Identification and Characterization of a Novel Follistatin-like Protein as a Binding Protein for the TGF-β Family

Abstract

Follistatin is an activin-binding protein that prevents activin from binding to its receptors and neutralizes its activity. Follistatin also binds bone morphogenetic proteins (BMPs). In this study, we report the identification of a novel follistatin-like protein from mouse. The mouse cDNA encodes a 256-residue precursor and most likely a mouse homologue of human FLRG, which was found at the breakpoint of the chromosomal rearrangement in a B-cell line. Whereas follistatin has three follistatin domains, which are presumed to be growth factor binding motifs, FLRG possesses only two follistatin domains. Northern blotting revealed that mRNAs for FLRG were abundantly expressed in heart, lung, kidney, and testis in mouse. The recombinant mouse FLRG proteins were found to have binding activity for both activin and bone morphogenetic protein-2. Like follistatin, FLRG has higher affinity for activin than for BMP-2. The FLRG protein inhibited activin-induced and BMP-2-induced transcriptional responses in a dose-dependent manner. Glutathione S-transferase fusion proteins encoding various regions of FLRG were produced and studied. Ligand blotting using 125 I-activin revealed that the COOHterminal region containing the second follistatin domain was able to bind activin. Our finding implies that cellular signaling by activin and BMPs is tightly regulated by multiple members of the follistatin family.

Periostin Secreted by Mesenchymal Stem Cells Supports Tendon Formation in an Ectopic Mouse Model

“…These factors have not been linked to tendon formation and their abundance in adult tendon has therefore not been tested previously. Fstl1 encodes a secreted glycoprotein that binds members of the TGF-b superfamily of growth/differentiation factors and is able to interfere with BMP2-induced transcriptional responses in a dose-dependent manner [14]. Its expression might be one of the reasons for the low osteogenic properties of tenogenic C3H10T½-BMP2/Smad8ca cells [5] and for tenogenic tissue in general [15].…”

“…cDNA synthesis and reverse transcriptionpolymerase chain reaction Five micrograms of total RNA from cell cultures was reverse transcribed with the cDNA synthesis kit (MMLV-RT; Life Technologies) and with oligo-dT (12)(13)(14)(15)(16)(17)(18) -primer. For tendon and muscle tissue, the entire material was used for cDNA synthesis, which was carried out after DNase treatment to remove genomic DNA according to the protocol of the supplier (Revert Aid First Strand cDNA synthesis Kit; Fermentas) using random hexamer primers.…”

FLRG, an activin-binding protein, is a new target of TGFβ transcription activation through Smad proteins

“…Follistatin, through its binding to activin, modulates its biological activity in a broad range of cells. This suggests that FLRG may constitute another partner in the regulation of the biological activity of activin (Tsuchida et al, 2000). TGFb increases the expression of both activin ligands, FLRG and follistatin (Maguer-Satta et al, 2001).…”

Follistatin-related gene expression, but not follistatin expression, is decreased in human endometrial adenocarcinoma

“…Recently, a new protein that shares the characteristic 10-cysteine structures of follistatin domains was cloned from a B cell leukemia line based on its overexpression in response to a chromosomal translocation (10). Referred to as follistatin-related gene (FLRG) on the basis of the striking sequence homology with follistatin (11), it also shares a similar Nterminal domain with follistatin, a region that is crucial for the binding to activin (12) and, like follistatin, it binds strongly to activin (11,13,14).…”