Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus with a double-stranded DNA capable of establishing latent infection in the host cell. During latency, only a limited number of viral genes are expressed in infected host cells, and that helps the virus to evade host immune cell response. During primary infection, the KSHV genome is chromatinized and maintained as an episome, which is tethered to the host chromosome via Latency Associated Nuclear Antigen (LANA). The KSHV episome undergoes the same chromatin modification with the host cell chromosome and, therefore, is regulated by various epigenetic modifications, such as DNA methylation, histone methylation, and histone acetylation. The KSHV genome is also organized in a spatiotemporal manner by forming genomic loops, which enable simultaneous and coordinated control of dynamic gene transcription, particularly during the lytic replication phase. The genome-wide approaches and advancing bioinformatic tools have increased the resolution of studies on the dynamic transcriptional control and our understanding of KSHV latency-lytic switch regulation. We will summarize our current understanding of the epigenetic gene regulation on the KSHV chromatin.

Ferredoxin 1 and 2 (FDX1/2) constitute an evolutionarily conserved FDX family of iron-sulfur cluster-containing proteins. FDX1/2 are cognate substrates of ferredoxin reductase and serve as conduits for electron transfer from NADPH to a set of proteins involved in biogenesis of corticosteroids, hemes, iron-sulfur cluster, and lipoylated proteins. Fdx1 is essential for embryonic development and lipid homeostasis. Herein, Fdx2-deficient mice were generated to explore the physiological role of FDX2. Interestingly, unlike Fdx1-null embryos, which were dead at embryonic day 10.5 to 13.5, Fdx2-null mice were viable. Both Fdx2-null and Fdx2-heterozygous mice had a short lifespan and were susceptible to spontaneous tumors and steatohepatitis. Moreover, FDX2 deficiency increased, whereas overexpression of FDX2 decreased cytoplasmic accumulation of lipid droplets. Consistently, FDX2 deficiency led to accumulation of cholesterol and triglycerides. Mechanistically, FDX2 deficiency suppressed expression of cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) and activated master lipid transcription regulators sterol regulatory element-binding proteins 1/2, thus leading to altered lipid metabolism. Untargeted lipidomic analysis showed that FDX2 deficiency led to altered biosynthesis of various lipid classes, including cardiolipins, cholesterol, ceramides, triglycerides, and fatty acids. In summary, these findings underline an indispensable role of FDX2 in tumor suppression and lipid homeostasis at both cellular and organismal levels without being a prerequisite for embryonic development.

ABSTRACT Protein knock-down with an inducible degradation system is a powerful tool to study proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposi’s Sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-AID (mAID) tag at the LANA N-terminus with KSHV BAC16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-LANA. Incubation with 5-phenyl-indole-3-acetic acid (5-Ph-IAA), a derivative of natural auxin, rapidly degraded LANA within 1.5 h. In contrast to our hypothesis, depletion of LANA did not trigger lytic reactivation but rather decreased inducible lytic gene expression when we stimulated reactivation with a combination of ORF50 protein expression and sodium butyrate treatment. Decreased overall lytic gene induction seemed to associate with a rapid loss of KSHV genomes in the absence of LANA. The rapid loss of viral genomic DNA was blocked by treatment with lysosomal inhibitor chloroquine. Furthermore, siRNA-mediated knockdown of cellular innate immune proteins, cyclic AMP-GMP synthase (cGAS) and Stimulator of Interferon Genes (STING), and other autophagy-related genes rescued the degradation of viral genomic DNA upon LANA depletion. These results suggest that LANA is actively protecting viral genomic DNA from sensing by cGAS-STING signaling axis, and add novel insights into the role of LANA in latency maintenance. IMPORTANCE KSHV LANA plays a wide variety of roles in latency maintenance and lytic gene expression. We adopted the inducible protein knockdown approach and revealed that depletion of LANA induced rapid degradation of viral genomic DNA. The viral genome degradation was rescued by inhibition of the cellular innate immune pathway and autophagy. These observations suggest that LANA might play a role in hiding KSHV episome from cellular innate immune DNA sensors. Our study thus provides novel insights into the role of LANA in latency maintenance.

Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a latent infection, and viral genes are poised to be transcribed in the latent chromatin. In the poised chromatins, KSHV latency-associated nuclear antigen (LANA) interacts with cellular chromodomain-helicase-DNA-binding protein 4 (CHD4) and inhibits viral promoter activation. CHD4 is known to regulate cell differentiation by preventing enhancers from activating promoters. Here, we identified a putative CHD4 inhibitor peptide (VGN73) from the LANA sequence corresponding to the LANA-CHD4 interaction surface. The VGN73 interacts with CHD4 at its PHD domain with a dissociation constant (KD) of 14 nM. Pre-treatment with VGN73 enhanced monocyte differentiation into macrophages and globally altered the repertoire of activated genes in U937 cells. Furthermore, the introduction of the peptide into the cancer cells induced caspase-mediated CHD4 cleavage, triggered cell death, and inhibited tumor growth in a xenograft mouse model. The VGN73 may facilitate cell differentiation therapy.

Kaposi sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the cell nucleus, but where KSHV episomal genomes are tethered and the mechanisms underlying KSHV lytic reactivation are unclear. Here, we study the nuclear microenvironment of KSHV episomes and show that the KSHV latency-lytic replication switch is regulated via viral long non-coding (lnc)RNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. KSHV episomes localize with CHD4 and ADNP proteins, components of the cellular ChAHP complex. The CHD4 and ADNP proteins occupy the 5'-region of the highly inducible lncRNAs and terminal repeats of the KSHV genome together with latency-associated nuclear antigen (LANA). Viral lncRNA binding competes with CHD4 DNA binding, and KSHV reactivation sequesters CHD4 from the KSHV genome, which is also accompanied by detachment of KSHV episomes from host chromosome docking sites. We propose a model in which robust KSHV lncRNA expression determines the latency-lytic decision by regulating LANA/CHD4 binding to KSHV episomes.

Protein knock-down with an inducible degradation system is a powerful tool to study proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposi’s Sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-AID (mAID) tag at the LANA N-terminus with KSHV BAC16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-tagged LANA. Incubation with 5-phenyl-indole-3-acetic acid (5-Ph-IAA), a derivative of natural auxin, rapidly degraded LANA protein within 1.5 hours. In contrast to our hypothesis, depletion of LANA not only failed to trigger lytic reactivation but rather decreased inducible lytic gene expression when we triggered reactivation with a combination of ORF50 protein expression and sodium butyrate treatment. Decreased overall lytic gene induction seemed to associate with a rapid loss of KSHV genomes in the absence of LANA. Furthermore, we found that small cell fractions harbor non-depletable LANA dots in the presence of 5-Ph-IAA. In the cell population containing degradation-resistant LANA, induction of lytic reactivation was strongly attenuated. These results suggest that (i) there are at least two populations of LANA dots in cells, (ii) local nuclear environment and its epigenetic effects on the episomes are heritable to daughter cells; this biological had substantial effects in degree of KSHV reactivation, and finally (iii) LANA may have an additional function in protecting KSHV episomes from degradation.IMPORTANCE KSHV LANA protein plays a wide variety of roles in latency maintenance and lytic gene expression. We adapted the inducible protein knockdown approach to examine its role directly, and revealed that there are cell populations that possess viral episomes insensitive to reactivation stimuli. Viral reactivation is known to be highly heterogenic, and our observations suggest that LANA tethering sites on host chromatin may play a critical role in determining diverse responsiveness to the stimuli. We also demonstrated that depletion of LANA leads to rapid reduction of viral genome, which suggests that LANA might be actively protecting latent viral genome from degradation. These results add novel insights into the role of LANA in latency maintenance and regulation of lytic reactivation.

Studies on “HIT&RUN” effects by viral protein are difficult when using traditional affinity precipitation-based techniques under dynamic conditions, because only proteins interacting at a specific instance in time can be precipitated by affinity purification. Recent advances in proximity labeling (PL) have enabled study of both static and dynamic protein-protein interactions. Here we applied PL method with recombinant Kaposi’s sarcoma-associated herpesvirus (KSHV). KSHV, a gamma-herpesvirus, uniquely encodes four interferon regulatory factors (IRFs 1-4) in the genome, and we identified KSHV vIRF-1 and vIRF-4 interacting proteins during reactivation. Fusion of mini-TurboID with vIRF-1 or vIRF-4 did not interfere with KSHV gene expression, DNA replication, or de novo infections. PL identified 213 and 70 proteins for vIRF-1 and vIRF-4 respectively, which possibly interact during KSHV reactivation, and 47 of those were shared between the two vIRFs; the list also includes three viral proteins, ORF17, thymidine kinase, and vIRF-4. Functional annotation of respective interacting proteins showed highly overlapping biological functions such as mRNA processing and transcriptional regulation by TP53. Involvement of commonly interacting 44 cellular proteins in innate immune regulation were examined by siRNAs, and we identified that splicing factor 3B (SF3B) family proteins were clearly involved in interferons transcription and suppressed KSHV reactivation. We propose that recombinant TurboID-KSHV is a powerful tool to probe key cellular proteins that play a role in KSHV replication, and selective splicing factors may have a function beyond connecting two exon sequences to regulate innate immune responses. Importance Viral protein interaction with a host protein shows at least two sides: (i) taking host protein functions for its own benefit and (ii) disruption of existing host protein complex formation to inhibit undesirable host responses. Due to use of affinity-precipitation approaches, the majority of our studies focused on how the virus takes advantage of the newly-formed protein interactions for its own replication. Proximity labeling (PL) however, can also highlight the transient and negative effects – those interactions which lead to dissociation from the existing protein complex. Here we highlight the power of PL in combination with recombinant KSHV to study viral host interactions.

