Cell type-specific control of protein synthesis and proliferation by FGF-dependent signaling to the translation repressor 4E-BP Rachel Ruoffa, Olga Katsaraa, and Victoria Kolupaevaa,1 aDepartment of Microbiology, New York University Langone Medical Center, New York, NY 10016 Edited by Nahum Sonenberg, McGill University, Montreal, Canada, and approved May 16, 2016 (received for review April 7, 2016) Regulation of protein synthesis plays a vital role in posttranscrip- the proliferation and differentiation of chondrocytes (4). Chon- tional modulation of gene expression. Translational control most drocyte homeostasis is regulated by a number of signaling molecules commonly targets the initiation of protein synthesis: loading 40S and transcription factors, and genetic studies identified FGF ribosome complexes onto mRNA and AUG start codon recognition. signaling as a crucial player in these processes (5). FGF molecules This step is initiated by eukaryotic initiation factor 4E (eIF4E) (the signal by activating FGF Receptors (FGFRs), and several FGFR m7GTP cap-binding protein), whose binding to eIF4G (a scaffolding mutations have been linked to autosomal, dominant bone subunit) and eIF4A (an ATP-dependent RNA helicase) leads to assem- morphogenetic disorders (6). Importantly, all of these mutations bly of active eIF4F complex. The ability of eIF4E to recognize the cap are “gain of function” mutations, resulting in excessive FGF is prevented by its binding to eIF4E binding protein (4E-BP), which signaling. It has been well established that the major effect of thereby inhibits cap-dependent translation by sequestering eIF4E. FGF signaling in chondrocytes is growth inhibition (7, 8), an The 4E-BP activity is, in turn, inhibited by mTORC1 [mTOR (the mech- effect which is in striking contrast to a universal proliferative anistic target of rapamycin) complex 1] mediated phosphorylation. response seen in nearly all cell types. We previously identified Here, we define a previously unidentified mechanism of mTOR-in- several downstream effectors of FGF signaling in chondrocytes dependent 4E-BP1 regulation that is used by chondrocytes upon FGF at the transcriptional level (9). However, the effect of FGF signaling. Chondrocytes are responsible for the formation of the signaling on protein synthesis has never been studied, and the CELL BIOLOGY skeleton long bones. Unlike the majority of cell types where FGF mechanism of this unique chondrocyte-specific response remains signaling triggers proliferation, chondrocytes respond to FGF with to be elucidated. inhibition. We establish that FGF specifically suppresses protein syn- Here, we investigated the impact of FGF signaling on protein thesis in chondrocytes, but not in any other cells of mesenchymal synthesis in chondrocytes, where it causes growth inhibition. origin. Furthermore, 4E-BP1 repressor activity is necessary not only Unexpectedly, we report for the first time to our knowledge that for suppression of protein synthesis, but also for FGF-induced cell- FGF signaling can inhibit protein synthesis. This unique re- cycle arrest. Importantly, FGF-induced changes in the 4E-BP1 activity sponse is acquired de novo during chondrogenesis because observed in cell culture are likewise detected in vivo and reflect the mesenchymal stem cells (MSC) from which chondrocytes derive action of FGF signaling on downstream targets during bone devel- do not respond in a similar manner. FGF-induced inhibition of opment. Thus, our findings demonstrate that FGF signaling differen- protein synthesis requires the activity of 4E-BP1, the canonical tially impacts protein synthesis through either stimulation or inhibitor of cap-dependent mRNA translation. We suggest a repression, in a cell-type–dependent manner, with 4E-BP1 being a previously unidentified mechanism of 4E-BP1 activation that is key player. implemented in the presence of active mTORC1 (mTOR com- plex 1). This mechanism would allow for selective and rapid 4E-BP | FGF | chondrocytes | translational control adjustments in the activity of mTOR targets in response to ex- tracellular cues such as FGF signaling. egulation of translation allows rapid and reversible adjust- Rments of mRNA translation in response to various physio- Significance logical and pathological conditions (1). The rate-limiting step in protein synthesis is initiation, during which the small ribosomal Translational control is a key component of gene regulation; subunit, bound to several translation initiation factors and initiator therefore, it is important to understand alterations to the trans- MettRNA,isrecruitedtomRNA.Ribosomal loading onto mRNAs lational machinery that occur during development. We identified is mediated by eukaryotic initiation factor 