miR-129-3p alleviates chondrocyte apoptosis in knee joint fracture-induced osteoarthritis through CPEB1.

BACKGROUND: Osteoarthritis (OA), a refractory disease, is one of the leading contributors for disability worldwide. Since chondrocyte is the only resident cell in cartilage, this study aims to explore the roles of miR-129-3p and CPEB1 in chondrocyte apoptosis in knee joint fracture-induced OA. METHODS: Cartilage was collected from 20 OA patients who underwent total knee replacement (OA group) and 20 patients with knee contusion (normal group). Then, miR-129-3p and CPEB1 levels in the cartilage were quantified by qRT-PCR. Primary rat chondrocytes in the knee were isolated and identified by toluidine blue staining and immunofluorescent staining of type II collagen. OA cellular models were induced by TNF-α treatment, in which miR-129-3p and CPEB1 expressions were assessed. Subsequently, cell viability, apoptosis, and the expression levels of apoptotic protein and caspase-3 were measured. Dual luciferase reporter assay identified the interaction between miR-129-3p and CPEB1. RESULTS: Patients in the OA group had decreased miR-129-3p expression and increased CPEB1 expression than those in the normal group. TNF-α treatment successfully induced the OA cellular model. Downregulated miR-129-3p and upregulated CPEB1 expressions were found in OA-treated chondrocytes. miR-129-3p overexpression or CPEB1 knockdown improved chondrocyte viability and attenuated apoptosis, and vice versa. miR-129-3p negatively regulated CPEB1, thus ameliorating apoptosis and enhancing cell viability. CONCLUSION: miR-129-3p negatively targeted CPEB1 to facilitate chondrocyte viability and hamper apoptosis.

Distinct roles of Pcf11 zinc-binding domains in pre-mRNA 3'-end processing.

New transcripts generated by RNA polymerase II (RNAPII) are generally processed in order to form mature mRNAs. Two key processing steps include a precise cleavage within the 3' end of the pre-mRNA, and the subsequent polymerization of adenosines to produce the poly(A) tail. In yeast, these two functions are performed by a large multi-subunit complex that includes the Cleavage Factor IA (CF IA). The four proteins Pcf11, Clp1, Rna14 and Rna15 constitute the yeast CF IA, and of these, Pcf11 is structurally the least characterized. Here, we provide evidence for the binding of two Zn2+ atoms to Pcf11, bound to separate zinc-binding domains located on each side of the Clp1 recognition region. Additional structural characterization of the second zinc-binding domain shows that it forms an unusual zinc finger fold. We further demonstrate that the two domains are not mandatory for CF IA assembly nor RNA polymerase II transcription termination, but are rather involved to different extents in the pre-mRNA 3'-end processing mechanism. Our data thus contribute to a more complete understanding of the architecture and function of Pcf11 and its role within the yeast CF IA complex.

Essential role for non-canonical poly(A) polymerase GLD4 in cytoplasmic polyadenylation and carbohydrate metabolism.

Regulation of gene expression at the level of cytoplasmic polyadenylation is important for many biological phenomena including cell cycle progression, mitochondrial respiration, and learning and memory. GLD4 is one of the non-canonical poly(A) polymerases that regulates cytoplasmic polyadenylation-induced translation, but its target mRNAs and role in cellular physiology is not well known. To assess the full panoply of mRNAs whose polyadenylation is controlled by GLD4, we performed an unbiased whole genome-wide screen using poy(U) chromatography and thermal elution. We identified hundreds of mRNAs regulated by GLD4, several of which are involved in carbohydrate metabolism including GLUT1, a major glucose transporter. Depletion of GLD4 not only reduced GLUT1 poly(A) tail length, but also GLUT1 protein. GLD4-mediated translational control of GLUT1 mRNA is dependent of an RNA binding protein, CPEB1, and its binding elements in the 3΄ UTR. Through regulating GLUT1 level, GLD4 affects glucose uptake into cells and lactate levels. Moreover, GLD4 depletion impairs glucose deprivation-induced GLUT1 up-regulation. In addition, we found that GLD4 affects glucose-dependent cellular phenotypes such as migration and invasion in glioblastoma cells. Our observations delineate a novel post-transcriptional regulatory network involving carbohydrate metabolism and glucose homeostasis mediated by GLD4.

