title: Hydroxyflutamide affects connexin 43 via the activation of PI3K/Akt-dependent pathway but has no effect on the crosstalk between PI3K/Akt and ERK1/2 pathways at the Raf-1 kinase level in primary rat Sertoli cells.
abstract: We investigated the effects of 2-hydroxyflutamide (HF), an active metabolite of the anti-androgen flutamide, on the activation of the phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) in rat Sertoli cells. Sertoli cells, isolated from 20-day-old rat testes, were cultured in vitro and treated with HF, testosterone, or HF+testosterone. Studies by western blotting demonstrated that HF inhibited the testosterone-mediated increase in c-Src activity (p<0.05). In contrast, Akt phosphorylation was augmented within 5 min after HF treatment (p<0.01). This effect was accompanied by a rapid upregulation in PTEN phosphorylation (p<0.001). Despite no changes in Raf-1 phosphorylation, HF increased extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation (p<0.001), indicating that the effect of the anti-androgen on ERK1/2 was independent of PI3K/Akt-pathway activation at this level. Since HF inhibited the testosterone-mediated increase in c-Src activity, it is likely that activation of both Akt and ERK1/2 occurred in a p-Src independent manner. Activation of PI3K/Akt-pathway by HF resulted in the reduced level of Sertoli cell functional marker, connexin 43 (p<0.01). Collectively, these data provide evidence that HF rapidly and transiently affects the protein kinase-dependent signaling pathways, acting both as an antagonist and agonist. Moreover, using testes of flutamide-treated rats for 7 days, we demonstrated that the anti-androgen can modulate the protein kinase-dependent pathways in long term by enhancing Akt and ERK1/2 protein expression (p<0.05).
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Chojnacka
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Zarzycka
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Hejmej
authors.affiliation: Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10065, USA.
authors.forename: Dolores D
authors.initials: DD
authors.lastname: Mruk
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Gorowska
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Kotula-Balak
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Klimek
authors.affiliation: Department of Endocrinology, Institute of Zoology, Jagiellonian University, Krakow, Poland. Electronic address: barbara.bilinska@uj.edu.pl.
authors.forename: Barbara
authors.initials: B
authors.lastname: Bilinska
mesh_terms: D000726:Androgen Antagonists / Q000494:pharmacology*; D000728:Androgens / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism*; D005485:Flutamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D008297:Male; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017208:Rats, Wistar; D012708:Sertoli Cells / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013739:Testosterone / Q000494:pharmacology
chemical_list: D000726:Androgen Antagonists; D000728:Androgens; D018031:Connexin 43; D005485:Flutamide; D019869:Phosphatidylinositol 3-Kinases; D011494:Protein Kinases; D013739:Testosterone; C580592:Gja1 protein, rat; C014290:hydroxyflutamide
keywords: Cx43; ERK1/2; Hydroxyflutamide; PI3K/Akt; Primary Sertoli cells; Rat; c-Src