4F (eIF4F), eIF4A, and a unique mTOR (the mechanistic target of rapamycin)-independent eIF4B. By binding to the cap structure of mRNAs, eIF4F positions mechanism of 4E-BP (a repressor of cap-dependent translation) the ribosomal preinitiation complex onto the 5′ end of mRNA and regulation that is utilized by chondrocytes in response to FGF sig- facilitates scanning toward the 5′ proximal AUG codon. One of the naling. It is well established that FGF signaling leads to inhibition of key mechanisms controlling protein synthesis involves modulating chondrocyte proliferation, and is crucial for bone development. We the activity of the m7GTP cap-binding protein eIF4E by a family found that FGF specifically suppresses protein synthesis in chon- of eIF4E binding proteins (4E-BPs). The ability of 4E-BPs to drocytes by activating 4E-BP. Importantly, FGF-induced changes in sequester eIF4E from eIF4F complexes depends on their phos- the translation apparatus observed in cell culture are similarly de- phorylation status (2). Hypophosphorylated 4E-BP binds eIF4E to tected in vivo, and reflect the action of FGF signaling on down- repress cap-dependent mRNA translation, whereas phosphoryla- stream targets during bone development. tion results in its release from eIF4E, which can then form func- tional eIF4F complexes. Phosphorylation of 4E-BP is mediated Author contributions: V.K. designed research; R.R., O.K., and V.K. performed research; R.R., by the serine/threonine kinase mTOR (the mechanistic target of O.K., and V.K. analyzed data; and O.K. and V.K. wrote the paper. rapamycin), which is activated by nutrients and numerous growth The authors declare no conflict of interest. factors, including fibroblast growth factors (FGFs) (3). This article is a PNAS Direct Submission. FGFs are critical regulators of many developmental processes, 1To whom correspondence should be addressed. Email: [email protected]. including bone development. Long bones and vertebrae are This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. formed through endochondral ossification, a process that requires 1073/pnas.1605451113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1605451113 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Results cells into osteoblasts, chondrocytes, and adipocytes (Fig. S1D)(13, FGF Signaling Down-Regulates Protein Synthesis in Chondrocytes. We 14). After 11 d in differentiation media the cells were treated with D first analyzed the effect of FGF on protein synthesis in chondrocytes FGF and total protein synthesis was analyzed. As shown in Fig. 1 , using Rat ChondroSarcoma (RCS) cells. These cells exhibit most FGF treatment inhibited protein synthesis only in the cells com- properties of proliferating chondrocytes, including FGF response, mitted to the chondrocyte lineage, supporting our hypothesis that and allow genetic manipulations that cannot be performed in pri- FGF-induced down-regulation of protein synthesis is chondrocyte- mary chondrocytes, whose life span in culture is limited. The specific and likely acquired during chondrocyte differentiation. changes in total protein synthesis in FGF-treated and untreated cells were measured after pulse-labeling with 35SMet/Cysmix.In FGF Signaling in Chondrocytes Activates 4E-BP1. To elucidate the parallel, we monitored FGF-induced changes in the cell cycle by mechanism of this chondrocyte-specific effect of FGF on protein FACScan analysis. Two additional cell lines with FGF proliferative synthesis we analyzed overall abundance and phosphorylation status response were included: OB1 cells (immortalized osteoblasts) (10) of 4E-BP1, the repressor of cap-dependent translation. There are four well-described 4E-BP phosphorylation sites: Thr37 and Thr46 and C3H10T1/2 cells that are functionally similar to MSC (11) and are able to differentiate into multiple lineages. Relative S-phase are phosphorylated first, followed by Thr70 and Ser65. mTORC1 complexes, comprised of mTOR kinase, Raptor, and GβL, medi- levels of OB1 and C H T1/2 cells were increased following FGF 3 10 ate 4E-BP inactivation (15). Differentially phosphorylated treatment (1.5-fold and threefold, respectively, at 24 h), whereas 4E-BP isoforms can be separated on high-density SDS PAGE proliferation of RCS cells was inhibited (Fig. S1A). The rate of total according to their phosphorylation marks with the uppermost protein synthesis in OB1 cells was virtually unaffected and slightly band representing fully phosphorylated, Ser65 positive, in- up-regulatedinC3H10T1/2 cells (1.3-fold). Importantly, FGF sup- A – capable of eIF4E binding, 4E-BP. As shown in Fig. 2 , pressed the rate of protein synthesis in RCS cells by 70 75% after dephosphorylated 4E-BP1 was detected as early as after 4 h of 24