Cellular and viral determinants of retroviral nuclear entry.

Retroviruses must integrate their cDNA into the host genome to generate proviruses. Viral DNA-protein complexes interact with cellular proteins and produce pre-integration complexes, which carry the viral genome and cross the nuclear pore channel to enter the nucleus and integrate viral DNA into host chromosomal DNA. If the reverse transcripts fail to integrate, linear or circular DNA species such as 1- and 2-long terminal repeats are generated. Such complexes encounter numerous cellular proteins in the cytoplasm, which restrict viral infection and protect the nucleus. To overcome host cell defenses, the pathogens have evolved several evasion strategies. Viral proteins often contain nuclear localization signals, allowing entry into the nucleus. Among more than 1000 proteins identified as required for HIV infection by RNA interference screening, karyopherins, cleavage and polyadenylation specific factor 6, and nucleoporins have been predominantly studied. This review discusses current opinions about the synergistic relationship between the viral and cellular factors involved in nuclear import, with focus on the unveiled mysteries of the host-pathogen interaction, and highlights novel approaches to pinpoint therapeutic targets.

Global Analysis of CPEBs Reveals Sequential and Non-Redundant Functions in Mitotic Cell Cycle.

CPEB (Cytoplasmic Polyadenylation Element Binding) proteins are a family of four RNA-binding proteins that regulate the translation of maternal mRNAs controlling meiotic cell cycle progression. But CPEBs are not limited to the transcriptionally silent germline; they are also expressed, in various combinations, in somatic cells, yet their role in regulation of mitosis-related gene expression is largely unknown. Deregulation of CPEB1 and CPEB4 have been linked to tumor development. However, a systematic analysis addressing their requirements for the temporal regulation of mitotic gene expression has yet to be performed. This study addresses the requirements of each of the four CPEBs for mitotic phase transitions, with a particular focus on cytoplasmic polyadenylation and translational regulation. We demonstrate that CPEB3 is the only member dispensable for mitotic cell division, whereas the other three members, CPEB1, 2, and 4, are essential to successful mitotic cell division. Thus, CPEB1 is required for prophase entry, CPEB2 for metaphase and CPEB4 for cytokinesis. These three CPEBs have sequential non-redundant functions that promote the phase-specific polyadenylation and translational activation of CPE-regulated transcripts in the mitotic cell cycle.

Cytoplasmic polyadenylation element binding proteins in development, health, and disease.

The cytoplasmic polyadenylation element binding (CPEB) proteins are sequence-specific mRNA binding proteins that control translation in development, health, and disease. CPEB1, the founding member of this family, has become an important model for illustrating general principles of translational control by cytoplasmic polyadenylation in gametogenesis, cancer etiology, synaptic plasticity, learning, and memory. Although the biological functions of the other members of this protein family in vertebrates are just beginning to emerge, it is already evident that they, too, mediate important processes, such as cancer etiology and higher cognitive function. In Drosophila, the CPEB proteins Orb and Orb2 play key roles in oogenesis and in neuronal function, as do related proteins in Caenorhabditis elegans and Aplysia. We review the biochemical features of the CPEB proteins, discuss their activities in several biological systems, and illustrate how understanding CPEB activity in model organisms has an important impact on neurological disease.

FIP1L1 presence in FIP1L1-RARA or FIP1L1-PDGFRA differentially contributes to the pathogenesis of distinct types of leukemia.