In previous studies, we have shown that expression of a viral lncRNA, polyadenylated nuclear RNA (PAN RNA) is essential for inducible viral genomic looping and distal gene activation during Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation. Here we identify viral lncRNA binding proteins, and show that an underlying molecular mechanism regulating the KSHV latency-lytic replication switch is via a viral lncRNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. Proximity biotin labeling, single cell transcriptomics, and siRNA screening along with complementation studies identified that CHD4's enzymatic activity silences viral gene expression by preventing transcription factory formation. Furthermore, Capture Hi-C, Cleavage Under Targets and Release Using Nuclease (CUT&RUN), and proteomics approaches together identify KSHV episome docking sites on host chromosomes and colocalization with a CHD4 protein complex, ChAHP, at epigenetically active genomic regions. PAN RNA binds and competes with CHD4 DNA binding in vitro, and KSHV episomes detached from these host genomic loci sites when reactivation is triggered. Our studies suggest that CHD4 exhibits strong repressor function by preventing inducible genomic looping, and is therefore important for the ability of KSHV to establish and maintain latency in a "poised" state at selected host genomic loci.

Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes a latent infection in the cell nucleus, but where KSHV episomal genomes are tethered and the mechanisms underlying KSHV lytic reactivation are unclear. Here, we study the nuclear microenvironment of KSHV episomes and show that the KSHV latency-lytic replication switch is regulated via viral long non-coding (lnc)RNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. KSHV episomes localize with a CHD4 complex, ChAHP, at epigenetically active genomic regions and tethers frequently near centromeric regions of host chromosomes. The ChAHP complex also occupies the 5’-region of a highly-inducible lncRNAs and terminal repeats of KSHV genome with latency-associated nuclear antigen (LANA). Viral lncRNA binding competes with CHD4 DNA binding , and KSHV reactivation is accompanied by the detachment of KSHV episomes from host chromosome docking sites We propose a model in which elevated lncRNA expression determines the KSHV latency-lytic decision by regulating LANA/ChAHP DNA binding at inducible viral enhancers.

In herpesvirus replicating cells, host cell gene transcription is frequently down-regulated because important transcriptional apparatuses are appropriated by viral transcription factors. Here, we show a small peptide derived from the Kaposi’s sarcoma-associated herpesvirus transactivator (K-Rta) sequence, which attenuates cellular MYC expression, reduces cell proliferation, and selectively kills cancer cell lines in both tissue culture and a xenograft tumor mouse model. Mechanistically, the peptide functions as a decoy to block the recruitment of coactivator complexes consisting of Nuclear receptor coactivator 2 (NCOA2), p300, and SWI/SNF proteins to the MYC promoter in primary effusion lymphoma cells. Thiol(SH)-linked alkylation for the metabolic sequencing of RNA ( SLAM seq ) with target-transcriptional analyses further confirm that the viral peptide directly attenuates MYC and MYC-target gene expression. This study thus provides a unique tool to control MYC activation, which may be used as a therapeutic payload to treat MYC-dependent diseases such as cancers and autoimmune diseases.

