Chaperone-E3 Ligase Complex HSP70-CHIP Mediates Ubiquitination of Ribosomal Protein S3.

In addition to its role in ribosome biogenesis, ribosomal protein S3 (RPS3), a component of the 40S ribosomal subunit, has been suggested to possess several extraribosomal functions, including an apoptotic function. In this study, we demonstrated that in the mouse brain, the protein levels of RPS3 were altered by the degree of nutritional starvation and correlated with neuronal apoptosis. After endurable short-term starvation, the apoptotic function of RPS3 was suppressed by Akt activation and Akt-mediated T70 phosphorylation, whereas after prolonged starvation, the protein levels of RPS3 notably increased, and abundant neuronal death occurred. These events coincided with ubiquitination and subsequent degradation of RPS3, controlled by HSP70 and the cochaperone E3 ligase: carboxy terminus of heat shock protein 70-interacting protein (CHIP). Thus, our study points to an extraribosomal role of RPS3 in balancing neuronal survival or death depending on the degree of starvation through CHIP-mediated polyubiquitination and degradation.

Cytoplasmic ribosomal protein S3 (rpS3) plays a pivotal role in mitochondrial DNA damage surveillance.

Ribosomal protein S3 (rpS3) is known to play critical roles in ribosome biogenesis and DNA repair. When cellular ROS levels increase, the mitochondrial genes are highly vulnerable to DNA damage. Increased ROS induces rpS3 accumulation in the mitochondria for DNA repair while significantly decreasing the cellular protein synthesis. For the entrance into the mitochondria, the accumulation of rpS3 was regulated by interaction with HSP90, HSP70, and TOM70. Pretreatment with geldanamycin, which binds to the ATP pocket of HSP90, significantly decreased the interaction of rpS3 with HSP90 and stimulated the accumulation of rpS3 in the mitochondria. Furthermore, cellular ROS was decreased and mtDNA damage was rescued when levels of rpS3 were increased in the mitochondria. Therefore, we concluded that when mitochondrial DNA damages accumulate due to increased levels of ROS, rpS3 accumulates in the mitochondria to repair damaged DNA due to the decreased interaction between rpS3 and HSP90 in the cytosol.

Ribosomal protein S3 is secreted as a homodimer in cancer cells.

Protein secretion is a general phenomenon by which cells communicate with the extracellular environment. Secretory proteins, including hormones, enzymes, toxins, and antimicrobial peptides have various functions in extracellular environments. Here, we determined that ribosomal protein S3 (rpS3) is homodimerized and secreted in several cancer cell lines such as HT1080 (human fibrosarcoma) and MPC11 (mouse plasmacytoma). Moreover, we found that the secreted rpS3 protein increased in doxorubicin-resistant MPC11 cells compared to that in MPC11 cells. In addition, we also detected that the level of secreted rpS3 increased in more malignant cells, which were established with continuous exposure of cigarette smoke condensate. These findings suggest that the secreted rpS3 protein is an indicator of malignant tumors.

Mitochondrial genome diversity of Balamuthia mandrillaris revealed by a fatal case of granulomatous amoebic encephalitis.

Introduction: Balamuthia (B.) mandrillaris is a free-living amoeba that can cause rare yet fatal granulomatous amoebic encephalitis (GAE). However, efficacious treatment for GAE is currently unavailable, especially when genomic studies on B. mandrillaris are limited. Methods: In this study, B. mandrillaris strain KM-20 was isolated from the brain tissue of a GAE patient, and its mitochondrial genome was de novo assembled using high-coverage Nanopore long reads and Illumina short reads. Results and Discussion: Phylogenetic and comparative analyses revealed a range of diversification in the mitochondrial genome of KM-20 and nine other B. mandrillaris strains. According to the mitochondrial genome alignment, one of the most variable regions was observed in the ribosomal protein S3 (rps3), which was caused by an array of novel protein tandem repeats. The repeating units in the rps3 protein tandem region present significant copy number variations (CNVs) among B. mandrillaris strains and suggest KM-20 as the most divergent strain for its highly variable sequence and highest copy number in rps3. Moreover, mitochondrial heteroplasmy was observed in strain V039, and two genotypes of rps3 are caused by the CNVs in the tandem repeats. Taken together, the copy number and sequence variations of the protein tandem repeats enable rps3 to be a perfect target for clinical genotyping assay for B. mandrillaris. The mitochondrial genome diversity of B. mandrillaris paves the way to investigate the phylogeny and diversification of pathogenic amoebae.