FIP1-like 1 (FIP1L1) is associated with two leukemogenic fusion genes: FIP1L1-retinoic acid receptor alpha (RARA) and FIP1L1-platelet-derived growth factor receptor alpha (PDGFRA). Analyses of a series of deletion mutants revealed that the FIP1 motif in FIP1L1-RARA plays a pivotal role in its homodimerization and transcriptional repressor activity. However, in FIP1L1-PDGFRA, the C-terminal PDGFRA portion possesses the ability of forming a homodimer by itself, making FIP1L1 dispensable for constitutive activation of this kinase. Both the full-length and the C-terminal PDGFRA portion of FIP1L1-PDGFRA could transform the IL-3-dependent hematopoietic cell line, BAF-B03. Moreover, when either the full-length or the C-terminal PDGFRA portion of FIP1L1-PDGFRA was introduced in these cells, they grew in the absence of IL-3. The cells having the C-terminal PDGFRA portion of FIP1L1-PDGFRA, however, were partially IL-3 dependent, whereas the cells having the full-length FIP1L1-PDGFRA became completely IL-3 independent for their growth. Taken together, these results show that FIP1L1 differentially contributes to the pathogenesis of distinct types of leukemia.

The expression of CPEB proteins is sequentially regulated during zebrafish oogenesis and embryogenesis.

In many species, there is little transcription in the mature oocyte, and zygotic transcription does not begin immediately after fertilization. In zebrafish, zygotic transcription is not initiated until the mid-blastula transition, thus the production of new proteins during oogenesis and early embryogenesis is dependent on the translation of maternal mRNAs. In a growing number of species, the translation of key maternal transcripts is coupled to their cytoplasmic polyadenylation. One family of RNA-binding proteins implicated in this process is the cytoplasmic polyadenylation element (CPE)-binding proteins (CPEBs), which bind to a sequence in the 3'-untranslated regions of regulated transcripts and mediate their storage/repression or translation. In several species, there is evidence for two classes of CPEBs, a larger oocyte-type and a smaller CPEB that functions during embryogenesis. This appears to be the case in zebrafish as well, and we now provide evidence suggesting that the oocyte-type CPEB (zorba) regulates the translation of the embryonic-type (ElrA) by keeping the ElrA transcript in storage until fertilization. When zorba levels fall, ElrA protein is then produced and available to regulate the translation of additional mRNAs during embryogenesis. We have also identified a potential target of ElrA, the maternal mRNA for hnRNPab, which is a potential homolog of the Drosophila gene squid, whose product plays a role in patterning the Drosophila oocyte and embryo. These data suggest that during zebrafish embryogenesis, cytoplasmic polyadenylation mediates a cascade of translational control whose final targets play central patterning roles during embryogenesis.

Genetic and acute CPEB1 depletion ameliorate fragile X pathophysiology.

Fragile X syndrome (FXS), the most common cause of inherited mental retardation and autism, is caused by transcriptional silencing of FMR1, which encodes the translational repressor fragile X mental retardation protein (FMRP). FMRP and cytoplasmic polyadenylation element-binding protein (CPEB), an activator of translation, are present in neuronal dendrites, are predicted to bind many of the same mRNAs and may mediate a translational homeostasis that, when imbalanced, results in FXS. Consistent with this possibility, Fmr1(-/y); Cpeb1(-/-) double-knockout mice displayed amelioration of biochemical, morphological, electrophysiological and behavioral phenotypes associated with FXS. Acute depletion of CPEB1 in the hippocampus of adult Fmr1(-/y) mice rescued working memory deficits, demonstrating reversal of this FXS phenotype. Finally, we find that FMRP and CPEB1 balance translation at the level of polypeptide elongation. Our results suggest that disruption of translational homeostasis is causal for FXS and that the maintenance of this homeostasis by FMRP and CPEB1 is necessary for normal neurologic function.

Translational control of cell growth and malignancy by the CPEBs.

The cytoplasmic polyadenylation element binding proteins (CPEBs) associate with specific sequences in mRNA 3' untranslated regions to promote translation. They do so by inducing cytoplasmic polyadenylation, which requires specialized poly(A) polymerases. Aberrant expression of these proteins correlates with certain types of cancer, indicating that cytoplasmic RNA 3' end processing is important in the control of growth. Several CPEB-regulated mRNAs govern cell cycle progression, regulate senescence, establish cell polarity, and promote tumorigenesis and metastasis. In this Opinion article, we discuss the emerging evidence that indicates a key role for the CPEBs in cancer biology.

