Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB,c-Jun N-terminal kinase,and p38 mitogen-activated protein kinase

Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V–fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.     

Lipopolysaccharide (LPS) Promotes Apoptosis in Human Breast Epithelial × Breast Cancer Hybrids,but Not in Parental Cells

Toll-like receptors (TLRs) belong to the group of pathogen recognition receptors known to play a crucial role in the innate immune system. In cancer, TLR expression is still debated controversially due to contradictory results reporting that both induction of apoptosis as well as tumor progression could depend on TLR signaling, whereby recent data rather indicate a pro-tumorigenic effect. The biological phenomenon of cell fusion has been associated with cancer progression due to findings revealing that fusion-derived hybrid cells could exhibit properties like an increased metastatogenic capacity and an increased drug resistance. Thus, M13MDA435 hybrid cell lines, which derived from spontaneous fusion events between human M13SV1-EGFP-Neo breast epithelial cells and human MDA-MB-435-Hyg breast cancer cells, were investigated. Cultivation of cells in the presence of the TLR4 ligand LPS potently induced apoptosis in all hybrid clones, but not in parental cells, which was most likely attributed to differential kinetics of the TLR4 signal transduction cascade. Activation of this pathway concomitant with NF-κB nuclear translocation and TNF-α expression was solely observed in hybrid cells. However, induction of LPS mediated apoptosis was not TNF-α dependent since TNF-α neutralization was not correlated to a decreased amount of dead cells. In addition to TNF-α, LPS also caused IFN-β expression in hybrid clones 1 and 3. Interestingly, hybrid clones differ in the mode of LPS induced apoptosis. While neutralization of IFN-β was sufficient to impair the LPS induced apoptosis in M13MDA435-1 and -3 hybrids, the amount of apoptotic M13MDA435-2 and -4 hybrid cells remained unchanged in the presence of neutralizing IFN-β antibodies. In summary, the fusion of non-LPS susceptible parental human breast epithelial cells and human breast cancer cells gave rise to LPS susceptible hybrid cells, which is in view with the cell fusion hypothesis that hybrid cells could exhibit novel properties.     

Multi-Scale In Vivo Systems Analysis Reveals the Influence of Immune Cells on TNF-α-Induced Apoptosis in the Intestinal Epithelium

Intestinal epithelial cells exist within a complex environment that affects how they interpret and respond to stimuli. We have applied a multi-scale in vivo systems approach to understand how intestinal immune cells communicate with epithelial cells to regulate responses to inflammatory signals. Multivariate modeling analysis of a large dataset composed of phospho-signals, cytokines, and immune cell populations within the intestine revealed an intimate relationship between immune cells and the epithelial response to TNF-α. Ablation of lymphocytes in the intestine prompted a decrease in the expression of MCP-1, which in turn increased the steady state number of intestinal plasmacytoid dendritic cells (pDCs). This change in the immune compartment affected the intestinal cytokine milieu and subsequent epithelial cell signaling network, with cells becoming hypersensitive to TNF-α-induced apoptosis in a way that could be predicted by mathematical modeling. In summary, we have uncovered a novel cellular network that regulates the response of intestinal epithelial cells to inflammatory stimuli in an in vivo setting.     

Involvement of PI3K/AKT and MAPK Pathways for TNF-α Production in SiHa Cervical Mucosal Epithelial Cells Infected with Trichomonas vaginalis

Trichomonas vaginalis; induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-α production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-α production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-α production was significantly decreased compared to the control; however, TNF-α reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-α production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-α production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.     

Membrane Type 1–Matrix Metalloproteinase/Akt Signaling Axis Modulates TNF-α-Induced Procoagulant Activity and Apoptosis in Endothelial Cells

Membrane type 1–matrix metalloproteinase (MT1-MMP) functions as a signaling molecule in addition to a proteolytic enzyme. Our hypothesis was that MT1-MMP cooperates with protein kinase B (Akt) in tumor necrosis factor (TNF)-α-induced signaling pathways of vascular responses, including tissue factor (TF) procoagulant activity and endothelial apoptosis, in cultured human aortic endothelial cells (ECs). TNF-α (10 ng/mL) induced a decrease in Akt phosphorylation within 60 minutes in ECs. A chemical inhibitor of MMP, TIMP-2 and selective small interfering RNA (siRNA)-mediated suppression of MT1-MMP reversed TNF-α-triggered transient decrease of Akt phosphorylation within 60 minutes, suggesting that MT1-MMP may be a key regulator of Akt phosphorylation in TNF-α-stimulated ECs. In the downstream events, TNF-α increased TF antigen and activity, and suppressed the expression of thrombomodulin (TM) antigen. Inhibition of Akt markedly enhanced TNF-α-induced expression of TF antigen and activity, and further reduced the expression of TM antigen. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-α. Moreover, TNF-α induced apoptosis of ECs through Akt- and forkhead box protein O1 (FoxO1)-dependent signaling pathway and nuclear factor-kB (NF-kB) activation. Knockdown of MT1-MMP by siRNA reversed apoptosis of ECs by inhibiting TNF-α-induced Akt-dependent regulation of FoxO1 in TNF-α-stimulated ECs. Immunoprecipitation demonstrated that TNF-α induced the changes in the associations between the cytoplasmic fraction of MT1-MMP and Akt in ECs. In conclusion, we show new evidence that MT1-MMP/Akt signaling axis is a key modifier for TNF-α-induced signaling pathways for modulation of procoagulant activity and apoptosis of ECs.     