RPS3 Promotes the Metastasis and Cisplatin Resistance of Adenoid Cystic Carcinoma.

Background: Adenoid cystic carcinoma (ACC) is a malignant tumor in salivary gland tissue, that is characterized by strong invasiveness and lung metastasis, leading to poor survival rates. RPS3 is been reported to be associated with the biological functions of tumor cells. This study explored the regulatory effect of RPS3 in ACC to provide new therapeutic targets for ACC therapy. Methods: We reviewed the clinical and pathologic data of 73 ACC patients. The expression of RPS3 was examined in ACC by immunohistochemistry. Transwell, wound healing, half-maximal inhibitory concentration (IC50) and other experiments were used to determine the regulatory effect of RPS3 on ACC functions. Coimmunoprecipitation and mass spectrometry analysis were used to detect the binding proteins of RPS3, mechanisms by which RPS3/STAT1/NF-kB signaling regulates ACC behavior were assessed using western blotting (WB), qPCR, etc. To explore the regulatory effect of RPS3 on ACC in vivo, we constructed nude mouse sciatic nerve infiltration model and a lung metastasis model for studies. Results: High RPS3 expression was associated with metastasis and a poor prognosis in ACC patients. Inhibition of RPS3 expression reduced ACC migration, invasion and cisplatin resistance, and overexpression of RPS3 promoted ACC migration, invasion and cisplatin resistance. Further experiments revealed that RPS3 can activate the STAT1/NF-kB signaling pathway and regulate ACC behavior through binding to STAT1. The incidence of sciatic nerve infiltration and lung metastasis in nude mice after RPS3 knockdown was lower than that of the control group in vivo. Conclusion: RPS3 is highly expressed and associated with the prognosis and survival of ACC patients. The RPS3/STAT1/NF-kB pathway may play an important regulatory role in ACC migration, invasion and chemoresistance. As a new therapeutic target of ACC, its clinical application value is worthy of attention and further exploration.

PRRSV Induces HMGB1 Phosphorylation at Threonine-51 Residue to Enhance Its Secretion.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces secretion of high mobility group box 1 (HMGB1) to mediate inflammatory response that is involved in the pulmonary injury of infected pigs. Our previous study indicates that protein kinase C-delta (PKC-delta) is essential for HMGB1 secretion in PRRSV-infected cells. However, the underlying mechanism in HMGB1 secretion induced by PRRSV infection is still unclear. Here, we discovered that the phosphorylation level of HMGB1 in threonine residues increased in PRRSV-infected cells. A site-directed mutagenesis study showed that HMGB1 phosphorylation at threonine-51 was associated with HMGB1 secretion induced by PRRSV infection. Co-immunoprecipitation (co-IP) of HMGB1 failed to precipitate PKC-delta, but interestingly, mass spectrometry analysis of the HMGB1 co-IP product showed that PRRSV infection enhanced HMGB1 binding to ribosomal protein S3 (RPS3), which has various extra-ribosomal functions. The silencing of RPS3 by siRNA blocked HMGB1 secretion induced by PRRSV infection. Moreover, the phosphorylation of HMGB1 at threonine-51 was correlated with the interaction between HMGB1 and RPS3. In vivo, PRRSV infection also increased RPS3 levels and nuclear accumulation in pulmonary alveolar macrophages. These results demonstrate that PRRSV may induce HMGB1 phosphorylation at threonine-51 and increase its interaction with RPS3 to enhance HMGB1 secretion. This finding provides insights into the pathogenesis of PRRSV infection.