Knockdown of pre-mRNA cleavage factor Im 25 kDa promotes neurite outgrowth.

Mammalian precursor mRNA (pre-mRNA) cleavage factor I (CFIm) plays important roles in the selection of poly(A) sites in a 3'-untranslated region (3'-UTR), producing mRNAs with variable 3' ends. Because 3'-UTRs often contain cis elements that impact stability or localization of mRNA or translation, alternative polyadenylation diversifies utilization of primary transcripts in mammalian cells. However, the physiological role of CFIm remains unclear. CFIm acts as a heterodimer comprising a 25kDa subunit (CFIm25) and one of the three large subunits-CFIm59, CFIm68, or CFIm72. CFIm25 binds directly to RNA and introduces and anchors the larger subunit. To examine the physiological roles of CFIm, we knocked down the CFIm25 gene in neuronal cells using RNA interference. Knockdown of CFIm25 increased the number of primary dendrites of developing hippocampal neurons and promoted nerve growth factor (NGF)-induced neurite extension from rat pheochromocytoma PC12 cells without affecting the morphology of proliferating PC12 cells. On the other hand, CFIm25 knockdown did not influence constitutively active or dominantly negative RhoA suppression or promotion of NGF-induced neurite extension from PC12 cells, respectively. Taken together, our results indicate that endogenous CFIm may promote neuritogenesis in developing neurons by coordinating events upstream of NGF-induced RhoA inactivation.

The CPEB-family of proteins, translational control in senescence and cancer.

Cytoplasmic elongation of the poly(A) tail was originally identified as a mechanism to activate maternal mRNAs, stored as silent transcripts with short poly(A) tails, during meiotic progression. A family of RNA-binding proteins named CPEBs, which recruit the translational repression or cytoplasmic polyadenylation machineries to their target mRNAs, directly mediates cytoplasmic polyadenylation. Recent years have witnessed an explosion of studies showing that CPEBs are not only expressed in a variety of somatic tissues, but have essential functions controlling gene expression in time and space in the adult organism. These "new" functions of the CPEBs include regulating the balance between senescence and proliferation and its pathological manifestation, tumor development. In this review, we summarize current knowledge on the functions of the CPEB-family of proteins in the regulation of cell proliferation, their target mRNAs and the mechanism controlling their activities.

Identification of JAK2 as a mediator of FIP1L1-PDGFRA-induced eosinophil growth and function in CEL.

The Fip1-like1 (FIP1L1)-platelet-derived growth factor receptor alpha fusion gene (F/P) arising in the pluripotent hematopoietic stem cell (HSC),causes 14% to 60% of patients with hypereosinophilia syndrome (HES). These patients, classified as having F/P (+) chronic eosinophilic leukemia (CEL), present with clonal eosinophilia and display a more aggressive disease phenotype than patients with F/P (-) HES patients. The mechanisms underlying predominant eosinophil lineage targeting and the cytotoxicity of eosinophils in this leukemia remain unclear. Given that the Janus tyrosine kinase (JAK)/signal transducers and activators of transcription (Stat) signaling pathway is key to cytokine receptor-mediated eosinophil development and activated Stat3 and Stat5 regulate the expression of genes involved in F/P malignant transformation, we investigated whether and how JAK proteins were involved in the pathogenesis of F/P-induced CEL. F/P activation of JAK2, Stat3 and Stat5, were confirmed in all the 11 F/P (+) CEL patients examined. In vitro inhibition of JAK2 in EOL-1, primary F/P(+) CEL cells (PC) and T674I F/P Imatinib resistant cells(IR) by either JAK2-specific short interfering RNA (siRNA) or the tryphostin derivative AG490(AG490), significantly reduced cellular proliferation and induced cellular apoptosis. The F/P can enhance the IL-5-induced JAK2 activation, and further results indicated that JAK2 inhibition blocked IL-5-induced cellular migration and activation of the EOL-1 and PC cells in vitro. F/P-stimulation of the JAK2 suppressed cells led to a significantly reduction in Stat3 activation, but relatively normal induction of Stat5 activation. Interestingly, JAK2 inhibition also reduced PI3K, Akt and NF-κB activity in a dose-dependent manner, and suppressed expression levels of c-Myc and Survivin. These results strongly suggest that JAK2 is activated by F/P and is required for F/P stimulation of cellular proliferation and infiltration, possibly through induction of c-Myc and Survivin expression via activation of multiple signaling pathways, including NF-κB, Stat3, and PI3K/Akt.

