Faculty Bibliography

Objectives Degeneration of articular cartilage is central to OA pathology; however, the molecular mechanisms leading to these irreversible changes are still poorly understood. Here, we investigated how changes in the chondrocyte translational apparatus may contribute to the pathology of OA. Methods Normal and OA human knee cartilage was used to analyze the activity of different components of the translational machinery. Chondrocytes isolated from lesional and non-lesional areas of OA cartilage were used to estimate relative rate of protein synthesis by metabolic labeling. Experimental OA was induced by transection of the anterior cruciate ligament in rats to investigate changes in the translational apparatus associated with OA. The role of IL-1beta signaling was assessed in vitro using rat articular chondrocytes. Expression of mRNAs was analyzed by qPCR and protein levels by immunohistochemistry and Western blotting. Results We identified several novel traits of OA chondrocytes, including upregulation of the Serine/Threonine kinases AKT2 and AKT3 at the post-transcriptional level and increased rate of total protein synthesis, likely due to inactivation of 4E-BP1, a known repressor of cap-dependent translation. We found that 4E-BP1 inactivation is mTOR-dependent and crucial for upregulation of protein synthesis in general and in particular for MMP13 and ADAMTS5 expression. In addition, IL-1beta treatment led to 4E-BP1 inactivation and upregulation of protein synthesis in articular chondrocytes. Conclusions Precise control of protein synthesis is vital for cartilage homeostasis and its dysregulation contributes to the molecular pathology of OA. Our study therefore identifies a novel set of potential therapeutic targets

Regulation of protein synthesis plays a vital role in posttranscriptional modulation of gene expression. Translational control most commonly targets the initiation of protein synthesis: loading 40S ribosome complexes onto mRNA and AUG start codon recognition. This step is initiated by eukaryotic initiation factor 4E (eIF4E) (the m7GTP cap-binding protein), whose binding to eIF4G (a scaffolding subunit) and eIF4A (an ATP-dependent RNA helicase) leads to assembly of active eIF4F complex. The ability of eIF4E to recognize the cap is prevented by its binding to eIF4E binding protein (4E-BP), which thereby inhibits cap-dependent translation by sequestering eIF4E. The 4E-BP activity is, in turn, inhibited by mTORC1 [mTOR (the mechanistic target of rapamycin) complex 1] mediated phosphorylation. Here, we define a previously unidentified mechanism of mTOR-independent 4E-BP1 regulation that is used by chondrocytes upon FGF signaling. Chondrocytes are responsible for the formation of the skeleton long bones. Unlike the majority of cell types where FGF signaling triggers proliferation, chondrocytes respond to FGF with inhibition. We establish that FGF specifically suppresses protein synthesis in chondrocytes, but not in any other cells of mesenchymal origin. Furthermore, 4E-BP1 repressor activity is necessary not only for suppression of protein synthesis, but also for FGF-induced cell-cycle arrest. Importantly, FGF-induced changes in the 4E-BP1 activity observed in cell culture are likewise detected in vivo and reflect the action of FGF signaling on downstream targets during bone development. Thus, our findings demonstrate that FGF signaling differentially impacts protein synthesis through either stimulation or repression, in a cell-type-dependent manner, with 4E-BP1 being a key player.

Fibroblast growth factor (FGF)-induced growth arrest of chondrocytes is a unique cell type-specific response which contrasts with the proliferative response of most cell types and underlies several genetic skeletal disorders caused by activating FGF receptor (FGFR) mutations. We have shown that one of the earliest key events in FGF-induced growth arrest is dephosphorylation of the retinoblastoma protein (Rb) family member p107 by protein phosphatase 2A (PP2A), a ubiquitously expressed multisubunit phosphatase. In this report, we show that the PP2A-B55alpha holoenzyme (PP2A containing the B55alpha subunit) is responsible for this phenomenon. Only the B55alpha (55-kDa regulatory subunit, alpha isoform) regulatory subunit of PP2A was able to bind p107, and this interaction was induced by FGF in chondrocytes but not in other cell types. Small interfering RNA (siRNA)-mediated knockdown of B55alpha prevented p107 dephosphorylation and FGF-induced growth arrest of RCS (rat chondrosarcoma) chondrocytes. Importantly, the B55alpha subunit bound with higher affinity to dephosphorylated p107. Since the p107 region interacting with B55alpha is also the site of cyclin-dependent kinase (CDK) binding, B55alpha association may also prevent p107 phosphorylation by CDKs. FGF treatment induces dephosphorylation of the B55alpha subunit itself on several serine residues that drastically increases the affinity of B55alpha for the PP2A A/C dimer and p107. Together these observations suggest a novel mechanism of p107 dephosphorylation mediated by activation of PP2A through B55alpha dephosphorylation. This mechanism might be a general signal transduction pathway used by PP2A to initiate cell cycle arrest when required by external signals.

The retinoblastoma/pocket protein family is one of the master regulators of the eukaryotic cell cycle. It includes the retinoblastoma protein (Rb) and the related p107 and p130 proteins. The importance of the Rb pathway for homeostasis and tumour suppression is evident from the fact that inactivating mutations in Rb are frequently associated with many cancers. Rbs regulate the cell cycle by controlling the activity of the E2F family of transcription factors. The activity of Rb proteins themselves is modulated by their phosphorylation status at several Ser/Thr residues: phosphorylation by cyclin-dependent kinases inactivates Rb proteins and positively influences the transcription of genes necessary for cell cycle progression. Although the mechanisms of cyclin-dependent kinase-mediated inactivation of Rb proteins are understood in great detail, our knowledge of the process that counteracts Rb phosphorylation is still quite limited. The present review focuses on the Ser/Thr phosphatases that are responsible for the dephosphorylation and thus activation of Rb proteins. Two major scenarios are considered: (a) when pocket proteins are dephosphorylated during regular cell cycle progression and (b) when rapid dephosphorylation is dictated by external stress or growth inhibitory conditions, such as oxidative stress, UV radiation or other DNA-damaging stimuli, and cell differentiation factors. It transpires that protein phosphatase 1 and protein phosphatase 2A can efficiently modulate pocket protein activity in a highly context-dependent manner and both are tightly regulated by the presence of different regulatory subunits or interacting proteins.