Fucosylated Proteome Profiling Identifies a Fucosylated, Non-Ribosomal, Stress-Responsive Species of Ribosomal Protein S3.

Alterations in genes encoding for proteins that control fucosylation are known to play causative roles in several developmental disorders, such as Dowling-Degos disease 2 and congenital disorder of glycosylation type IIc (CDGIIc). Recent studies have provided evidence that changes in fucosylation can contribute to the development and progression of several different types of cancers. It is therefore important to gain a detailed understanding of how fucosylation is altered in disease states so that interventions may be developed for therapeutic purposes. In this report, we find that fucosylation occurs on many intracellular proteins. This is an interesting finding, as the fucosylation machinery is restricted to the secretory pathway and is thought to predominately affect cell-membrane-bound and secreted proteins. We find that Ribosomal protein S3 (RPS3) is fucosylated in normal tissues and in cancer cells, and that the extent of its fucosylation appears to respond to stress, including MAPK inhibitors, suggesting a new role in posttranslational protein function. Our data identify a new ribosome-independent species of fucosylated RPS3 that interacts with proteins involved in posttranscriptional regulation of RNA, such as Heterogeneous nuclear ribonucleoprotein U (HNRNPU), as well as with a predominance of non-coding RNAs. These data highlight a novel role for RPS3, which, given previously reported oncogenic roles for RPS3, might represent functions that are perturbed in pathologies such as cancer. Together, our findings suggest a previously unrecognized role for fucosylation in directly influencing intracellular protein functions.

High RPS3A expression correlates with low tumor immune cell infiltration and unfavorable prognosis in hepatocellular carcinoma patients.

Despite the use of immune checkpoint blockade (ICB) therapy for hepatocellular carcinoma (HCC), developing more effective immunotherapy and predicting HCC's response to ICB therapy remain top priorities. Ribosomal protein S3A (RPS3A) is a multifunctional molecule, but its association with tumor immune cell infiltration and prognosis in HCC patients is unknown. Thus, we aimed to investigate the correlation of RPS3A with HCC immune cell infiltration and prognosis to explore novel therapeutic strategies and prognostic biomarkers for this disease. Here, we showed that RPS3A expression levels were higher in HCC cell lines and samples than in normal hepatocytes and adjacent tumor-free tissues, respectively, and patients with high RPS3A expression had worse overall and recurrence-free survival durations than did patients with low expression. Moreover, single-sample gene set enrichment analysis (ssGSEA) and immunohistochemistry demonstrated a strongly negative correlation between RPS3A expression and tumor immune cell infiltration. Meanwhile, RPS3A was revealed to be positively correlated with that of most examined immune checkpoint molecules. GSEA also suggested that high RPS3A expression promoted the biological processes related to tumorigenesis, metastasis, and immunosuppression. Finally, RPS3A-based nomograms were constructed and exhibited better predictive accuracy for HCC prognosis and more net clinical benefits when compared with traditional prognosis-prediction staging systems. In short, these findings suggest that high RPS3A expression correlates with low tumor immune cell infiltration and poor prognosis in HCC patients. Furthermore, RPS3A-based nomograms are robust HCC prognostic predictors. RPS3A therefore may serve as a therapeutic target in and predict the efficacy of ICB therapy for HCC.

Knockdown of ribosomal protein S3 causes preimplantation developmental arrest in mice.

Ribosomal protein S3 (RpS3), a member of the ribosome 40S subunit, has conventional ribosomal function and additional extraribosomal functions. The aim of the present study was to analyze the expression and localization of RpS3 and its function in early embryogenesis in mice. RpS3 mRNA and protein were expressed in multiple mouse tissues. In the ovary, RpS3 protein was ubiquitously and highly expressed in oocytes and granulosa cells. After ovulation and fertilization, RpS3 mRNA and protein were detected in oocytes and preimplantation embryos. Furthermore, RpS3 protein was localized in the cytoplasm of oocytes and preimplantation embryos. Moreover, knockdown of RpS3 in zygotes led to a significantly decreased rate of blastocyst formation. These results provide the first evidence for a novel function of RpS3 in regulating early embryonic development in mice.