Molecular mechanisms of Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation have been studied primarily by measuring the total or average activity of an infected cell population, which often consists of a mixture of both nonresponding and reactivating cells that in turn contain KSHVs at various stages of replication. Studies on KSHV gene regulation at the individual cell level would allow us to better understand the basis for this heterogeneity, and new preventive measures could be developed based on findings from nonresponding cells exposed to reactivation stimuli. Here, we generated a recombinant reporter virus, which we named "Rainbow-KSHV," that encodes three fluorescence-tagged KSHV proteins (mBFP2-ORF6, mCardinal-ORF52, and mCherry-LANA). Rainbow-KSHV replicated similarly to a prototype reporter-KSHV, KSHVr.219, and wild-type BAC16 virus. Live imaging revealed unsynchronized initiation of reactivation and KSHV replication with diverse kinetics between individual cells. Cell fractionation revealed temporal gene regulation, in which early lytic gene expression was terminated in late protein-expressing cells. Finally, isolation of fluorescence-positive cells from nonresponders increased dynamic ranges of downstream experiments 10-fold. Thus, this study demonstrates a tool to examine heterogenic responses of KSHV reactivation for a deeper understanding of KSHV replication. IMPORTANCE Sensitivity and resolution of molecular analysis are often compromised by the use of techniques that measure the ensemble average of large cell populations. Having a research tool to nondestructively identify the KSHV replication stage in an infected cell would not only allow us to effectively isolate cells of interest from cell populations but also enable more precise sample selection for advanced single-cell analysis. We prepared a recombinant KSHV that can report on its replication stage in host cells by differential fluorescence emission. Consistent with previous host gene expression studies, our experiments reveal the highly heterogenic nature of KSHV replication/gene expression at individual cell levels. The utilization of a newly developed reporter-KSHV and initial characterization of KSHV replication in single cells are presented.

BACKGROUND/AIMS:Directional migration of corneal epithelial cells is essential for healing of corneal wounds, which is a robust response mediated by biochemical and bioelectrical cues. Naturally occurring electric fields at corneal wounds provide a powerful guidance cue for directional cell migration, as does extracellular ATP. Our recent large-scale siRNA library screening identified a role for purinergic signaling in the electric field-guided migration (galvanotaxis/electrotaxis) of human corneal epithelial (hTCEpi) cells.METHODS:We examined the effect of extracellular ATP on galvanotaxis of hTCEpi cells. Galvanotactic cell migration was recorded by video microscopy, and directedness and migration speed was calculated. The role of purinergic receptors in galvanotaxis regulation was evaluated by pharmacological inhibition or knocking down of P2X and P2Y receptors.RESULTS:Addition of ATP enhanced galvanotaxis, and most remarkably sensitized galvanotaxis response to very low level of electric fields in the physiological range (10-30 mV/mm). The stimulatory effect of extracellular ATP was diminished by apyrase treatment. Importantly, cells stimulated with extracellular ATP migrated with significantly increased directedness and speed, which were diminished by knocking down or pharmacological inhibition of P2X and P2Y receptors. Inhibition of pannexin-1 (ATP permeable channel) significantly impaired galvanotaxis. Moreover, pharmacological inhibition of ectoATPase enhanced galvanotaxis.CONCLUSION:Extracellular ATP and physiological electric fields synergistically enhanced the galvanotaxis response of hTCEpi cells. hTCEpi cells are likely to secrete ATP actively, and purinergic signaling is down-regulated by ecto-ATPases. Both P2X and P2Y receptors coordinately play a role for galvanotaxis of hTCEpi cells.

Proper control of cell migration is critically important in many biologic processes, such as wound healing, immune surveillance, and development. Much progress has been made in the initiation of cell migration; however, little is known about termination and sometimes directional reversal. During active cell migration, as in wound healing, development, and immune surveillance, the integrin expression profile undergoes drastic changes. Here, we uncovered the extensive regulatory and even opposing roles of integrins in directional cell migration in electric fields (EFs), a potentially important endogenous guidance mechanism. We established cell lines that stably express specific integrins and determined their responses to applied EFs with a high throughput screen. Expression of specific integrins drove cells to migrate to the cathode or to the anode or to lose migration direction. Cells expressing αMβ2, β1, α2, αIIbβ3, and α5 migrated to the cathode, whereas cells expressing β3, α6, and α9 migrated to the anode. Cells expressing α4, αV, and α6β4 lost directional electrotaxis. Manipulation of α9 molecules, one of the molecular directional switches, suggested that the intracellular domain is critical for the directional reversal. These data revealed an unreported role for integrins in controlling stop, go, and reversal activity of directional migration of mammalian cells in EFs, which might ensure that cells reach their final destination with well-controlled speed and direction.-Zhu, K., Takada, Y., Nakajima, K., Sun, Y., Jiang, J., Zhang, Y., Zeng, Q., Takada, Y., Zhao, M. Expression of integrins to control migration direction of electrotaxis.

