Potentiation of Smad-mediated transcriptional activation by the RNA-binding protein RBPMS

Abstract

Smad2, Smad3 and Smad4 proteins are considered to be key mediators of transforming growth factor-β (TGF-β) signaling. However, the identities of the Smad partners mediating TGF-β signaling are not fully understood. Here, we show that RNA-binding protein with multiple splicing (RBPMS), a member of the RNA-binding protein family, physically interacts with Smad2, Smad3 and Smad4 both in vitro and in vivo. The presence of TGF-β increases the binding of RBPMS with these Smad proteins. Consistent with the binding results, overexpression of RBPMS enhances Smad-dependent transcriptional activity in a TGF-β-dependent manner, whereas knockdown of RBPMS decreases this activity. RBPMS interacts with TGF-β receptor type I (TβR-I), increases phosphorylation of C-terminal SSXS regions in Smad2 and Smad3, and promotes the nuclear accumulation of the Smad proteins. Moreover, RBPMS fails to enhance the transcriptional activity of Smad2 and Smad3 that lack the C-terminal phosphorylation sites. Our data provide the first evidence for an RNA-binding protein playing a role in regulation of Smad-mediated transcriptional activity and suggest that RBPMS stimulates Smad-mediated transactivation possibly through enhanced phosphorylation of Smad2 and Smad3 at the C-terminus and promotion of the nuclear accumulation of the Smad proteins.

Human four-and-a-half LIM family members suppress tumor cell growth through a TGF-β–like signaling pathway

“…Since FHL1 regulates TGF-β-responsive transcription in the presence or absence of TGF-β, we sought to determine whether FHL modulation of TGF-β-responsive transcription is TGF-β- and TGF-β receptor-independent. As shown in Figure 3A, TGF-β-neutralizing antibody did not affect FHL1 modulation of p3TP-Lux reporter activity, although it completely abolished reporter activity regulated by RNA-binding protein with multiple splicing (RBPMS), a protein that regulates this reporter activity in a TGF-β-dependent manner (22). Similar results were seen in the hepatoma cell lines, Hep3B and SMMC7721 (data not shown).…”

“…The Matchmaker 2-hybrid system (Clontech) was used to isolate proteins that interacted with the Smad4 bait protein. The bait plasmid and a mammary cDNA library were sequentially transformed into AH109 yeast cells as previously described (22).…”

“…Cells were transfected and lysed in lysis buffer. Subcellular fractionation and coimmunoprecipitation were performed as previously described (22).…”

“…Similar results were observed in the hepatoma cell lines, Hep3B and SMMC7721, and the embryonic kidney cell line, 293T (data not shown). Moreover, using a lac repressor-lac operator recognition system (22), FHL1 directly enhanced the transcriptional activity of lac-Smad2, lac-Smad3, or lac-Smad4 in a TGF-β-independent manner ( Figure 2D).…”

Expression of TGF-β Signaling Regulator RBPMS (RNA-Binding Protein With Multiple Splicing) Is Regulated by IL-1β and TGF-β Superfamily Members, and Decreased in Aged and Osteoarthritic Cartilage

“…Recently, a protein called RNA-binding protein with multiple splicing (RBPMS) has been shown to physically interact with Smads and to enhance C-terminal phosphorylation of Smad2 and Smad3, further enhancing transcriptional activity. 14 Since TGF-β signaling is crucial for maintaining healthy cartilage in a Smad-dependent manner, we have studied whether RBPMS is expressed in articular chondrocytes and whether this expression is modulated by growth factors such as TGF-β itself and BMP2, and by an inflammatory mediator (interleukin-1β ). In addition, we analyzed RBPMS expression in different age groups of C57BL/6N mice (6 months and 20 months) and in a model of experimental OA in mice to explore a putative role in maintenance of articular cartilage.…”