TNF-α synergises with IFN-γ to induce caspase-8-JAK1/2-STAT1-dependent death of intestinal epithelial cells

Rewiring of host cytokine networks is a key feature of inflammatory bowel diseases (IBD) such as Crohn’s disease (CD). Th1-type cytokines—IFN-γ and TNF-α—occupy critical nodes within these networks and both are associated with disruption of gut epithelial barrier function. This may be due to their ability to synergistically trigger the death of intestinal epithelial cells (IECs) via largely unknown mechanisms. In this study, through unbiased kinome RNAi and drug repurposing screens we identified JAK1/2 kinases as the principal and nonredundant drivers of the synergistic killing of human IECs by IFN-γ/TNF-α. Sensitivity to IFN-γ/TNF-α-mediated synergistic IEC death was retained in primary patient-derived intestinal organoids. Dependence on JAK1/2 was confirmed using genetic loss-of-function studies and JAK inhibitors (JAKinibs). Despite the presence of biochemical features consistent with canonical TNFR1-mediated apoptosis and necroptosis, IFN-γ/TNF-α-induced IEC death was independent of RIPK1/3, ZBP1, MLKL or caspase activity. Instead, it involved sustained activation of JAK1/2-STAT1 signalling, which required a nonenzymatic scaffold function of caspase-8 (CASP8). Further modelling in gut mucosal biopsies revealed an intercorrelated induction of the lethal CASP8-JAK1/2-STAT1 module during ex vivo stimulation of T cells. Functional studies in CD-derived organoids using inhibitors of apoptosis, necroptosis and JAKinibs confirmed the causative role of JAK1/2-STAT1 in cytokine-induced death of primary IECs. Collectively, we demonstrate that TNF-α synergises with IFN-γ to kill IECs via the CASP8-JAK1/2-STAT1 module independently of canonical TNFR1 and cell death signalling. This non-canonical cell death pathway may underpin immunopathology driven by IFN-γ/TNF-α in diverse autoinflammatory diseases such as IBD, and its inhibition may contribute to the therapeutic efficacy of anti-TNFs and JAKinibs.Subject terms: Necroptosis, Cell death and immune response, Interferons, Tumour-necrosis factors, Crohn''s disease     

AMP-18 Targets p21 to Maintain Epithelial Homeostasis

Dysregulated homeostasis of epithelial cells resulting in disruption of mucosal barrier function is an important pathogenic mechanism in inflammatory bowel diseases (IBD). We have characterized a novel gastric protein, Antrum Mucosal Protein (AMP)-18, that has pleiotropic properties; it is mitogenic, anti-apoptotic and can stimulate formation of tight junctions. A 21-mer synthetic peptide derived from AMP-18 exhibits the same biological functions as the full-length protein and is an effective therapeutic agent in mouse models of IBD. In this study we set out to characterize therapeutic mechanisms and identify molecular targets by which AMP-18 maintains and restores disrupted epithelial homeostasis in cultured intestinal epithelial cells and a mouse model of IBD. Tumor necrosis factor (TNF)-α, a pro-inflammatory cytokine known to mediate gastrointestinal (GI) mucosal injury in IBD, was used to induce intestinal epithelial cell injury, and study the effects of AMP-18 on apoptosis and the cell cycle. An apoptosis array used to search for targets of AMP-18 in cells exposed to TNF-α identified the cyclin-dependent kinase inhibitor p21^WAF1/CIP1. Treatment with AMP-18 blunted increases in p21 expression and apoptosis, while reversing disturbed cell cycle kinetics induced by TNF-α. AMP-18 appears to act through PI3K/AKT pathways to increase p21 phosphorylation, thereby reducing its nuclear accumulation to overcome the antiproliferative effects of TNF-α. In vitamin D receptor-deficient mice with TNBS-induced IBD, the observed increase in p21 expression in colonic epithelial cells was suppressed by treatment with AMP peptide. The results indicate that AMP-18 can maintain and/or restore the homeostatic balance between proliferation and apoptosis in intestinal epithelial cells to protect and repair mucosal barrier homeostasis and function, suggesting a therapeutic role in IBD.     