Quercetin has multiple potential to control various cell function keeping our body condition healthy. In this review article, we describe the molecular mechanism on how quercetin exerts its action on blood pressure, neurite elongation and epithelial ion transport based from a viewpoint of cytosolic Cl- environments, which is recently recognized as an important signaling factor in various types of cells. Recent studies show various roles of cytosolic Cl- in regulation of blood pressure and neurite elongation, and prevention from bacterial and viral infection. We have found the stimulatory action of quercetin on Cl- transporter, Na+-K+-2Cl(-) cotransporter 1 (NKCC1; an isoform of NKCC), which has been recognized as one of the most interesting, fundamental actions of quercetin. In this review article, based on this stimulatory action of quercetin on NKCC1, we introduce the molecular mechanism of quercetin on: 1) blood pressure, 2) neurite elongation, and 3) epithelial Cl- secretion including tight junction forming in epithelial tissues. 1) Quercetin induces elevation of the cytosolic Cl- concentration via activation of NKCC1, leading to anti-hypertensive action by diminishing expression of epithelial Na+ channel (ENaC), a key ion channel involved in renal Na+ reabsorption, while quercetin has no effects on the blood pressure with normal salt intake. 2) Quercetin also has stimulatory effects on neurite elongation by elevating the cytosolic Cl- concentration via activation of NKCC1 due to tubulin polymerization facilitated through Cl--induced inhibition of GTPase. 3) Further, in lung airway epithelia quercetin stimulates Cl- secretion by increasing the driving force for Cl- secretion via elevation of the cytosolic Cl- concentration: this leads to water secretion, participating in prevention of our body from bacterial and viral infection. In addition to transcellular ion transport, quercetin regulates tight junction function via enhancement of tight junction integrity by modulating expression and assembling tight junction-forming proteins. Based on these observations, it is concluded that quercetin is a useful medicinal compound keeping our body to be in healthy condition.

We investigated suppression of the slow growth of an Escherichia coli ispA null mutant lacking farnesyl diphosphate (FPP) synthase (i.e. IspA) by plasmids carrying prenyl diphosphate synthase genes. The growth rates of ispA mutant-transformants harboring a medium-copy number plasmid that carries ispA or ispB were almost the same as that of the wild-type strain. Although the level of FPP in the transformant with the ispA plasmid was almost the same as that in the wild-type strain, the level in the transformant with the ispB plasmid was as low as that in the ispA mutant. Purified octaprenyl diphosphate synthase (IspB) could condense isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) to form octaprenyl diphosphate and nonaprenyl diphosphate. It is possible that suppression of the slow growth of the ispA mutant by ispB was due to condensation of IPP not only with FPP but also with DMAPP by octaprenyl diphosphate synthase.

Chloride ion (Cl-) is one of the most abundant anions in our body. Increasing evidence suggests that Cl- plays fundamental roles in various cellular functions. We have previously reported that electroneutral cation-chloride cotransporters, such as Na+-K+-2Cl- cotransporter 1 (NKCC1) and K+-Cl- cotransporter 1 (KCC1), are involved in neurite outgrowth during neuronal differentiation. In the present study, we studied if there is correlation between intracellular Cl- concentrations ([Cl-]i) and the length of growing neurites. We measured [Cl-]i in the cell body and growing neurite tips using halide-sensitive fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), revealing that [Cl-]i in the tip of growing neurite was higher than that in cell body in a single cell. Importantly, there was a significant positive correlation between the length of growing neurite and [Cl-]i in neurite tip. Bumtanide (BMT), an inhibitor of NKCC1, significantly inhibited neurite outgrowth and decreased [Cl-]i in neurite tip. The results obtained in the present study and our previous studies together strongly suggest that high [Cl-]i in neurite tip region is crucial for efficient neurite outgrowth.

Chloride ion (Cl–) is one of fundamental anions in our body. Recent studies have revealed that Cl– influences various cellular functions such as cell cycle progression, proliferation, and adhesion/migration of cells. Here we focused on the effect of intracellular Cl– on neurite outgrowth. During differentiation, neuronal cells undergo dramatic shape changes. Typically, neuronal cells extend long membranous processes, named neurites. We have confirmed that, in rat pheochromocytoma PC12 cells, NGF–induced neurite outgrowth requires uptake of Cl– into the intracellular space via Na+–K+–2Cl– cotransporter 1 (NKCC1). In addition, NGF–induced neurite outgrowth was enhanced by inhibition of K+–Cl– cotransporter 1 (KCC1), which was another type of Cl– cotransporter mediating excretion of K+ and Cl– from intracellular space. In growing neurite of neuronal cells, it is suggested thatα/β–tubulin heterodimers assemble to form microtubule, and assembly of microtubule promotes neurite elongation. We also demonstrated that Cl– affected intrinsic GTPase activity of tubulin and the action of Cl– on intrinsic GTPase activity of tubulin would be one of important factors regulating tubulin polymerization (formation of microtubule). These observations show that novel regulatory mechanisms forming microtubule by intracellular Cl– and that intracellular Cl– would be an important factor regulating the neurite outgrowth.

