Regulation of FSHβ induction in LβT2 cells by BMP2 and an Activin A/BMP2 chimera, AB215

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   Figure 1

   AB215 and BMP2 inhibit Activin A signaling to varying degrees in HEK293T cells. (A) The sequences of Activin A, BMP2, and AB215 are shown illustrating the segmental composition of AB215 and the location of the type 1 receptor- and type 2 receptor-binding epitopes. HEK293T cells were transfected with ID1-Luc (B and F), ARE-Lux (C, D, E and G), and constitutively active BMP type 1 receptors (CA-ALK2, CA-ALK3, or CA-ALK6) (F and G). Cells were treated with the indicated doses of BMP2, AB215, or Activin A (B, D, E and G) in the absence or presence of the ALK2/3/6 inhibitor LDN193189 (150 nM, dotted lines). All the assays were carried out in triplicate with five independent experiments and transfection differences were normalized using β-galactosidase. Curves were fitted using the Prism Software (GraphPad, San Diego, CA, USA) and shown in mean±s.d. RLU, relative light units.
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   Figure 2

   AB215 but not BMP2 inhibits Activin A signaling in LβT2 cells. HEK293T cells were transfected with ID1-Luc (A and E), ARE-Lux (B, C, D and F) and constitutively active BMP type 1 receptors (CA-ALK2, CA-ALK3, or CA-ALK6) (E and F), and treated with the indicated doses of BMP2, AB215, or Activin A (A, C, D and F) in the absence or presence of the ALK2/3/6 inhibitor LDN193189 (150 nM, dotted lines). All the assays were done in triplicate with five independent experiments and transfection differences were normalized using β-galactosidase. Curves were fitted using the Prism Software (GraphPad) and shown in mean±s.d. RLU, relative light units. The T-test was employed in (B) and one-way ANOVA with Dunnett's post hoc test was employed in (E and F).
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   Figure 3

   AB215 and BMP2 have opposing effects on Activin A induction of FSHβ. LβT2 cells were transfected with the −1900/+1 mFSHB-Luc construct and treated with the indicated doses of AB215, BMP2, and Activin A in the absence or presence of LDN193189 (150 nM, dotted lines; A, B and C). All the assays were carried out in triplicate with five independent experiments, and transfection differences were normalized using β-galactosidase. Curves were fitted using the Prism Software and shown in mean±s.d. RLU, relative light unites. Alternatively, FSHB mRNA levels were measured by RT-PCR after transfecting LβT2 cells with CA-ALK2, CA-ALK3, or CA-ALK6 and treating them as indicated with Activin A (D) or after treating untransfected cells with Activin A, BMP2, and/or AB215 as indicated (E). LβT2 cells were also treated with Activin A, BMP2, and/or AB215 as indicated and the resulting cell lysates were analyzed for the presence of phospho-Smad2 and phospho-Smad3 (F). Significance has been calculated using one-way ANOVA with Dunnett's (D) and Tukey's (E) post hoc tests and data are shown in means±s.d.
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   Figure 4

   AB215 and BMP2 signaling in HEK293T cells and LβT2 cells. Diagram illustrating the signaling and crosstalk of Activin A, AB215, and BMP2 in HEK293T cells and LβT2 cells. BMP pathway activation inhibits Activin A signaling in HEK293T cells but either has no inhibitory effect (ARE-Lux induction) or enhances (FSHβ induction) Activin A signaling in LβT2 cells. AB215 inhibits Activin A signaling in both cell lines while BMP2 partially inhibits Activin A signaling in HEK293T cells but not in LβT2 cells. Full colour version of this figure available via http://dx.doi.org/10.1530/JOE-14-0317.

• © 2014 Society for Endocrinology