Alizarin,a nature compound,inhibits the growth of pancreatic cancer cells by abrogating NF-κB activation

The current performance of nature compounds in antitumor field is gradually attracted more and more attention, we discovered a nature active ingredient alizarin possess potent natural reductive NF-κB activity to against pancreatic cancer. However, the preclinical pharmacology and therapeutic effect, and the underlying mechanisms of alizarin in inhibiting pancreatic cancer are still unclear. After high-throughput screening, this is the first report that alizarin can induce a potent inhibitory effect against pancreatic cancer cells. Alizarin induced cell cycle arrest and promoted cell apoptosis by inhibiting TNF-α-stimulated NF-κB activity and nuclear translocation, and inactivated its related TNF-α-TAK1-NF-κB signaling cascade followed by downregulation of NF-κB target genes involved in cell apoptosis (Bcl-2, Bcl-xL, XIAP) and in the cell cycle and growth (cyclin D, c-myc). Due to the abrogation of NF-κB activity, combination of alizarin and gemcitabine exerted a better inhibitory effect on pancreatic cancer. In summary, natural component alizarin, inhibited cell proliferation and induced apoptosis in vitro and in vivo through targeting of the NF-κB signaling cascade with minimal toxicity, which combine with gemcitabine, can significantly enhance the antitumor capability, playing a synergistic effect. Therefore, alizarin may play a role in reversing gemcitabine resistance caused by overactivated NF-κB in clinical application in the future.     

Type I Interferons Control Proliferation and Function of the Intestinal Epithelium

Wnt pathway-driven proliferation and renewal of the intestinal epithelium must be tightly controlled to prevent development of cancer and barrier dysfunction. Although type I interferons (IFN) produced in the gut under the influence of microbiota are known for their antiproliferative effects, the role of these cytokines in regulating intestinal epithelial cell renewal is largely unknown. Here we report a novel role for IFN in the context of intestinal knockout of casein kinase 1α (CK1α), which controls the ubiquitination and degradation of both β-catenin and the IFNAR1 chain of the IFN receptor. Ablation of CK1α leads to the activation of both β-catenin and IFN pathways and prevents the unlimited proliferation of intestinal epithelial cells despite constitutive β-catenin activity. IFN signaling contributes to the activation of the p53 pathway and the appearance of apoptotic and senescence markers in the CK1α-deficient gut. Concurrent genetic ablation of CK1α and IFNAR1 leads to intestinal hyperplasia, robust attenuation of apoptosis, and rapid and lethal loss of barrier function. These data indicate that IFN play an important role in controlling the proliferation and function of the intestinal epithelium in the context of β-catenin activation.     

Effects of Forced Expression of an NH2-terminal Truncated β-Catenin on Mouse Intestinal Epithelial Homeostasis

β-Catenin functions as a downstream component of the Wnt/Wingless signal transduction pathway and as an effector of cell–cell adhesion through its association with cadherins. To explore the in vivo effects of β-catenin on proliferation, cell fate specification, adhesion, and migration in a mammalian epithelium, a human NH₂-terminal truncation mutant (ΔN89β-catenin) was expressed in the 129/Sv embryonic stem cell–derived component of the small intestine of adult C57Bl/6–ROSA26↔ 129/Sv chimeric mice. ΔN89β-Catenin was chosen because mutants of this type are more stable than the wild-type protein, and phenocopy activation of the Wnt/Wingless signaling pathway in Xenopus and Drosophila. ΔN89β-Catenin had several effects. Cell division was stimulated fourfold in undifferentiated cells located in the proliferative compartment of the intestine (crypts of Lieberkühn). The proliferative response was not associated with any discernible changes in cell fate specification but was accompanied by a three- to fourfold increase in crypt apoptosis. There was a marked augmentation of E-cadherin at the adherens junctions and basolateral surfaces of 129/Sv (ΔN89β-catenin) intestinal epithelial cells and an accompanying slowing of cellular migration along crypt-villus units. 1–2% of 129/Sv (ΔN89β-catenin) villi exhibited an abnormal branched architecture. Forced expression of ΔN89β-catenin expression did not perturb the level or intracellular distribution of the tumor suppressor adenomatous polyposis coli (APC). The ability of ΔN89β-catenin to interact with normal cellular pools of APC and/or augmented pools of E-cadherin may have helped prevent the 129/Sv gut epithelium from undergoing neoplastic transformation during the 10-mo period that animals were studied. Together, these in vivo studies emphasize the importance of β-catenin in regulating normal adhesive and signaling functions within this epithelium.     