The DNA repair domain of human rpS3 protects against photoaging by removing cyclobutane pyrimidine dimers.

Ribosomal protein S3 (rpS3) has endonuclease activity for DNA repair. In particular, rpS3 cleaves the phosphodiester bonds of damaged DNA. In this study, we show that the repair domain of rpS3 spans amino acids 144-189. We fused rpS3 with the transactivator of transcription (TAT) sequence to introduce the rpS3 repair domain into cells. We find that the TAT-rpS3 (aa: 144-189) peptide cleaves UV-induced cyclobutane pyrimidine dimers (CPDs) in cells. We also reveal that the TAT-rpS3 peptide reduces matrix metalloproteinase-1 (MMP-1) induction in UV-irradiated fibroblasts and increases cell migration activity. Taken together, our study suggests that penetration of the rpS3 repair domain into cells can cleave UV-induced CPDs and reduce MMP-1 expression induced by UV.

A novel TLR4 binding protein, 40S ribosomal protein S3, has potential utility as an adjuvant in a dendritic cell-based vaccine.

BACKGROUND: Dendritic cells (DCs) are professional antigen presenting cells (APCs), which can activate antigen-specific CD8+ T cell immunity, resulting in tumor clearance. Immature DCs are usually stimulated by various adjuvants through their immune receptors. Among them, Toll-like receptor 4 (TLR4) has an important role in activating DCs to cause their maturation. In fact, TLR4 is well-known to induce innate and adaptive immune responses against various external microbial or internal damage associated molecular patterns (DAMP). LPS is widely regarded as a strong stimulator of TLR4 signaling. However, LPS is inappropriate for use in humans since it is an endotoxin. Unfortunately, other TLR4 ligands such as HMGB1 or heat shock proteins have weak adjuvant effects. Therefore, there is a need to identify novel, biocompatible, strong, TLR4 ligands. METHODS: 40S ribosomal protein S3 (RPS3) was screened through pull-down assay using TLR4. BMDCs from wild type (WT) and TLR4 knock-out mice were treated by RPS3 to identify the activation and maturation of DCs. T cell generation including memory T cells, tumor prevention, and treatment experiments were performed with BMDCs based vaccination. Also, human DCs originated from patients were treated by RPS3 to confirm the activation and maturation of DCs. RESULTS: In this study, we identified 40S ribosomal protein S3 (RPS3) through a pull-down assay using a variety of human cancer cell-derived proteins that could bind to TLR4. RPS3 was released from tumor cells following treatment with an anticancer drug, and it was shown that the released RPS3 binds to TLR4. Recombinant RPS3 induced maturation and activation of DCs, and following pulsing with tumor specific antigens, these DCs could be used as a vaccine to significantly increase tumor specific CD8+IFN-γ+ T cells, and provide both tumor prevention and tumor treatment effects. The effect of RPS3 on DC maturation and its utility as a vaccine were shown to be dependent on TLR4 using TLR4 knockout mice. CONCLUSIONS: This study therefore proved that human cancer cell-derived RPS3, a novel TLR4 ligand, has great potential as an adjuvant in tumor-specific antigen DC-based vaccines.

Detection of 8-oxoguanine and apurinic/apyrimidinic sites using a fluorophore-labeled probe with cell-penetrating ability.