Weak electric fields guide cell migration, known as galvanotaxis/electrotaxis. The sensor(s) cells use to detect the fields remain elusive. Here we perform a large-scale screen using an RNAi library targeting ion transporters in human cells. We identify 18 genes that show either defective or increased galvanotaxis after knockdown. Knockdown of the KCNJ15 gene (encoding inwardly rectifying K(+) channel Kir4.2) specifically abolishes galvanotaxis, without affecting basal motility and directional migration in a monolayer scratch assay. Depletion of cytoplasmic polyamines, highly positively charged small molecules that regulate Kir4.2 function, completely inhibits galvanotaxis, whereas increase of intracellular polyamines enhances galvanotaxis in a Kir4.2-dependent manner. Expression of a polyamine-binding defective mutant of KCNJ15 significantly decreases galvanotaxis. Knockdown or inhibition of KCNJ15 prevents phosphatidylinositol 3,4,5-triphosphate (PIP3) from distributing to the leading edge. Taken together these data suggest a previously unknown two-molecule sensing mechanism in which KCNJ15/Kir4.2 couples with polyamines in sensing weak electric fields.

Potassium chloride cotransporters (KCCs) mediate electroneutrally-coupled transport of K(+) and Cl(-), and play crucial roles in various cell functions including regulation of cell volume and homeostasis of cellular Cl(-)content. Four isoforms of KCCs (KCC1, 2, 3, and 4) have been identified. KCC1 is ubiquitously expressed, whereas KCC2 is mainly expressed in neuronal cells of central nervous system. KCC3 is highly expressed in heart, skeletal muscle, kidney, lung and placenta. KCC4 is mainly expressed in epithelial cells. In this study, we investigated roles of KCCs in NGF-induced neurite outgrowth of rat pheochromocytoma PC12 cells. The most abundantly expressed isoform in PC12 cells was KCC1. Inhibition of KCCs using [(dihydronindenyl)oxy] alkanoic acid (DIOA), an inhibitor of KCCs, enhanced the NGF-induced neurite outgrowth of PC12 cells in a dose-dependent manner. Treatment of PC12 cells with NGF significantly decreased mRNA expression of KCC1, whereas other isoforms, KCC2-4, showed no changes in their mRNA expression in response to NGF treatment. Knockdown of KCC1 using small interfering RNA (siRNA) enhanced the NGF-induced neurite outgrowth. These results suggest that KCC1 negatively regulates the NGF-induced neurite outgrowth of PC12 cells.

In growing neurite of neuronal cells, it is suggested that alpha/beta-tubulin heterodimers assemble to form microtubule, and assembly of microtubule promotes neurite elongation. On the other hand, recent studies reveal importance of intracellular Cl- in regulation of various cellular functions such as cell cycle progression, differentiation, cell migration, and elongation of neurite in neuronal cells. In this study, we investigated effects of Cl- on in vitro tubulin polymerization. We found that efficiency of in vitro tubulin polymerization (the number of microtubule) was higher (3 to 5-fold) in Cl--containing solutions than that in Cl--free solutions containing Br- or NO3-. On the other hand, GTPase activity of tubulin was lower (2/3-fold) in Cl--containing solutions than that in Cl--free solutions containing Br- or NO3- Efficiency of in vitro tubulin polymerization in solutions containing a non-hydrolyzable analogue of GTP (GpCpp) instead of GTP was much higher than that in the presence of GTP. Effects of replacement of GTP with GpCpp on in vitro tubulin polymerization was weaker in Cl- solutions (10-fold increases) than that in Br- or NO3- solutions (20-fold increases), although the efficiency of in vitro tubulin polymerization in Cl- solutions containing GpCpp was still higher than that in Br- or NO3- solutions containing GpCpp. Our results suggest that a part of stimulatory effects of Cl- on in vitro tubulin polymerization is mediated via an inhibitory effect on GTPase activity of tubulin, although Cl- would also regulate in vitro tubulin polymerization by factors other than an inhibitory effect on GTPase activity. (c) 2012 Elsevier Inc. All rights reserved.