Translational control of TWIST1 expression in MCF-10A cell lines recapitulating breast cancer progression.

TWIST1 is a highly conserved basic helix-loop-helix transcription factor that promotes epithelial-mesenchymal transition (EMT). Its misregulation has been observed in various types of tumors. Using the MCF-10A-series of cell lines that recapitulate the early stages of breast cancer formation and EMT, we found TWIST1 to be upregulated during EMT and downregulated early in carcinogenesis. The TWIST1 3'UTR contains putative regulatory elements, including miRNA target sites and two cytoplasmic polyadenylation elements (CPE). We found that miR-580, CPEB1, and CPEB2 act as negative regulators of TWIST1 expression in a sequence-specific and additive/cooperative manner.

Involvement of hGLD-2 in cytoplasmic polyadenylation of human p53 mRNA.

Cytoplasmic polyadenylation is a post-transcriptional mechanism regulating mRNA stability and translation. The human p53 3'-untranslated region (3'-UTR) contains two regions similar to cytoplasmic polyadenylation elements (CPEs) just upstream of the poly(A) hexanucleotide. Evaluation of the p53 CPE-like elements was performed by luciferase reporter assays, qPCR, and poly(A) assays. Herein, we report the down regulation of a luciferase reporter fused to the p53 3'-UTR, when human CPE-binding protein 1 (hCPEB1) is overexpressed. This inhibition is partially rescued when hCPEB1fused to hGLD-2 [a human cytoplasmic poly(A) polymerase] is overexpressed instead. The stability of a luciferase mRNA containing the p53 3'-UTR downstream, is decreased when hCPEB1 is overexpressed as seen by qPCR. Expression of hGLD-2 restores the mRNA stability. This is due to elongation of the poly(A) tail as seen by a PCR-based poly(A) test and in vitro poly(A) assay. Taken together, our results suggest that hCPEB1 and hGLD-2 are antagonizing factors regulating p53 mRNA stability.

The P-loop domain of yeast Clp1 mediates interactions between CF IA and CPF factors in pre-mRNA 3' end formation.

Cleavage factor IA (CF IA), cleavage and polyadenylation factor (CPF), constitute major protein complexes required for pre-mRNA 3' end formation in yeast. The Clp1 protein associates with Pcf11, Rna15 and Rna14 in CF IA but its functional role remained unclear. Clp1 carries an evolutionarily conserved P-loop motif that was previously shown to bind ATP. Interestingly, human and archaean Clp1 homologues, but not the yeast protein, carry 5' RNA kinase activity. We show that depletion of Clp1 in yeast promoted defective 3' end formation and RNA polymerase II termination; however, cells expressing Clp1 with mutant P-loops displayed only minor defects in gene expression. Similarly, purified and reconstituted mutant CF IA factors that interfered with ATP binding complemented CF IA depleted extracts in coupled in vitro transcription/3' end processing reactions. We found that Clp1 was required to assemble recombinant CF IA and that certain P-loop mutants failed to interact with the CF IA subunit Pcf11. In contrast, mutations in Clp1 enhanced binding to the 3' endonuclease Ysh1 that is a component of CPF. Our results support a structural role for the Clp1 P-loop motif. ATP binding by Clp1 likely contributes to CF IA formation and cross-factor interactions during the dynamic process of 3' end formation.

Cytoplasmic polyadenylation-element-binding protein (CPEB)1 and 2 bind to the HIF-1alpha mRNA 3'-UTR and modulate HIF-1alpha protein expression.

