experiments were approved by the Northern Stockholm Animal Ethics Committee (registration no. N192/03). Isolation and culture of Leydig cells Leydig cells were prepared from the immature rats by treatment of testes with collagenase as described earlier (9). Briefly, decapsulated testes were incubated with collagenase (0.25 mg/ml) for 20 min at 37 C, after which the crude mixture of interstitial cells was collected by centrifugation at 300 ⫻ g for 10 min, following by washing in HBSS con- 4629 4630 Endocrinology, October 2004, 145(10):4629 – 4634 taining 0.1% (wt/vol) BSA. To obtain purified Leydig cells, this crude cell suspension was loaded on top of a discontinuous Percoll gradient (consisting of layers of 20, 40, 60, and 90% Percoll in HBSS) and subsequently centrifuged at 800 ⫻ g for 20 min. The fractions enriched in Leydig cells thus obtained were then centrifuged in a continuous, selfgenerating density gradient starting with 60% Percoll at 20,000 ⫻ g for 30 min at 4 C. The purity of the Leydig cells was shown to be 90%, as determined by histochemical staining for 3␤-hydroxysteroid dehydrogenase (10). The cell viability, as assessed by Trypan blue exclusion, was greater than 90%. These purified Leydig cells were washed twice in DMEM-F12 and thereafter resuspended in DMEM-F12 supplemented with 15 mm HEPES (pH 7.4), 1 mg/ml BSA, 365 mg/liter glutamine, 100 IU/ml penicillin, and 100 ␮g/ml streptomycin. For culturing, 100 ␮l of a suspension containing 1.5 ⫻ 105 cells/ml were plated into each well of a 96-well Falcon plate (Falcon, Franklin Lake, NJ) and incubated for 24 h at 34 C. At this time point, fresh culture medium was added and the cells preincubated with the specific inhibitor of ERK, Uo126 (0.1–10 ␮m) for 30 min, before incubation with hCG (10 ng/ml), (Bu)2cAMP (1 mm), and/or 22R-OHC (10 ␮m) for 3 h. This range of concentrations of Uo126 was shown earlier to inhibit ERK activities without affecting a number of other protein kinases (11). 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