BACKGROUND: Reactive oxygen species (ROS) produce different lesions in DNA by ROS-induced DNA damage. Detection and quantification of 8-oxo-7,8-dihydroguanine (8-oxoG) within cells are important for study. Human ribosomal protein S3 (hRpS3) has a high binding affinity to 8-oxoG. In this study, we developed an imaging probe to detect 8-oxoG using a specific peptide from hRpS3. Transactivator (TAT) proteins are known to have cell-penetrating properties. Therefore, we developed a TAT-S3 probe by attaching a TAT peptide to our imaging probe. RESULTS: A DNA binding assay was conducted to confirm that our probe bound to 8-oxoG and apurinic/apyrimidinic (AP) sites. We confirmed that the TAT-S3 probe localized in the mitochondria, without permeabilization, and fluoresced in H2O2-treated HeLa cells and zebrafish embryos. Treatment with Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, reduced TAT-S3 probe fluorescence. Additionally, treatment with O8, an inhibitor of OGG1, increased probe fluorescence. A competition assay was conducted with an aldehyde reaction probe (ARP) and methoxyamine (MX) to confirm binding of TAT-S3 to the AP sites. The TAT-S3 probe showed competitive binding to AP sites with ARP and MX. CONCLUSIONS: These results revealed that the TAT-S3 probe successfully detected the presence of 8-oxoG and AP sites in damaged cells. The TAT-S3 probe may have applications for the detection of diseases caused by reactive oxygen species.

Esculentoside A specifically binds to ribosomal protein S3a and impairs LPS-induced signaling in macrophages.

Esculentoside A (EsA), a saponin isolated from Phytolacca esculenta, is reported as a potent suppressor of pro-inflammatory functions of macrophages. However, little is known about the target proteins of EsA for its anti-inflammatory activity. In the present study, to identify the intracellular target for EsA, affinity resins bearing immobilized EsA were used to capture binding proteins of EsA from RAW264.7 cell lysates. Mass spectrography and Western blot analysis of captured proteins indicated that ribosomal protein S3a preferentially bound to EsA affinity resin. Competition experiment further demonstrated that free EsA can disturb the specific interaction between recombinant RPS3a and affinity resin. Surface Plasmon Resonance analysis confirmed that EsA directly bound to RPS3a. Lentivirus-mediated RNAi RPS3a resulted in suppression of TNF-α and IL-6 production and impediment of signal transduction in LPS-stimulated RAW264.7 cells, indicating that RPS3a is required for LPS-triggered signaling during induction of pro-inflammatory cytokines. In addition, EsA inhibited the expression of inflammatory factors more strongly in the case of RPS3a interference. These results suggest that EsA exerts its anti-inflammatory activity by targeting RPS3a and impairing its signaling function. These new findings not only extended our understanding on the intracellular mechanisms of EsA, but also indicated RPS3a as an essential component for LPS-mediated pro-inflammatory signaling, thus implying RPS3a as a novel therapeutic target for anti-inflammatory therapy.

Ribosomal Protein S3 Gene Silencing Protects Against Cigarette Smoke-Induced Acute Lung Injury.

Chronic obstructive pulmonary disease (COPD) is estimated to be the third leading cause of death by 2030. Transcription factor NF-κB may play a critical role in COPD pathogenesis. Ribosomal protein S3 (RPS3), a 40S ribosomal protein essential for executing protein translation, has recently been found to interact with the NF-κB p65 subunit and promote p65 DNA-binding activity. We sought to study whether RPS3 gene silencing could protect against cigarette-smoke (CS)-induced acute lung injury in a mouse model. Effects of an intratracheal RPS3 siRNA in CS-induced lung injury were determined by measuring bronchoalveolar lavage (BAL) fluid cell counts, levels of inflammatory and oxidative damage markers, and NF-κB translocation. Lung RPS3 level was found to be upregulated for the first time with CS exposure, and RPS3 siRNA blocked CS-induced neutrophil counts in BAL fluid. RPS3 siRNA suppressed CS-induced lung inflammatory mediator and oxidative damage marker levels, as well as nuclear p65 accumulation and transcriptional activation. RPS3 siRNA was able to disrupt CS extract (CSE)-induced NF-κB activation in an NF-κB reporter gene assay. We report for the first time that RPS3 gene silencing ameliorated CS-induced acute lung injury, probably via interruption of the NF-κB activity, postulating that RPS3 is a novel therapeutic target for COPD.

Glutathionylation of Yersinia pestis LcrV and Its Effects on Plague Pathogenesis.

Glutathionylation, the formation of reversible mixed disulfides between glutathione and protein cysteine residues, is a posttranslational modification previously observed for intracellular proteins of bacteria. Here we show that Yersinia pestis LcrV, a secreted protein capping the type III secretion machine, is glutathionylated at Cys273 and that this modification promotes association with host ribosomal protein S3 (RPS3), moderates Y. pestis type III effector transport and killing of macrophages, and enhances bubonic plague pathogenesis in mice and rats. Secreted LcrV was purified and analyzed by mass spectrometry to reveal glutathionylation, a modification that is abolished by the codon substitution Cys273Ala in lcrV Moreover, the lcrVC273A mutation enhanced the survival of animals in models of bubonic plague. Investigating the molecular mechanism responsible for these virulence attributes, we identified macrophage RPS3 as a ligand of LcrV, an association that is perturbed by the Cys273Ala substitution. Furthermore, macrophages infected by the lcrVC273A variant displayed accelerated apoptotic death and diminished proinflammatory cytokine release. Deletion of gshB, which encodes glutathione synthetase of Y. pestis, resulted in undetectable levels of intracellular glutathione, and we used a Y. pestis ΔgshB mutant to characterize the biochemical pathway of LcrV glutathionylation, establishing that LcrV is modified after its transport to the type III needle via disulfide bond formation with extracellular oxidized glutathione.IMPORTANCEYersinia pestis, the causative agent of plague, has killed large segments of the human population; however, the molecular bases for the extraordinary virulence attributes of this pathogen are not well understood. We show here that LcrV, the cap protein of bacterial type III secretion needles, is modified by host glutathione and that this modification contributes to the high virulence of Y. pestis in mouse and rat models for bubonic plague. These data suggest that Y. pestis exploits glutathione in host tissues to activate a virulence strategy, thereby accelerating plague pathogenesis.

New role of human ribosomal protein S3: Regulation of cell cycle via phosphorylation by cyclin-dependent kinase 2.

Human ribosomal protein S3 (hRpS3) is a component of the 40S ribosomal subunit that associated in protein synthesis. hRpS3 has additional ribosomal functions such as DNA repair, transcription, metastasis, and apoptosis via interaction with numerous signaling molecules and has different modifications. Cyclin-dependent kinases (CDKs) are heterodimeric serine/threonine protein kinases that regulate cell cycle progression. Among its members, the Cdk1-cyclin B complex is known to control cell progression in the G2/M phase, while Cdk2-cyclin E/A complexes function in G1/S and S/G2 transition. In our previous study, we observed interaction between hRpS3 and Cdk1. The present study investigated the interaction between hRpS3 and Cdk2. Cdk2 phosphorylated hRps3 at amino acid residues S6 and T221 during the S-phase. Furthermore, hRpS3 knockdown delayed cell cycle progression by modulating the expression of cell cycle-related proteins, including cyclin B1 and cyclin E1. These findings suggest that hRpS3 is involved in Cdk2-mediated cell cycle regulation.

Ribosomal protein S3 (rpS3) secreted from various cancer cells is N-linked glycosylated.

Ribosomal protein S3 (rpS3) is a 243 amino acid component of the 40S ribosomal small subunit. It has multiple roles in translation and extra-ribosomal functions like apoptosis and DNA repair. RpS3 is secreted only in cancer cell lines. Presently, mass spectrometry analysis revealed rpS3 to be glycosylated at the Asn165 residue. A point mutation at this residue decreased secretion of rpS3 in cancer cell lines. Secretion was also inhibited by the endoplasmic reticulum (ER)-Golgi transport inhibitor Brefeldin A and by Tunicamycin, an inhibitor of N-linked glycosylation. N-linked glycosylation of rpS3 was confirmed as necessary for rpS3 secretion into culture media via the ER-Golgi dependent pathway. RpS3 bound to Concanavalin A, a carbohydrate binding lectin protein, while treatment with peptide-N-glycosidase F shifted the secreted rpS3 to a lower molecular weight band. In addition, the N165G mutant of rpS3 displayed reduced secretion compared to the wild-type. An in vitro binding assay detected rpS3 homodimer formation via the N-terminal region (rpS3:1-85) and a middle region (rpS3:95-158). The results indicate that the Asn 165 residue of rpS3 is a critical site for N-linked glycosylation and passage through the ER-Golgi secretion pathway.

Structural basis for Sfm1 functioning as a protein arginine methyltransferase.

SPOUT proteins constitute one class of methyltransferases, which so far are found to exert activity mainly towards RNAs. Previously, yeast Sfm1 was predicted to contain a SPOUT domain but can methylate ribosomal protein S3. Here we report the crystal structure of Sfm1, which comprises of a typical SPOUT domain and a small C-terminal domain. The active site is similar to that of protein arginine methyltransferases but different from that of RNA methyltransferases. In addition, Sfm1 exhibits a negatively charged surface surrounding the active site unsuitable for RNA binding. Our biochemical data show that Sfm1 exists as a monomer and has high activity towards ribosomal protein S3 but no activity towards RNA. It can specifically catalyze the methylation of Arg146 of S3 and the C-terminal domain is critical for substrate binding and activity. These results together provide the structural basis for Sfm1 functioning as a PRMT for ribosomal protein S3.

Ribosomal protein S3 regulates GLI2-mediated osteosarcoma invasion.

It has been reported that GLI2 promotes proliferation, migration, and invasion of mesenchymal stem cell and osteosarcoma cells. To examine the molecular mechanisms of GLI2-mediated osteosarcoma metastasis, we performed a microarray analysis. The gene encoding ribosomal protein S3 (RPS3) was identified as a target of GLI2. Real-time PCR revealed that RPS3 was upregulated in osteosarcoma cell lines compared with normal osteoblast cells. Knockdown of GLI2 decreased RPS3 expression, whereas forced expression of a constitutively active form of GLI2 upregulated the expression of RPS3. RPS3 knockdown by siRNA decreased the migration and invasion of osteosarcoma cells. Although forced expression of constitutively active GLI2 increased the migration of human mesenchymal stem cells, knockdown of RPS3 reduced the up-regulated migration. In contrast, forced expression of RPS3 increased migration and invasion of osteosarcoma cells. Moreover, reduction of migration by GLI2 knockdown was rescued by forced expression of RPS3. Immunohistochemical analysis showed that RPS3 expression was increased in primary osteosarcoma lesions with lung metastases compared with those without. These findings indicate that GLI2-RPS3 signaling may be a marker of invasive osteosarcoma and a therapeutic target for patients with osteosarcoma.

Caspase-3 cleaved p65 fragment dampens NF-κB-mediated anti-apoptotic transcription by interfering with the p65/RPS3 interaction.

Caspase-3-mediated p65 cleavage is believed to suppress nuclear factor-kappa B (NF-κB)-mediated anti-apoptotic transactivation in cells undergoing apoptosis. However, only a small percentage of p65 is cleaved during apoptosis, not in proportion to the dramatic reduction in NF-κB transactivation. Here we show that the p65(1-97) fragment generated by Caspase-3 cleavage interferes with ribosomal protein S3 (RPS3), an NF-κB "specifier" subunit, and selectively retards the nuclear translocation of RPS3, thus dampening the RPS3/NF-κB-dependent anti-apoptotic gene expression. Our findings reveal a novel cell fate determination mechanism to ensure cells undergo programed cell death through interfering with RPS3/NF-κB-conferred anti-apoptotic transcription by the fragment from partial p65 cleavage by activated Caspase-3.