The heterodimeric HIF (hypoxia-inducible factor)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homoeostasis. Besides the well described regulation of the HIF-1alpha subunit via hydroxylation-mediated protein stability in hypoxia, there are several indications of an additional translational control of the HIF-1alpha mRNA, especially after growth factor stimulation. We identified an interaction of CPEB (cytoplasmic polyadenylation-element-binding protein) 1 and CPEB2 with the 3'-UTR (untranslated region) of HIF-1alpha mRNA. Overexpression of CPEB1 and CPEB2 affected HIF-1alpha protein levels mediated by the 3'-UTR of HIF-1alpha mRNA. Stimulation of neuroblastoma SK-N-MC cells with insulin and thus activation of endogenous CPEBs increased the expression of a luciferase reporter gene fused to the 3'-UTR of HIF-1alpha as well as endogenous HIF-1alpha protein levels. This could be abrogated by treating the cells with CPEB1 or CPEB2 siRNAs (short interfering RNAs). Injection of HIF-1alpha cRNA into Xenopus oocytes verified the elongation of the poly(A)+ (polyadenylated) tail by cytoplasmic polyadenylation. Thus CPEB1 and CPEB2 are involved in the regulation of HIF-1alpha following insulin stimulation.

Dasatinib inhibits the growth and survival of neoplastic human eosinophils (EOL-1) through targeting of FIP1L1-PDGFRalpha.

OBJECTIVE: Chronic eosinophilic leukemia (CEL) is a myeloproliferative disorder characterized by molecular and/or cytogenetic evidence of clonality of eosinophils, marked eosinophilia, and organ damage. In many patients, the transforming mutation FIP1L1-PDGFRalpha and the related CHIC2 deletion are found. The respective oncoprotein, FIP1L1-PDGFRalpha, is considered to play a major role in malignant cell growth in CEL. The tyrosine kinase (TK) inhibitor imatinib (STI571) has been described to counteract the TK activity of FIP1L1-PDGFRalpha in most patients. However, not all patients with CEL show a response to imatinib. Therefore, several attempts have been made to identify other TK inhibitors that counteract growth of neoplastic eosinophils. MATERIALS AND METHODS: We provide evidence that dasatinib, a multi-targeted kinase inhibitor, blocks the growth and survival of EOL-1, an eosinophil leukemia cell line carrying FIP1L1-PDGFRalpha. RESULTS: The effects of dasatinib on proliferation of EOL-1 cells were dose-dependent, with an IC50 of 0.5 to 1 nM, which was found to be in the same range when compared to IC50 values produced with imatinib. Dasatinib was also found to induce apoptosis in EOL-1 cells in a dose-dependent manner (IC50: 1-10 nM). The apoptosis-inducing effects of dasatinib on EOL-1 cells were demonstrable by light microscopy, flow cytometry, and in a TUNEL assay. In Western blot experiments, dasatinib completely blocked the phosphorylation of FIP1L1-PDGFRalpha in EOL-1 cells. CONCLUSIONS: Dasatinib inhibits the growth of leukemic eosinophils through targeting of the disease-related oncoprotein FIP1L1-PDGFRalpha. Based on this observation, dasatinib may be considered as a new interesting treatment option for patients with CEL.

Chronic eosinophilic leukemias and the myeloproliferative variant of the hypereosinophilic syndrome.

Among patients with hypereosinophilia, a myeloproliferative variant is recognized. In many of these patients a diagnosis of eosinophilic leukemia can be made. The molecular mechanism is often a fusion gene, incorporating part of PDGFRA or PDGFRB, encoding anaberrant tyrosine kinase. Prompt diagnosis of such cases is important since specific tyrosine kinase inhibitor therapy is indicated.

Recent advances in eosinophil biology.

Eosinophils are pleiotropic multi-functional leukocytes involved in initiation and propagation of diverse inflammatory responses. Recent studies examining eosinophil biology have focused on delineating the molecular basis of FIP1L1/PDGRFalpha-fusion gene induced HES, the molecular steps involved in eosinophil recruitment in tumor-associated eosinophilia and EGID, and the role of eosinophils in asthma. In this review, these studies are summarized, focusing on the implications of these findings in the understanding the role of eosinophils in diseases.