FGF signaling inhibits chondrocyte proliferation and requires the function of the p107 and p130 members of the Rb protein family to execute growth arrest. p107 dephosphorylation plays a critical role in the chondrocyte response to FGF, as overexpression of cyclin D1/CDK4 complexes (the major p107 kinase) in rat chondrosarcoma (RCS) cells overcomes FGF-induced p107 dephosphorylation and growth arrest. In cells overexpressing cyclin D1/CDK4, FGF-induced downregulation of cyclin E/CDK2 activity was absent. To examine the role of cyclin E/CDK2 complexes in mediating FGF-induced growth arrest, this kinase was overexpressed in RCS cells. FGF-induced dephosphorylation of either p107 or p130 was not prevented by overexpressing cyclin E/CDK2 complexes. Unexpectedly, however, FGF-treated cells exhibited sustained proliferation even in the presence of hypophosphorylated p107 and p130. Both pocket proteins were able to form repressive complexes with E2F4 and E2F5 but these repressors were not translocated into the nucleus and therefore were unable to occupy their respective target DNA sites. Overexpressed cyclin E/CDK2 molecules were stably associated with p107 and p130 in FGF-treated cells in the context of E2F repressive complexes. Taken together, our data suggest a novel mechanism by which cyclin E/CDK2 complexes can promote cell cycle progression in the presence of dephosphorylated Rb proteins and provide a novel insight into the key Retinoblastoma/E2F/cyclin E pathway. Our data also highlight the importance of E2F4/p130 complexes for FGF-mediated growth arrest in chondrocytes.

Fibroblast growth factors (FGFs) negatively regulate long bone development by inhibiting the proliferation of chondrocytes that accumulate in the G(1) phase of the cycle following FGF treatment. Here we report that FGF also causes a striking but transient delay in mitotic entry in RCS chondrocytes by inactivating the cyclin B1-associated CDK1(CDC2) kinase. As a consequence of this inactivation, cells accumulate in the G(2) phase of the cycle for the first 4-6 hours of the treatment. Cyclin B1/CDK1 activity is then restored and cells reach a G(1) arrest. The reduced cyclin B1/CDK1 activity was accompanied by increased CDK1 inhibitory phosphorylation, likely caused by increased activity and expression of the Myt1 kinase. FGF1 also caused dephosphorylation of the CDC25C phosphatase, that however appears due the inactivation of cyclin B1/CDK1 complex in the CDK1 feedback loop, and not the activation of specific phosphatases. the inactivation of the cyclin B1/CDK1 complex is a direct effect of FGF signaling, and not a consequence of the G(2) arrest as it can be observed also in cells blocked at mitosis by Nocodazole. The Chk1 and AtM/ATR kinase are known to play essential roles in the G(2) checkpoint induced by DNA damage/genotoxic stress, but inhibition of Chk1 or ATM/ATR not only did not prevent, but rather potentiated the FGF-induced G(2) arrest. Additionally our results indicate that the transient G(2) arrest is induced by FGF in RCS cell through mechanisms that are independent of the G(1) arrest, and that the G(2) block is not strictly required for the sustained G(1) arrest but may provide a pausing mechanism that allows the FGF response to be fully established

The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNA(i)(Met) attachment to 40S ribosomal subunits, scanning, start codon selection and subunit joining. Its bacterial ortholog IF1 consists of an oligonucleotide/oligosaccharide-binding (OB) domain, whereas eIF1A additionally contains a helical subdomain, N-terminal tail (NTT) and C-terminal tail (CTT). The NTT and CTT both enhance ribosomal recruitment of eIF2/GTP/Met-tRNA(i)(Met), but have opposite effects on the stringency of start codon selection: the CTT increases, whereas the NTT decreases it. Here, we determined the position of eIF1A on the 40S subunit by directed hydroxyl radical cleavage. eIF1A's OB domain binds in the A site, similar to IF1, whereas the helical subdomain contacts the head, forming a bridge over the mRNA channel. The NTT and CTT both thread under Met-tRNA(i)(Met) reaching into the P-site. The NTT threads closer to the mRNA channel. In the proposed model, the NTT does not clash with either mRNA or Met-tRNA(i)(Met), consistent with its suggested role in promoting the 'closed' conformation of ribosomal complexes upon start codon recognition. In contrast, eIF1A-CTT appears to interfere with the P-site tRNA-head interaction in the 'closed' complex and is likely ejected from the P-site upon start codon recognition.

The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions (+)11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions (-)3/(-)4 to rpS5(S7p) and h23b, (-)6/(-)7 to rpS14(S11p), and (-)8-(-)11 to the 3'-terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions (+)7/(+)8 to the central region of h28, (+)4/(+)5 to rpS15(S19p), and (-)6 and (-)7/(-)10 to eukaryote-specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2alpha (eIF2alpha) contacts position (-)3, and now report that eIF3 interacts with positions (-)8-(-)17, forming an extension of the mRNA-binding channel that likely contributes to unique aspects of eukaryotic initiation.