Generation of FLAG-tagged Arx knock-in mouse model.

The Aristaless-related homeobox (ARX) is a paired-like homeodomain transcription factor playing important roles in brain development. Patients with mutations in ARX have a spectrum of neurodevelopmental disorders such as epilepsy, intellectual disability, and autism spectrum disorder, with or without structural abnormalities of the brain such as lissencephaly (smooth brain), microcephaly (small brain), and/or agenesis of the corpus callosum. Mouse models have provided important clues on the pathophysiologic roles of ARX in these disorders. However, successfully isolating specific in vivo complexes of ARX, with DNA and proteins, has remained as a challenge. To facilitate in vivo detection of ARX complexes, we generated a mouse line containing one epitope of FLAG-tag (1 × FLAG) targeted at the translational start site of the endogenous Arx gene using CRSPR/Cas9 strategy. Homozygous Flag-Arx mice are viable and fertile without gross abnormality, suggesting that the FLAG-tag does not perturb the normal function of ARX. Using a FLAG antibody, we successfully detected ARX with immunofluorescent staining and pulled down ARX in embryonic brain tissues. This Flag-Arx mouse line will be a useful tool to isolate ARX complexes from mouse tissues for many applications.

Piezo1-Regulated Mechanotransduction Controls Flow-Activated Lymphatic Expansion.

BACKGROUND: Mutations in PIEZO1 (Piezo type mechanosensitive ion channel component 1) cause human lymphatic malformations. We have previously uncovered an ORAI1 (ORAI calcium release-activated calcium modulator 1)-mediated mechanotransduction pathway that triggers lymphatic sprouting through Notch downregulation in response to fluid flow. However, the identity of its upstream mechanosensor remains unknown. This study aimed to identify and characterize the molecular sensor that translates the flow-mediated external signal to the Orai1-regulated lymphatic expansion. METHODS: Various mutant mouse models, cellular, biochemical, and molecular biology tools, and a mouse tail lymphedema model were employed to elucidate the role of Piezo1 in flow-induced lymphatic growth and regeneration. RESULTS: Piezo1 was found to be abundantly expressed in lymphatic endothelial cells. Piezo1 knockdown in cultured lymphatic endothelial cells inhibited the laminar flow-induced calcium influx and abrogated the flow-mediated regulation of the Orai1 downstream genes, such as KLF2 (Krüppel-like factor 2), DTX1 (Deltex E3 ubiquitin ligase 1), DTX3L (Deltex E3 ubiquitin ligase 3L,) and NOTCH1 (Notch receptor 1), which are involved in lymphatic sprouting. Conversely, stimulation of Piezo1 activated the Orai1-regulated mechanotransduction in the absence of fluid flow. Piezo1-mediated mechanotransduction was significantly blocked by Orai1 inhibition, establishing the epistatic relationship between Piezo1 and Orai1. Lymphatic-specific conditional Piezo1 knockout largely phenocopied sprouting defects shown in Orai1- or Klf2- knockout lymphatics during embryo development. Postnatal deletion of Piezo1 induced lymphatic regression in adults. Ectopic Dtx3L expression rescued the lymphatic defects caused by Piezo1 knockout, affirming that the Piezo1 promotes lymphatic sprouting through Notch downregulation. Consistently, transgenic Piezo1 expression or pharmacological Piezo1 activation enhanced lymphatic sprouting. Finally, we assessed a potential therapeutic value of Piezo1 activation in lymphatic regeneration and found that a Piezo1 agonist, Yoda1, effectively suppressed postsurgical lymphedema development. CONCLUSIONS: Piezo1 is an upstream mechanosensor for the lymphatic mechanotransduction pathway and regulates lymphatic growth in response to external physical stimuli. Piezo1 activation presents a novel therapeutic opportunity for preventing postsurgical lymphedema. The Piezo1-regulated lymphangiogenesis mechanism offers a molecular basis for Piezo1-associated lymphatic malformation in humans.

ß2-adrenergic receptor regulates ER-mitochondria contacts.

Interactions between the endoplasmic reticulum (ER) and mitochondria (Mito) are crucial for many cellular functions, and their interaction levels change dynamically depending on the cellular environment. Little is known about how the interactions between these organelles are regulated within the cell. Here we screened a compound library to identify chemical modulators for ER-Mito contacts in HEK293T cells. Multiple agonists of G-protein coupled receptors (GPCRs), beta-adrenergic receptors (ß-ARs) in particular, scored in this screen. Analyses in multiple orthogonal assays validated that ß2-AR activation promotes physical and functional interactions between the two organelles. Furthermore, we have elucidated potential downstream effectors mediating ß2-AR-induced ER-Mito contacts. Together our study identifies ß2-AR signaling as an important regulatory pathway for ER-Mito coupling and highlights the role of these contacts in responding to physiological demands or stresses.

Organogenesis and distribution of the ocular lymphatic vessels in the anterior eye.

Glaucoma surgeries, such as trabeculectomy, are performed to lower intraocular pressure to reduce risk of vision loss. These surgeries create a new passage in the eye that reroutes the aqueous humor outflow to the subconjunctival space, where the fluid is presumably absorbed by the conjunctival lymphatics. Here, we characterized the development and function of the ocular lymphatics using transgenic lymphatic reporter mice and rats. We found that the limbal and conjunctival lymphatic networks are progressively formed from a primary lymphatic vessel that grows from the nasal-side medial canthus region at birth. This primary lymphatic vessel immediately branches out, invades the limbus and conjunctiva, and bidirectionally encircles the cornea. As a result, the distribution of the ocular lymphatics is significantly polarized toward the nasal side, and the limbal lymphatics are directly connected to the conjunctival lymphatics. New lymphatic sprouts are produced mainly from the nasal-side limbal lymphatics, posing the nasal side of the eye as more responsive to fluid drainage and inflammatory stimuli. Consistent with this polarized distribution of the ocular lymphatics, a higher drainage efficiency was observed in the nasal side than the temporal side of the eye when injected with a fluorescent tracer. In contrast, blood vessels are evenly distributed at the anterior surface of the eyes. Also, we found that these distinct vascular distribution patterns were conserved in human eyes. Together, our study demonstrated that the ocular surface lymphatics are more densely present in the nasal side and uncovered the potential clinical benefits in selecting the nasal side as a glaucoma surgery site to improve fluid drainage.

The Lymphatic Cell Environment Promotes Kaposi Sarcoma Development by Prox1-Enhanced Productive Lytic Replication of Kaposi Sarcoma Herpes Virus.

Kaposi sarcoma is the most common cancer in human immunodeficiency virus-positive individuals and is caused by Kaposi sarcoma-associated herpesvirus (KSHV). It is believed that a small number of latently infected Kaposi sarcoma tumor cells undergo spontaneous lytic reactivation to produce viral progeny for infection of new cells. Here, we use matched donor-derived human dermal blood and lymphatic endothelial cells (BEC and LEC, respectively) to show that KSHV-infected BECs progressively lose viral genome as they proliferate. In sharp contrast, KSHV-infected LECs predominantly entered lytic replication, underwent cell lysis, and released new virus. Continuous lytic cell lysis and de novo infection allowed LEC culture to remain infected for a prolonged time. Because of the strong propensity of LECs toward lytic replication, LECs maintained virus as a population, despite the death of individual host cells from lytic lysis. The master regulator of lymphatic development, Prox1, bound the promoter of the RTA gene to upregulate its expression and physically interacted with RTA protein to coregulate lytic genes. Thus, LECs may serve as a proficient viral reservoir that provides viral progeny for continuous de novo infection of tumor origin cells, and potentially BECs and mesenchymal stem cells, which give rise to Kaposi sarcoma tumors. Our study reveals drastically different host cell behaviors between BEC and LEC and defines the underlying mechanisms of the lymphatic cell environment supporting persistent infection in Kaposi sarcoma tumors. SIGNIFICANCE: This study defines the mechanism by which Kaposi's sarcoma could be maintained by virus constantly produced by lymphatic cells in HIV-positive individuals.

Piezo1 incorporates mechanical force signals into the genetic program that governs lymphatic valve development and maintenance.

The lymphatic system plays crucial roles in tissue homeostasis, lipid absorption, and immune cell trafficking. Although lymphatic valves ensure unidirectional lymph flows, the flow itself controls lymphatic valve formation. Here, we demonstrate that a mechanically activated ion channel Piezo1 senses oscillating shear stress (OSS) and incorporates the signal into the genetic program controlling lymphatic valve development and maintenance. Time-controlled deletion of Piezo1 using a pan-endothelial Cre driver (Cdh5[PAC]-CreERT2) or lymphatic-specific Cre driver (Prox1-CreERT2) equally inhibited lymphatic valve formation in newborn mice. Furthermore, Piezo1 deletion in adult lymphatics caused substantial lymphatic valve degeneration. Piezo1 knockdown in cultured lymphatic endothelial cells (LECs) largely abrogated the OSS-induced upregulation of the lymphatic valve signature genes. Conversely, ectopic Piezo1 overexpression upregulated the lymphatic valve genes in the absence of OSS. Remarkably, activation of Piezo1 using chemical agonist Yoda1 not only accelerated lymphatic valve formation in animals, but also triggered upregulation of some lymphatic valve genes in cultured LECs without exposure to OSS. In summary, our studies together demonstrate that Piezo1 is the force sensor in the mechanotransduction pathway controlling lymphatic valve development and maintenance, and Piezo1 activation is a potentially novel therapeutic strategy for congenital and surgery-associated lymphedema.

Arx Expression Suppresses Ventralization of the Developing Dorsal Forebrain.

Early brain development requires a tight orchestration between neural tube patterning and growth. How pattern formation and brain growth are coordinated is incompletely understood. Previously we showed that aristaless-related homeobox (ARX), a paired-like transcription factor, regulates cortical progenitor pool expansion by repressing an inhibitor of cell cycle progression. Here we show that ARX participates in establishing dorsoventral identity in the mouse forebrain. In Arx mutant mice, ventral genes, including Olig2, are ectopically expressed dorsally. Furthermore, Gli1 is upregulated, suggesting an ectopic activation of SHH signaling. We show that the ectopic Olig2 expression can be repressed by blocking SHH signaling, implicating a role for SHH signaling in Olig2 induction. We further demonstrate that the ectopic Olig2 accounts for the reduced Pax6 and Tbr2 expression, both dorsal specific genes essential for cortical progenitor cell proliferation. These data suggest a link between the control of dorsoventral identity of progenitor cells and the control of their proliferation. In summary, our data demonstrate that ARX functions in a gene regulatory network integrating normal forebrain patterning and growth, providing important insight into how mutations in ARX can disrupt multiple aspects of brain development and thus generate a wide spectrum of neurodevelopmental phenotypes observed in human patients.

Ascorbate peroxidase proximity labeling coupled with biochemical fractionation identifies promoters of endoplasmic reticulum-mitochondrial contacts.

To maintain cellular homeostasis, subcellular organelles communicate with each other and form physical and functional networks through membrane contact sites coupled by protein tethers. In particular, endoplasmic reticulum (ER)-mitochondrial contacts (EMC) regulate diverse cellular activities such as metabolite exchange (Ca2+ and lipids), intracellular signaling, apoptosis, and autophagy. The significance of EMCs has been highlighted by reports indicating that EMC dysregulation is linked to neurodegenerative diseases. Therefore, obtaining a better understanding of the physical and functional components of EMCs should provide new insights into the pathogenesis of several neurodegenerative diseases. Here, we applied engineered ascorbate peroxidase (APEX) to map the proteome at EMCs in live HEK293 cells. APEX was targeted to the outer mitochondrial membrane, and proximity-labeled proteins were analyzed by stable isotope labeling with amino acids in culture (SILAC)-LC/MS-MS. We further refined the specificity of the proteins identified by combining biochemical subcellular fractionation to the protein isolation method. We identified 405 proteins with a 2.0-fold cutoff ratio (log base 2) in SILAC quantification from replicate experiments. We performed validation screening with a Split-Rluc8 complementation assay that identified reticulon 1A (RTN1A), an ER-shaping protein localized to EMCs as an EMC promoter. Proximity mapping augmented with biochemical fractionation and additional validation methods reported here could be useful to discover other components of EMCs, identify mitochondrial contacts with other organelles, and further unravel their communication.

Aristaless Related Homeobox (ARX) Interacts with ß-Catenin, BCL9, and P300 to Regulate Canonical Wnt Signaling.

Mutations in the Aristaless Related Homeobox (ARX) gene are associated with a spectrum of structural (lissencephaly) and functional (epilepsy and intellectual disabilities) neurodevelopmental disorders. How mutations in this single transcription factor can result in such a broad range of phenotypes remains poorly understood. We hypothesized that ARX functions through distinct interactions with specific transcription factors/cofactors to regulate unique target genes in different cell types. To identify ARX interacting proteins, we performed an unbiased proteomics screen and identified several components of the Wnt/ß-catenin signaling pathway, including ß-catenin (CTNNB1), B-cell CLL/lymphoma 9 (BCL9) and leucine rich repeat flightless interacting protein 2 (LRRFIP2), in cortical progenitor cells. Our data show that ARX positively regulates Wnt/ ß-catenin signaling and that the C-terminal domain of ARX interacts with the armadillo repeats in ß-catenin to promote Wnt/ß-catenin signaling. In addition, we found BCL9 and P300 also interact with ARX to modulate Wnt/ß-catenin signaling. These data provide new insights into how ARX can uniquely regulate cortical neurogenesis, and connect the function of ARX with Wnt/ß-catenin signaling.

Hereditary spastic paraplegia-linked REEP1 modulates endoplasmic reticulum/mitochondria contacts.

OBJECTIVE: Mutations in receptor expression enhancing protein 1 (REEP1) are associated with hereditary spastic paraplegias (HSPs). Although axonal degeneration is thought to be a predominant feature in HSP, the role of REEP1 mutations in degeneration is largely unknown. Previous studies have implicated a role for REEP1 in the endoplasmic reticulum (ER), whereas others localized REEP1 with mitochondria. We sought to resolve the cellular localization of REEP1 and further elucidate the pathobiology underlying REEP1 mutations in patients. METHODS: A combination of cellular imaging and biochemical approaches was used to refine the cellular localization of REEP1. Next, Reep1 mutations associated with HSP were functionally tested in neuritic growth and degeneration assays using mouse cortical culture. Finally, a novel assay was developed and used with wild-type and mutant Reep1s to measure the interactions between the ER and mitochondria. RESULTS: We found that REEP1 is present at the ER-mitochondria interface, and it contains subdomains for mitochondrial as well as ER localization. Knockdown of Reep1 and expression of pathological Reep1 mutations resulted in neuritic growth defects and degeneration. Finally, using our novel split-RLuc8 assay, we show that REEP1 facilitates ER-mitochondria interactions, a function diminished by disease-associated mutations. INTERPRETATION: Our data potentially reconcile the current conflicting reports regarding REEP1 being either an ER or a mitochondrial protein. Furthermore, our results connect, for the first time, the disrupted ER-mitochondria interactions to a failure in maintaining health of long axons in HSPs. Finally, the split-RLuc8 assay offers a new tool to identify potential drugs for multiple neurodegenerative diseases with ER-mitochondria interaction defects.

Arx together with FoxA2, regulates Shh floor plate expression.

Mutations in the Aristaless related homeodomain transcription factor (ARX) are associated with a diverse set of X-linked mental retardation and epilepsy syndromes in humans. Although most studies have been focused on its function in the forebrain, ARX is also expressed in other regions of the developing nervous system including the floor plate (FP) of the spinal cord where its function is incompletely understood. To investigate the role of Arx in the FP, we performed gain-of-function studies in the chick using in ovo electroporation, and loss-of-function studies in Arx-deficient mice. We have found that Arx, in conjunction with FoxA2, directly induces Sonic hedgehog (Shh) expression through binding to a Shh floor plate enhancer (SFPE2). We also observed that FoxA2 induces Arx through its transcriptional activation domain whereas Nkx2.2, induced by Shh, abolishes this induction. Our data support a feedback loop model for Arx function; through interactions with FoxA2, Arx positively regulates Shh expression in the FP, and Shh signaling in turn activates Nkx2.2, which suppresses Arx expression. Furthermore, our data are evidence that Arx plays a role as a context dependent transcriptional activator, rather than a primary inducer of Shh expression, potentially explaining how mutations in ARX are associated with diverse, and often subtle, defects.

Tmem64 modulates calcium signaling during RANKL-mediated osteoclast differentiation.

Osteoclast maturation and function primarily depend on receptor activator of NF-κB ligand (RANKL)-mediated induction of nuclear factor of activated T cells c1 (NFATc1), which is further activated via increased intracellular calcium ([Ca(2+)](i)) oscillation. However, the coordination mechanism that mediates Ca(2+) oscillation during osteoclastogenesis remains ill defined. Here, we identified transmembrane protein 64 (Tmem64) as a regulator of Ca(2+) oscillation during osteoclastogenesis. We found that Tmem64-deficient mice exhibit increased bone mass due in part to impaired osteoclast formation. Using in vitro osteoclast culture systems, we show here that Tmem64 interacts with sarcoplasmic endoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) and modulates its activity. Consequently, Tmem64 deficiency significantly diminishes RANKL-induced [Ca(2+)](i) oscillation, which results in reduced Ca(2+)/calmodulin-dependent protein kinases (CaMK) IV and mitochondrial ROS, both of which contribute to achieving the CREB activity necessary for osteoclast formation. These data demonstrate that Tmem64 is a positive modulator of osteoclast differentiation via SERCA2-dependent Ca(2+) signaling.

Analysis of subunit assembly and function of the Saccharomyces cerevisiae RNase H2 complex.

RNase H2 of Saccharomyces cerevisiae consists of three essential subunits (Rnh201, Rnh202 and Rnh203) and plays a critical role in the removal of RNA incorporated in duplex DNA. In the present study, we purified individual subunits and heterodimeric subcomplexes to examine the assembly and biochemical function of subunits of RNase H2 in vitro. Reconstitution experiments revealed that Rnh202 and Rnh203 first form a subcomplex, followed by the recruitment of Rnh201 to complete complex formation. Rnh201 alone or in combination with Rnh203 showed neither substrate-binding, nor catalytic activity, indicating that both activities of Rnh201 are latent until it becomes an integral part of the complex. However, Rnh202 by itself showed substrate-binding activity. RNase H2 containing mutant Rnh202 defective in substrate binding had decreased substrate-binding activity, indicating that Rnh202 contributes directly to substrate binding. Reconstitution of RNase H2 complexes with various mutant subunits allowed us to assess the influence of conserved amino acid residues in either Rnh201 or Rnh202 on substrate-binding and catalytic activities. We found that the substrate-binding activities of both Rnh201 and Rnh202 were critical for cleavage of the phosphodiester bond present between DNA and RNA in RNase H2 substrates.

Genetic and functional interactions between Mus81-Mms4 and Rad27.

The two endonucleases, Rad27 (yeast Fen1) and Dna2, jointly participate in the processing of Okazaki fragments in yeasts. Mus81-Mms4 is a structure-specific endonuclease that can resolve stalled replication forks as well as toxic recombination intermediates. In this study, we show that Mus81-Mms4 can suppress dna2 mutational defects by virtue of its functional and physical interaction with Rad27. Mus81-Mms4 stimulated Rad27 activity significantly, accounting for its ability to restore the growth defects caused by the dna2 mutation. Interestingly, Rad27 stimulated the rate of Mus81-Mms4 catalyzed cleavage of various substrates, including regressed replication fork substrates. The ability of Rad27 to stimulate Mus81-Mms4 did not depend on the catalytic activity of Rad27, but required the C-terminal 64 amino acid fragment of Rad27. This indicates that the stimulation was mediated by a specific protein-protein interaction between the two proteins. Our in vitro data indicate that Mus81-Mms4 and Rad27 act together during DNA replication and resolve various structures that can impede normal DNA replication. This conclusion was further strengthened by the fact that rad27 mus81 or rad27 mms4 double mutants were synergistically lethal. We discuss the significance of the interactions between Rad27, Dna2 and Mus81-Mms4 in context of DNA replication.

The MPH1 gene of Saccharomyces cerevisiae functions in Okazaki fragment processing.

Saccharomyces cerevisiae MPH1 was first identified as a gene encoding a 3' to 5' DNA helicase, which when deleted leads to a mutator phenotype. In this study, we isolated MPH1 as a multicopy suppressor of the dna2K1080E helicase-negative lethal mutant. Purified Mph1 stimulated the endonuclease activities of both Fen1 and Dna2, which act faithfully in the processing of Okazaki fragments. This stimulation required neither ATP hydrolysis nor the helicase activity of Mph1. Multicopy expression of MPH1 also suppressed the temperature-sensitive growth defects in cells expressing dna2Delta405N, which lacks the N-terminal 405 amino acids of Dna2. However, Mph1 did not stimulate the endonuclease activity of the Dna2Delta405N mutant protein. The stimulation of Fen1 by Mph1 was limited to flap-structured substrates; Mph1 hardly stimulated the 5' to 3' exonuclease activity of Fen1. Mph1 binds to flap-structured substrate more efficiently than to nicked duplex structures, suggesting that the stimulatory effect of Mph1 is exerted through its binding to DNA substrates. In addition, we found that Mph1 reversed the inhibitory effects of replication protein A on Fen1 activity. Our biochemical and genetic data indicate that the in vivo suppression of Dna2 defects observed with both dna2K1080E and dna2Delta405N mutants occur via stimulation of Fen1 activity. These findings suggest that Mph1 plays an important, although not essential, role in processing of Okazaki fragments by facilitating the formation of ligatable nicks.

Human replication factor C stimulates flap endonuclease 1.

Flap endonuclease 1 (FEN1) is the enzyme responsible for specifically removing the flap structure produced during DNA replication, repair, and recombination. Here we report that the human replication factor C (RFC) complex stimulates the nuclease activity of human FEN1 in an ATP-independent manner. Although proliferating cell nuclear antigen is also known to stimulate FEN1, less RFC was required for comparable FEN1 stimulation. Kinetic analyses indicate that the mechanism by which RFC stimulates FEN1 is distinct from that by proliferating cell nuclear antigen. Heat-denatured RFC or its subunit retained, fully or partially, the ability to stimulate FEN1. Via systematic deletion analyses, we have defined three specific regions of RFC4 capable of stimulating FEN1. The region of RFC4 with the highest activity spans amino acids 170-194 and contains RFC box VII. Four amino acid residues (i.e. Tyr-182, Glu-188, Pro-189, and Ser-192) are especially important for FEN1 stimulatory activity. Thus, RFC, via several stimulatory motifs per molecule, potently activates FEN1. This function makes RFC a critical partner with FEN1 for the processing of eukaryotic Okazaki fragments.

S1219 residue of 53BP1 is phosphorylated by ATM kinase upon DNA damage and required for proper execution of DNA damage response.

53BP1 is phosphorylated by the protein kinase ATM upon DNA damage. Even though several ATM phosphorylation sites in 53BP1 have been reported, those sites have little functional implications in the DNA damage response. Here, we show that ATM phosphorylates the S1219 residue of 53BP1 in vitro and that the residue is phosphorylated in cells exposed to ionizing radiation (IR). Transfection with siRNA targeting ATM abolished IR-induced phosphorylation at this residue, supporting the theory that this process is mediated by the kinase. To determine the functional relevance of this phosphorylation event, a U2OS cell line expressing S1219A mutant 53BP1 was established. IR-induced foci formation of MDC1 and gammaH2AX, DNA damage signaling molecules, was reduced in this cell line, implying that S1219 phosphorylation is required for recruitment of these molecules to DNA damage sites. