Inhibition of Nonsense-Mediated Decay Induces Nociceptive Sensitization through Activation of the Integrated Stress Response.

RNA stability is meticulously controlled. Here, we sought to determine whether an essential post-transcriptional regulatory mechanism plays a role in pain. Nonsense-mediated decay (NMD) safeguards against translation of mRNAs that harbor premature termination codons and controls the stability of ∼10% of typical protein-coding mRNAs. It hinges on the activity of the conserved kinase SMG1. Both SMG1 and its target, UPF1, are expressed in murine DRG sensory neurons. SMG1 protein is present in both the DRG and sciatic nerve. Using high-throughput sequencing, we examined changes in mRNA abundance following inhibition of SMG1. We confirmed multiple NMD stability targets in sensory neurons, including ATF4. ATF4 is preferentially translated during the integrated stress response (ISR). This led us to ask whether suspension of NMD induces the ISR. Inhibition of NMD increased eIF2-α phosphorylation and reduced the abundance of the eIF2-α phosphatase constitutive repressor of eIF2-α phosphorylation. Finally, we examined the effects of SMG1 inhibition on pain-associated behaviors. Peripheral inhibition of SMG1 results in mechanical hypersensitivity in males and females that persists for several days and priming to a subthreshold dose of PGE2. Priming was fully rescued by a small-molecule inhibitor of the ISR. Collectively, our results indicate that suspension of NMD promotes pain through stimulation of the ISR.SIGNIFICANCE STATEMENT Nociceptors undergo long-lived changes in their plasticity which may contribute to chronic pain. Translational regulation has emerged as a dominant mechanism in pain. Here, we investigate the role of a major pathway of RNA surveillance called nonsense-mediated decay (NMD). Modulation of NMD is potentially beneficial for a broad array of diseases caused by frameshift or nonsense mutations. Our results suggest that inhibition of the rate-limiting step of NMD drives behaviors associated with pain through activation of the ISR. This work reveals complex interconnectivity between RNA stability and translational regulation and suggests an important consideration in harnessing the salubrious benefits of NMD disruption.

The RNA-Binding Protein HuR Is Integral to the Function of Nociceptors in Mice and Humans.

HuR is an RNA-binding protein implicated in RNA processing, stability, and translation. Previously, we examined protein synthesis in dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We found that the HuR consensus binding element was enriched in transcripts with elevated translation. HuR is expressed in the soma of nociceptors and their axons. Pharmacologic inhibition of HuR with the small molecule CMLD-2 reduced the activity of mouse and human sensory neurons. Peripheral administration of CMLD-2 in the paw or genetic elimination of HuR from sensory neurons diminished behavioral responses associated with NGF- and IL-6-induced allodynia in male and female mice. Genetic disruption of HuR altered the proximity of mRNA decay factors near a key neurotrophic factor (TrkA). Collectively, the data suggest that HuR is required for local control of mRNA stability and reveals a new biological function for a broadly conserved post-transcriptional regulatory factor.SIGNIFICANCE STATEMENT Nociceptors undergo long-lived changes in excitability, which may contribute to chronic pain. Noxious cues that promote pain lead to rapid induction of protein synthesis. The underlying mechanisms that confer specificity to mRNA control in nociceptors are unclear. Here, we identify a conserved RNA-binding protein called HuR as a key regulatory factor in sensory neurons. Using a combination of genetics and pharmacology, we demonstrate that HuR is required for signaling in nociceptors. In doing so, we report an important mechanism of mRNA control in sensory neurons that ensures appropriate nociceptive responses to inflammatory mediators.

Global analyses of mRNA expression in human sensory neurons reveal eIF5A as a conserved target for inflammatory pain.

Nociceptors are a type of sensory neuron that are integral to most forms of pain. Targeted disruption of nociceptor sensitization affords unique opportunities to prevent pain. An emerging model for nociceptors are sensory neurons derived from human stem cells. Here, we subjected five groups to high-throughput sequencing: human induced pluripotent stem cells (hiPSCs) prior to differentiation, mature hiPSC-derived sensory neurons, mature co-cultures containing hiPSC-derived astrocytes and sensory neurons, mouse dorsal root ganglion (DRG) tissues, and mouse DRG cultures. Co-culture of nociceptors and astrocytes promotes expression of transcripts enriched in DRG tissues. Comparisons of the hiPSC models to tissue samples reveal that many key transcripts linked to pain are present. Markers indicative of a range of neuronal subtypes present in the DRG were detected in mature hiPSCs. Intriguingly, translation factors were maintained at consistently high expression levels across species and culture systems. As a proof of concept for the utility of this resource, we validated expression of eukaryotic initiation factor 5A (eIF5A) in DRG tissues and hiPSC samples. eIF5A is subject to a unique posttranslational hypusine modification required for its activity. Inhibition of hypusine biosynthesis prevented hyperalgesic priming by inflammatory mediators in vivo and diminished hiPSC activity in vitro. Collectively, our results illuminate the transcriptomes of hiPSC sensory neuron models. We provide a demonstration for this resource through our investigation of eIF5A. Our findings reveal hypusine as a potential target for inflammation associated pain in males.

Intercellular Arc Signaling Regulates Vasodilation.

Injury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in murine dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of translation in vitro and in vivo Inflammatory cues promote local translation of Arc in the skin. Arc-deficient male mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles (EVs), we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found that the excessive thermal responses and vasodilation observed in Arc defective mice are rescued by injection of Arc-containing EVs into the skin. Our findings suggest that activity-dependent production of Arc in afferent fibers regulates neurogenic inflammation potentially through intercellular signaling.SIGNIFICANCE STATEMENT Nociceptors play prominent roles in pain and inflammation. We examined rapid changes in the landscape of nascent translation in cultured dorsal root ganglia (DRGs) treated with a combination of inflammatory mediators using ribosome profiling. We identified several hundred transcripts subject to rapid preferential translation. Among them is the immediate early gene (IEG) Arc. We provide evidence that Arc is translated in afferent fibers in the skin. Arc-deficient mice display several signs of exaggerated inflammation which is normalized on injection of Arc containing extracellular vesicles (EVs). Our work suggests that noxious cues can trigger Arc production by nociceptors which in turn constrains neurogenic inflammation in the skin.

Nociceptor Translational Profiling Reveals the Ragulator-Rag GTPase Complex as a Critical Generator of Neuropathic Pain.

Nociceptors, sensory neurons in the DRG that detect damaging or potentially damaging stimuli, are key drivers of neuropathic pain. Injury to these neurons causes activation of translation regulation signaling, including the mechanistic target of rapamycin complex 1 (mTORC1) and mitogen-activated protein kinase interacting kinase (MNK) eukaryotic initiation factor (eIF) 4E pathways. This is a mechanism driving changes in excitability of nociceptors that is critical for the generation of chronic pain states; however, the mRNAs that are translated to lead to this plasticity have not been elucidated. To address this gap in knowledge, we used translating ribosome affinity purification in male and female mice to comprehensively characterize mRNA translation in Scn10a-positive nociceptors in chemotherapy-induced neuropathic pain (CIPN) caused by paclitaxel treatment. This unbiased method creates a new resource for the field, confirms many findings in the CIPN literature and also find extensive evidence for new target mechanisms that may cause CIPN. We provide evidence that an underlying mechanism of CIPN is sustained mTORC1 activation driven by MNK1-eIF4E signaling. RagA, a GTPase controlling mTORC1 activity, is identified as a novel target of MNK1-eIF4E signaling. This demonstrates a novel translation regulation signaling circuit wherein MNK1-eIF4E activity drives mTORC1 via control of RagA translation. CIPN and RagA translation are strongly attenuated by genetic ablation of eIF4E phosphorylation, MNK1 elimination or treatment with the MNK inhibitor eFT508. We identify a novel translational circuit for the genesis of neuropathic pain caused by chemotherapy with important implications for therapeutics.SIGNIFICANCE STATEMENT Neuropathic pain affects up to 10% of the population, but its underlying mechanisms are incompletely understood, leading to poor treatment outcomes. We used translating ribosome affinity purification technology to create a comprehensive translational profile of DRG nociceptors in naive mice and at the peak of neuropathic pain induced by paclitaxel treatment. We reveal new insight into how mechanistic target of rapamycin complex 1 is activated in neuropathic pain pointing to a key role of MNK1-eIF4E-mediated translation of a complex of mRNAs that control mechanistic target of rapamycin complex 1 signaling at the surface of the lysosome. We validate this finding using genetic and pharmacological techniques. Our work strongly suggests that MNK1-eIF4E signaling drives CIPN and that a drug in human clinical trials, eFT508, may be a new therapeutic for neuropathic pain.

Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).

RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA.

Functionally distinct roles for eEF2K in the control of ribosome availability and p-body abundance.

Processing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splitting in vitro. Collectively, the data suggest that eEF2K defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons.

A role for translational regulation by S6 kinase and a downstream target in inflammatory pain.

BACKGROUND AND PURPOSE: Translational controls pervade neurobiology. Nociceptors play an integral role in the detection and propagation of pain signals. Nociceptors can undergo persistent changes in their intrinsic excitability. Pharmacological disruption of nascent protein synthesis diminishes acute and chronic forms of pain-associated behaviours. However, the targets of translational controls that facilitate plasticity in nociceptors are unclear. EXPERIMENTAL APPROACH: We used ribosome profiling to probe the translational landscape in dorsal root ganglion (DRG) neurons from male Swiss-Webster mice, after treatment with nerve growth factor and IL-6. Expression dynamics of c-Fos were followed with immunoblotting and immunohistochemistry. The involvement of ribosomal protein S6 kinase 1 (S6K1), a downstream component of mTOR signalling, in the control of c-Fos levels was assessed with low MW inhibitors of S6K1 (DG2) or c-Fos (T-5224), studying their effects on nociceptor activity in vitro using multielectrode arrays (MEAs) and pain behaviour in vivo in Swiss-Webster mice using the hyperalgesic priming model. KEY RESULTS: c-Fos was expressed in sensory neurons. Inflammatory mediators that promote pain in both humans and rodents promote c-Fos translation. The mTOR effector S6K1 is essential for c-Fos biosynthesis. Inhibition of S6K1 or c-Fos with low MW compounds diminished mechanical and thermal hypersensitivity in response to inflammatory cues. Additionally, both inhibitors reduced evoked nociceptor activity. CONCLUSION AND IMPLICATIONS: Our data show a novel role of S6K1 in modulating the rapid response to inflammatory mediators, with c-Fos being one key downstream target. Targeting the S6 kinase pathway or c-Fos is an exciting new avenue for pain-modulating compounds.

A peptide encoded within a 5' untranslated region promotes pain sensitization in mice.

ABSTRACT: Translational regulation permeates neuronal function. Nociceptors are sensory neurons responsible for the detection of harmful stimuli. Changes in their activity, termed plasticity, are intimately linked to the persistence of pain. Although inhibitors of protein synthesis robustly attenuate pain-associated behavior, the underlying targets that support plasticity are largely unknown. Here, we examine the contribution of protein synthesis in regions of RNA annotated as noncoding. Based on analyses of previously reported ribosome profiling data, we provide evidence for widespread translation in noncoding transcripts and regulatory regions of mRNAs. We identify an increase in ribosome occupancy in the 5' untranslated regions of the calcitonin gene-related peptide (CGRP/Calca). We validate the existence of an upstream open reading frame (uORF) using a series of reporter assays. Fusion of the uORF to a luciferase reporter revealed active translation in dorsal root ganglion neurons after nucleofection. Injection of the peptide corresponding to the calcitonin gene-related peptide-encoded uORF resulted in pain-associated behavioral responses in vivo and nociceptor sensitization in vitro. An inhibitor of heterotrimeric G protein signaling blocks both effects. Collectively, the data suggest pervasive translation in regions of the transcriptome annotated as noncoding in dorsal root ganglion neurons and identify a specific uORF-encoded peptide that promotes pain sensitization through GPCR signaling.

Antisense DNA parameters derived from next-nearest-neighbor analysis of experimental data.

BACKGROUND: The enumeration of tetrameric and other sequence motifs that are positively or negatively correlated with in vivo antisense DNA effects has been a useful addition to the arsenal of information needed to predict effective targets for antisense DNA control of gene expression. Such retrospective information derived from in vivo cellular experiments characterizes aspects of the sequence dependence of antisense inhibition that are not predicted by nearest-neighbor (NN) thermodynamic parameters derived from in vitro experiments. However, quantitation of the antisense contributions of motifs is problematic, since individual motifs are not isolated from the effects of neighboring nucleotides, and motifs may be overlapping. These problems are circumvented by a next-nearest-neighbor (NNN) analysis of antisense DNA effects in which the overlapping nature of nearest-neighbors is taken into account. RESULTS: Next-nearest-neighbor triplet combinations of nucleotides are the simplest that include overlapping sequence effects and therefore can encompass interactions beyond those of nearest neighbors. We used singular value decomposition (SVD) to fit experimental data from our laboratory in which phosphorothioate-modified antisense DNAs (S-DNAs) 20 nucleotides long were used to inhibit cellular protein expression in 112 experiments involving four gene targets and two cell lines. Data were fitted using a NNN model, neglecting end effects, to derive NNN inhibition parameters that could be combined to give parameters for a set of 49 sequences that represents the inhibitory effects of all possible overlapping triplet interactions in the cellular targets of these antisense S-DNAs. We also show that parameters to describe subsets of the data, such as the mRNAs being targeted and the cell lines used, can be included in such a derivation. While NNN triplet parameters provided an adequate model to fit our data, NN doublet parameters did not. CONCLUSIONS: The methodology presented illustrates how NNN antisense inhibitory information can be derived from in vivo cellular experiments. Subsequent calculations of the antisense inhibitory parameters for any mRNA target sequence automatically take into account the effects of all possible overlapping combinations of nearest-neighbors in the sequence. This procedure is more robust than the tallying of tetrameric motifs that have positive or negative antisense effects. The specific parameters derived in this work are limited in their applicability by the relatively small database of experiments that was used in their derivation.

Inhibition of Poly(A)-binding protein with a synthetic RNA mimic reduces pain sensitization in mice.

Nociceptors rely on cap-dependent translation to rapidly induce protein synthesis in response to pro-inflammatory signals. Comparatively little is known regarding the role of the regulatory factors bound to the 3' end of mRNA in nociceptor sensitization. Poly(A)-binding protein (PABP) stimulates translation initiation by bridging the Poly(A) tail to the eukaryotic initiation factor 4F complex associated with the mRNA cap. Here, we use unbiased assessment of PABP binding specificity to generate a chemically modified RNA-based competitive inhibitor of PABP. The resulting RNA mimic, which we designated as the Poly(A) SPOT-ON, is more stable than unmodified RNA and binds PABP with high affinity and selectivity in vitro. We show that injection of the Poly(A) SPOT-ON at the site of an injury can attenuate behavioral response to pain. Collectively, these results suggest that PABP is integral for nociceptive plasticity. The general strategy described here provides a broad new source of mechanism-based inhibitors for RNA-binding proteins and is applicable for in vivo studies.

Fetal hemoglobin induction by histone deacetylase inhibitors involves generation of reactive oxygen species.

