The TATA binding protein associated factor 4b (TAF4b) mediates FSH stimulation of the IGFBP-3 promoter in cultured porcine ovarian granulosa cells

Abstract

We have established the gene for IGF binding protein-3 (IGFBP-3) as a target for FSH action. FSH effects on this gene require the PKA pathway as well as the PI-3 kinase and MAPK pathways. At the IGFBP-3 promoter, FSH effects depend on a site for TATA box binding protein (TBP) and formation of a high molecular weight transcription complex. To further elucidate FSH effects on the downstream events involving the TBP site, we cloned a pig TAF4b cDNA into a P-Flag expression vector. By co-transfecting granulosa cells with the IGFBP-3 promoter, we found that TAF4b mimics and enhances FSH induction of IGFBP-3 reporter activity. Using RT-PCR we showed that FSH stimulates expression of TAF4b. This would suggest that the role of TAF4b in follicular development is regulated by FSH. TAF4b may thus be the TFIID component that binds to the TBP site on the IGFBP-3 promoter and is essential for FSH induction of IGFBP-3.

Figure 1

A box shade comparison of the pig peptide sequence translated from the pig TAF4b cDNA sequence determined by the open reading frame finder with that of sequence from the mouse (Mu, Accession # XM_128905), rat (Accession # XM_226146), predicted human (Hu-1, accession # XM_290809), and cloned human partial sequence (Hu-2, Accession # Y09321). The pig peptide sequence has 79% homology to the mouse, 89% to the predicted human, 87% to the cloned human, and 76% homology to the rat.

Figure 2

Luciferase activity of granulosa cells cotransfected with a −191/+9 IGFBP-3 promoter construct and a TAFb expression vector. Cells were cultured in 10% FBS for 18h and transfected with either a −191/+9 IGFBP-3 promoter construct of a combination of the IGFBP-3 construct and a TAF4b expression vector in PFLAG, using the Lipofectin method. Cells were maintained in 10% FBS until 95% confluent. They were then incubated in serum-free medium overnight before being exposed to 100 ng/ml FSH for 3h. Control cells were treated with an equal volume of PBS. Beta galactosidase was used as an internal control. All transfections were done in triplicate and repeated 3 times with different batches of granulose cells. Results are mean±SEM.

Figure 3

Expression of TAF4b mRNA by granulosa cells following FSH treatment. Granulosa cells were cultured to 95% confluence, serum starved overnight and treated with 100 ng/ml FSH for 0, 30, 60, 180 or 360 minutes. Total RNA was extracted and subjected to RT-PCR. Primers for the housekeeping gene GAPDH were used as internal controls. Densitometry was used to quantify the TAF4b mRNA levels relative to the GAPDH mRNA at each time point. The blot is representative of 3 different blots, with different batches of granulose cells.

Figure 4

Relative luciferase activity of granulosa cells transiently transfected with TAFb reporter constructs. TAF4b promoter constructs were generated by PCR using human genomic DNA as template and primers designed from the human TAF4b promoter sequence (Genbank accession # AC121320). Seeded granulosa cells were cultured in 10% FBS for 18 h and transfected with two TAF4b human promoter constructs using the Lipofectin method. A promoterless vector was used as a negative control. At 95% confluence, transfected cells were serum starved overnight and exposed to FSH for 3 h. Control cells were treated with an equal volume of PBS. All granulosa cells were co-transfected with plasmid for the housekeeping gene beta galactosidase as an internal control. All assays were done at least three times in triplicate. Results are reported as mean±SEM.

Figure 5

Luciferase activity of granulosa cells transfected with a −626/+36 TAF4b promoter construct exposed to FSH following a one h pre-incubation in 10 μM of the PKA inhibitor H89, 10 μM of the PI-3 kinase inhibitor LY294002 or 5 μM of the ERK1/2 inhibitor U0126. Cells were seeded in 10% FBS for 18 h, transfected in serum-free medium using the Lipofectin method for 6h, and further cultured in 10% FBS. All cells were co-transfected with beta galactosidase as an internal control. At 95% confluency, the cells were serum-starved overnight and pre-incubated in the inhibitors for 1h before being exposed to 100 ng/ml FSH for 3h. A negative control group without FSH was included. Transfection were done in triplicate and repeated at least 3 times.

Figure 6

Western blot analysis and kinase assays of total protein lysate from granulosa cells treated with FSH. Granulosa cells were cultured to 95% confluence, serum starved overnight and treated with 100 ng/ml FSH for 0, 10, 30, 60, or 180 minutes in serum-free medium. In panel A, total protein lysate was probed with antibodies directed against B-Raf and Raf 1. In panel B total protein was immunoprecipitated against B-Raf or Raf1 antibodies. A kinase assay was performed using His₆-MEK1 as substrate. In panel C, total protein was probed with antibodies directed against P-Raf1 antibodies (ser 259 and ser 338 respectively). Membranes were stripped and reprobed with antibodies against total Raf1 to validate loading intensity.

Figure 7

Summary of FSH signaling pathways employed by FSH pig granulosa cells in the regulation of IGFBP-3 FSH target genes. Binding of FSH to its receptor, FSHR activates adenylyl cyclase via coupling with the heterodimeric G proteins. The resulting increase in cAMP activates both PKA and the PI-3 kinase. Activation of PKA causes dissociation of the catalytic subunit, which enters the nucleus and phosphorylates transcription factors such as CREB. The phosphorylated CREB then recruits the CREB binding protein homolog p300. Phospho-CREB-p300 associates with Sp1, Sp3 and TAF4b to form a complex that stimulates IGFBP-3 transcription. Solid lines represent pathways that have been documented in granulosa cells, while dashed lines represent pathways suggested by our data. A similar figure has been previously published (Ongeri et al., 2005).