Sertoli-secreted FGF-2 induces PFKFB4 isozyme expression in mouse spermatogenic cells by activation of the MEK/ERK/CREB pathway.
Sertoli cells play a central role in the control and maintenance of spermatogenesis by secreting growth factors, in response to hormonal stimulation, that participate in the paracrine regulation of this process. In this study, we investigated how the hormonal regulation of spermatogenesis modulates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) isozyme expression in two mouse spermatogenic cell lines, GC-1 spg and GC-2 spd (ts). For this purpose, TM4 Sertoli cells were used to obtain conditioned medium that was treated or not with dihydrotestosterone for 2 days [dihydrotestosterone conditioned medium (TCM) and basal conditioned medium (BCM), respectively]. We observed an increase in the expression of PFKFB4 along with a decrease in PFKFB3 in spermatogenic cell lines treated with TCM. These effects were inhibited by the antiandrogen drug flutamide and by heat-inactivated TCM, indicating the protein nature of the TCM mediator and its dependence on Sertoli cell stimulation by dihydrotestosterone. In addition, adult rat testes treated with the GnRH antagonist Degarelix exhibited a reduction in the expression of PFKFB4 in germ cells. Addition of exogenous FGF-2 mimicked the changes in the Pfkfb gene expression, whereas neutralizing antibodies against FGF-2 abolished them. Interestingly, similar effects on Pfkfb gene expression were observed using different MAPK inhibitors (U-0126, PD-98059, and H-89). Luciferase analysis of Pfkfb4 promoter constructs demonstrated that a putative CRE-binding sequence located at -1,463 relative to the transcription start site is required to control Pfkfb4 gene expression after TCM treatment. Pulldown assays showed the binding of the CREB transcription factor to this site. Altogether, these results show how the paracrine regulation orchestrated by Sertoli cells in response to testosterone controls glycolysis in germ cells.
Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, E-08907, L'Hospitalet de Llobregat, Barcelona, Spain.
Marta
M
Gómez
Anna
A
Manzano
Agnes
A
Figueras
Francesc
F
Viñals
Francesc
F
Ventura
Jose Luis
JL
Rosa
Ramon
R
Bartrons
Àurea
À
Navarro-Sabaté
D000726:Androgen Antagonists / Q000494:pharmacology; D000818:Animals; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D002460:Cell Line; D013196:Dihydrotestosterone / Q000037:antagonists & inhibitors / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D004794:Enzyme Repression / Q000187:drug effects; D016222:Fibroblast Growth Factor 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D019899:Paracrine Communication / Q000187:drug effects; D025481:Phosphofructokinase-2 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012708:Sertoli Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013093:Spermatogonia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D000726:Androgen Antagonists; D057134:Antibodies, Neutralizing; C506672:PFKFB4 protein, human; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D013196:Dihydrotestosterone; D016222:Fibroblast Growth Factor 2; C107345:PFKFB3 protein, human; D025481:Phosphofructokinase-2