The TTYH3/MK5 Positive Feedback Loop regulates Tumor Progression via GSK3-β/β-catenin signaling in HCC

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, and identification of novel targets is necessary for its diagnosis and treatment. This study aimed to investigate the biological function and clinical significance of tweety homolog 3 (TTYH3) in HCC. TTYH3 overexpression promoted cell proliferation, migration, and invasion and inhibited HCCM3 and Hep3B cell apoptosis. TTYH3 promoted tumor formation and metastasis in vivo. TTYH3 upregulated calcium influx and intracellular chloride concentration, thereby promoting cellular migration and regulating epithelial-mesenchymal transition-related protein expression. The interaction between TTYH3 and MK5 was identified through co-immunoprecipitation assays and protein docking. TTYH3 promoted the expression of MK5, which then activated the GSK3β/β-catenin signaling pathway. MK5 knockdown attenuated the activation of GSK3β/β-catenin signaling by TTYH3. TTYH3 expression was regulated in a positive feedback manner. In clinical HCC samples, TTYH3 was upregulated in the HCC tissues compared to nontumor tissues. Furthermore, high TTYH3 expression was significantly correlated with poor patient survival. The CpG islands were hypomethylated in the promoter region of TTYH3 in HCC tissues. In conclusion, we identified TTYH3 regulates tumor development and progression via MK5/GSK3-β/β-catenin signaling in HCC and promotes itself expression in a positive feedback loop.     

Differential Modulation of TNF-α–Induced Apoptosis by Neisseria meningitidis

Infections by Neisseria meningitidis show duality between frequent asymptomatic carriage and occasional life-threatening disease. Bacterial and host factors involved in this balance are not fully understood. Cytopathic effects and cell damage may prelude to pathogenesis of isolates belonging to hyper-invasive lineages. We aimed to analyze cell–bacteria interactions using both pathogenic and carriage meningococcal isolates. Several pathogenic isolates of the ST-11 clonal complex and carriage isolates were used to infect human epithelial cells. Cytopathic effect was determined and apoptosis was scored using several methods (FITC-Annexin V staining followed by FACS analysis, caspase assays and DNA fragmentation). Only pathogenic isolates were able to induce apoptosis in human epithelial cells, mainly by lipooligosaccharide (endotoxin). Bioactive TNF-α is only detected when cells were infected by pathogenic isolates. At the opposite, carriage isolates seem to provoke shedding of the TNF-α receptor I (TNF-RI) from the surface that protect cells from apoptosis by chelating TNF-α. Ability to induce apoptosis and inflammation may represent major traits in the pathogenesis of N. meningitidis. However, our data strongly suggest that carriage isolates of meningococci reduce inflammatory response and apoptosis induction, resulting in the protection of their ecological niche at the human nasopharynx.     

Tuning of Protein Kinase Circuitry by p38α Is Vital for Epithelial Tissue Homeostasis

The epithelium of mucosal and skin surfaces serves as a permeability barrier and affords mechanisms for local immune defense. Crucial to the development and maintenance of a properly functioning epithelium is the balance of cell proliferation, differentiation, and death. Here we show that this balance depends on cross-regulatory interactions among multiple protein kinase-mediated signals and their coordinated transmission. From an investigation of conditional gene knock-out mice, we find that epithelial-specific loss of the protein kinase p38α leads to aberrant activation of TAK1, JNK, EGF receptor, and ERK in distinct microanatomical areas of the intestines and skin. Consequently, the epithelial tissues display excessive proliferation, inadequate differentiation, and sensitivity to apoptosis. These anomalies leave the tissue prone to damage and collapse at the trigger of an environmental insult. The vulnerability of p38α-deficient epithelium predicts adverse effects of long term pharmacological p38α inhibition; yet such limitations could be overcome by concomitant blockade of one or more of the dysregulated protein kinase signaling pathways.     

NOP14 regulates the growth,migration, and invasion of colorectal cancer cells by modulating the NRIP1/GSK-3β/β-catenin signaling pathway

Colorectal cancer (CRC) is the third most common cancer diagnosed worldwide. Recently, nucleolar complex protein 14 (NOP14) has been discovered to play a critical role in cancer development and progression, but the mechanisms of action of NOP14 in colorectal cancer remain to be elucidated. In this study, we used collected colorectal cancer tissues and cultured colorectal cancer cell lines (SW480, HT29, HCT116, DLD1, Lovo), and measured the mRNA and protein expression levels of NOP14 in colorectal cancer cells using qPCR and Western blotting. GFP-NOP14 was constructed and siRNA fragments against NOP14 were synthesized to investigate the importance of NOP14 for the development of colorectal cells. Transwell migration assays were used to measure cell invasion and migration, CCK-8 kits were used to measure cell activity, and flow cytometry was applied to the observation of apoptosis. We found that both the mRNA and protein levels of NOP14 were significantly upregulated in CRC tissues and cell lines. Overexpression of GFP-NOP14 markedly promoted the growth, migration, and invasion of the CRC cells HT19 and SW480, while genetic knockdown of NOP14 inhibited these behaviors. Overexpression of NOP14 promoted the expression of NRIP1 and phosphorylated inactivation of GSK-3β, leading to the upregulation of β-catenin. Genetic knockdown of NOP14 had the opposite effect on NRIP1/GSK-3/β-catenin signals. NOP14 therefore appears to be overexpressed in clinical samples and cell lines of colorectal cancer, and promotes the proliferation, growth, and metastasis of colorectal cancer cells by modulating the NRIP1/GSK-3β/β-catenin signaling pathway.Key words: Colorectal cancer, NOP14, proliferation, migration, invasion     

δ‐Tocotrienol sensitizes and re‐sensitizes ovarian cancer cells to cisplatin via induction of G1 phase cell cycle arrest and ROS/MAPK‐mediated apoptosis

ObjectivesAmong gynaecologic malignancies, ovarian cancer (OC) represents the leading cause of death for women worldwide. Current OC treatment involves cytoreductive surgery followed by platinum‐based chemotherapy, which is associated with severe side effects and development of drug resistance. Therefore, new therapeutic strategies are urgently needed. Herein, we evaluated the anti‐tumour effects of Vitamin E‐derived δ‐tocotrienol (δ‐TT) in two human OC cell lines, IGROV‐1 and SKOV‐3 cells.Materials and MethodsMTT and Trypan blue exclusion assays were used to assess δ‐TT cytotoxicity, alone or in combination with other molecules. δ‐TT effects on cell cycle, apoptosis, ROS generation and MAPK phosphorylation were investigated by flow cytometry, Western blot and immunofluorescence analyses. The synergism between δ‐TT and chemotherapy was evaluated by isobologram analysis.ResultsWe demonstrated that δ‐TT could induce cell cycle block at G1‐S phase and mitochondrial apoptosis in OC cell lines. In particular, we found that the proapoptotic activity of δ‐TT correlated with mitochondrial ROS production and subsequent JNK and p38 activation. Finally, we observed that the compound was able to synergize with cisplatin, not only enhancing its cytotoxicity in IGROV‐1 and SKOV‐3 cells but also re‐sensitizing IGROV‐1/Pt1 cell line to its anti‐tumour effects.Conclusionsδ‐TT triggers G1 phase cell cycle arrest and ROS/MAPK‐mediated apoptosis in OC cells and sensitizes them to platinum treatment, thus representing an interesting option for novel chemopreventive/therapeutic strategies for OC.     

Transforming Growth Factor β Regulates P-Body Formation through Induction of the mRNA Decay Factor Tristetraprolin

Transforming growth factor β (TGF-β) is a potent growth regulator and tumor suppressor in normal intestinal epithelium. Likewise, epithelial cell growth is controlled by rapid decay of growth-related mRNAs mediated through 3′ untranslated region (UTR) AU-rich element (ARE) motifs. We demonstrate that treatment of nontransformed intestinal epithelial cells with TGF-β inhibited ARE-mRNA expression. This effect of TGF-β was promoted through increased assembly of cytoplasmic RNA processing (P) bodies where ARE-mRNA localization was observed. P-body formation was dependent on TGF-β/Smad signaling, as Smad3 deletion abrogated P-body formation. In concert with increased P-body formation, TGF-β induced expression of the ARE-binding protein tristetraprolin (TTP), which colocalized to P bodies. TTP expression was necessary for TGF-β-dependent P-body formation and promoted growth inhibition by TGF-β. The significance of this was observed in vivo, where colonic epithelium deficient in TGF-β/Smad signaling or TTP expression showed attenuated P-body levels. These results provide new insight into TGF-β''s antiproliferative properties and identify TGF-β as a novel mRNA stability regulator in intestinal epithelium through its ability to promote TTP expression and subsequent P-body formation.