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. 2017 Mar 6;7:42937. doi: 10.1038/srep42937

Figure 4. Molecular mechanisms and pathways involved in IGSF1 deficiency.

Figure 4

In HEK293FT, IGSF1 represses the activity of both a human FSHB −875/+120 minimal promoter (pFSHB) (A) and its potent surrogate CAGA-luc promoter (pCAGA)(B), in a dose-response manner. (C) IGSF1 stimulates basal human TRHR promoter (pTRHR) activity in a dose-dependent manner. (D) A dose-response curve of Activin (2–80 ng/ml) demonstrates the potency of IGSF1 blunting the response to Activin. (E) IGSF1 is more potent than a dominant negative SMAD2 mutant (DSMAD2) and is able to repress the constitutive activity of an activating mutant of the Activin receptor (caALK4) in the absence of Activin. SB431542, antagonist of the receptor, was added in all conditions to show the functionality of the pCAGA response, blunted when this inhibitor was present. (F) In pituitary gonadotropes, RC-4B/C1, pCAGA response to activin was huge -circa 18 times over control- and reduced to 5 times in the presence of IGSF1 (left). A western blot shows induction of Fshb expression with Activin that was blunted in the presence of IGSF1 (right). (G) Activin does not induce the human TRHR promoter nor alters the basal stimulation in the presence of IGSF1. (H) TGFβ represses the luciferase activity of the TRHR promoter in a dose-response manner. Repression is reversed by IGSF1. (I) Endogenous expression of Trhr in pituitary GH4C1 cells by qRT-PCR (left). Trhr mRNA is repressed by TGFβ (black bars) and this is reversed by the TGFβ-inhibitor SB431542 (dark grey bars). IGSF1 blocks such repression as efficiently as the inhibitor. Prolactin (Prl) mRNA, another gene repressed by TGFβ, was not affected by the presence of IGSF1 and was equally repressed by TGFβ in its presence. SB431542 blocked TGFβ action in both genes. (J) C-terminal phosphorylation of SMAD2 (p-Smad2), mediated by TGFβRII/RI complex upon TGFβ addition, was markedly reduced by IGSF1 in pituitary GH4C1. Extracts were taken after one hour with TGFβ or Vehicle. SB431542, the TGFβRI inhibitor, blocks p-SMAD2 independently of the absence/presence of IGSF1. n.s.: not significant, *p < 0.05, **p < 0.01, ***p < 0.005.