OBJECTIVE: Several compounds, including butyrate and trichostatin A, have been shown to activate gamma-gene expression via p38 mitogen-activated protein kinase (MAPK) signaling. In eukaryotic cells, reactive oxygen species (ROS) act as signaling molecules to mediate phosphorylation of tyrosine kinases such as p38 MAPK to regulate gene expression. Therefore, we determined the role of the reactive oxygen species hydrogen peroxide (H(2)O(2)) in drug-mediated fetal hemoglobin (HbF) induction. METHODS: H(2)O(2) levels were measured using 2',7'-dichlorofluorescein-diacetate in K562 cells after drug treatments. To confirm a role for H(2)O(2) in HbF induction, studies were completed with the mitochondrial respiratory chain inhibitor myxothiazole, which prevents ROS generation. The ability of myxothiazole to block gamma-globin mRNA accumulation and HbF induction was measured in K562 cells and burst-forming unit-erythroid colonies respectively using quantitative real-time PCR and alkaline denaturation. RESULTS: Butyrate and trichostastin A stimulated p38 MAPK phosphorylation via a H(2)O(2)-dependent mechanism. Pretreatment with myxothiazole to inhibit ROS formation or SB203580 to impede p38 MAPK signaling attenuated gamma-gene activation in K562 cells and HbF induction in erythroid progenitors. However, myxothiazole had no effect on the ability of hydroxyurea to induce HbF. CONCLUSION: The findings presented herein support a ROS-p38 MAPK cell signaling mechanism for HbF induction by butyrate and trichostatin A.

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio.

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.

Cancer-Specific Production of N-Acetylaspartate via NAT8L Overexpression in Non-Small Cell Lung Cancer and Its Potential as a Circulating Biomarker.

In order to identify new cancer-associated metabolites that may be useful for early detection of lung cancer, we performed a global metabolite profiling of a non-small cell lung cancer (NSCLC) line and immortalized normal lung epithelial cells from the same patient. Among several metabolites with significant cancer/normal differences, we identified a unique metabolic compound, N-acetylaspartate (NAA), in cancer cells-undetectable in normal lung epithelium. NAA's cancer-specific detection was validated in additional cancer and control lung cells as well as selected NSCLC patient tumors and control tissues. NAA's cancer specificity was further supported in our analysis of NAA synthetase (gene symbol: NAT8L) gene expression levels in The Cancer Genome Atlas: elevated NAT8L expression in approximately 40% of adenocarcinoma and squamous cell carcinoma cases (N = 577), with minimal expression in all nonmalignant lung tissues (N = 74). We then showed that NAT8L is functionally involved in NAA production of NSCLC cells through siRNA-mediated suppression of NAT8L, which caused selective reduction of intracellular and secreted NAA. Our cell culture experiments also indicated that NAA biosynthesis in NSCLC cells depends on glutamine availability. For preliminary evaluation of NAA's clinical potential as a circulating biomarker, we developed a sensitive NAA blood assay and found that NAA blood levels were elevated in 46% of NSCLC patients (N = 13) in comparison with age-matched healthy controls (N = 21) among individuals aged 55 years or younger. Taken together, these results indicate that NAA is produced specifically in NSCLC tumors through NAT8L overexpression, and its extracellular secretion can be detected in blood. Cancer Prev Res; 9(1); 43-52. ©2015 AACR.

The reduction of Raf-1 protein by phosphorothioate ODNs and siRNAs targeted to the same two mRNA sequences.

Two sets of 20-mer phosphorothioate-modified oligodeoxynucleotide DNAs (sODN) and 21-mer or 22-mer small interfering RNAs (siRNAs), targeted to the same coding sites in raf-1 mRNA, were compared for their abilities to reduce the amount of endogenously expressed Raf-1 protein in T24 cells. The amount of Raf-1 protein was monitored by careful quantitation of Western blots. We found that the siRNAs were somewhat less effective than the S-ODNs in reducing the Raf-1 protein level 20 hours after a 4-hour transfection. The siRNA duplexes were characterized by circular dichroism (CD) spectra, and melting temperatures (Tm) were obtained for the siRNA duplexes and DNA x RNA hybrids formed by the S-ODNs. The S-ODNs differed in their effectiveness, the S-ODN that formed the more stable hybrid being the more effective in reducing the Raf-1 protein level, but the two siRNAs were equally effective despite a difference in Tm of about 20 degrees C. Finally, the siRNAs and S-ODNs had a comparable nonspecific effect on a nontargeted (Bcl-2) protein. Our data add to others in the literature that show it can be difficult to select siRNAs that are more effective than antisense ODNs in downregulating endogenously expressed proteins.

FK228 Analogues Induce Fetal Hemoglobin in Human Erythroid Progenitors.

Fetal hemoglobin (HbF) improves the clinical severity of sickle cell disease (SCD), therefore, research to identify HbF-inducing agents for treatment purposes is desirable. The focus of our study is to investigate the ability of FK228 analogues to induce HbF using a novel KU812 dual-luciferase reporter system. Molecular modeling studies showed that the structure of twenty FK228 analogues with isosteric substitutions did not disturb the global structure of the molecule. Using the dual-luciferase system, a subgroup of FK228 analogues was shown to be inducers of HbF at nanomolar concentrations. To determine the physiological relevance of these compounds, studies in primary erythroid progenitors confirmed that JMA26 and JMA33 activated HbF synthesis at levels comparable to FK228 with low cellular toxicity. These data support our lead compounds as potential therapeutic agents for further development in the treatment of SCD.

Identification of fetal hemoglobin-inducing agents using the human leukemia KU812 cell line.

Fetal hemoglobin (HbF) ameliorates the clinical severity of sickle cell disease; therefore continued research to identify efficacious HbF-inducing agents is desirable. In this study, we investigated KU812 leukemia cells that express the fetal γ-globin and adult ß-globin genes, as a system for screening and discovery of novel HbF inducers. KU812 cells were analyzed in the presence or absence of fetal bovine serum and then expression levels of the globin genes, cell surface markers and transcription factors were quantified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). For comparison, primary erythroid cells were grown in a two-phase liquid culture system. After drug inductions for 48-72 h, globin mRNA and HbF levels were quantified by RT-qPCR and enzyme-linked immunosorbent assay, respectively. Erythroid markers and transcription factors expression levels in KU812 cells were comparable to days 7-14 erythroid cells. We also tested several drugs including butyrate, trichostatin A, scriptaid, suberoylanilide hydroxamic acid and hydroxyurea, which induced γ-globin in KU812 cells; however, some agents also induced ß-globin. A novel agent STI-571 was studied in the system, which non-selectively induced the globin genes. Additional studies showed comparable globin gene response patterns in KU812 and primary erythroid cells after treatments with the various drug inducers. Mechanisms of drug-mediated γ-globin induction in KU812 cells require signaling through the p38 mitogen-activated protein kinase pathway similar to that previously demonstrated in primary erythroid cells. These data suggest that KU812 cells serve as a good screening system to identify potential HbF inducers for the treatment of ß-hemoglobinopathies.

Hydroxyurea generates nitric oxide in human erythroid cells: mechanisms for gamma-globin gene activation.

Hydroxyurea (HU) induces fetal hemoglobin synthesis through activation of cyclic guanine monophosphate (cGMP) signaling. Studies in sickle cell patients demonstrated increased circulating nitric oxide (NO) levels after oral HU treatment. However, the direct measurement of NO in erythroid cells and its role in fetal hemoglobin induction have not been defined. Therefore, we quantified the level of nitrate and nitrite (NOx) generated by HU in human erythroid progenitors in the presence of three nitric oxide synthase inhibitors (NOS), including N(G)-monomethyl-L-arginine (L-NMMA). In addition, cGMP levels were measured in the presence or absence of the pathway inhibitor 1H-(1,2,4)ox-adiazolo(4,3-a)quinoxalin-1-one, which blocks soluble guanylyl cyclase formation. HU treatment increased NOx levels and gamma-globin transcription in K562 and primary erythroid cells, which was augmented when HU was combined with L-NMMA. Pretreatment with the cGMP pathway inhibitor reversed gamma-gene activation by HU. These data demonstrate the direct stimulation of cellular NO and cGMP signaling in erythroid progenitors by HU as a possible mechanism for gamma-globin gene activation.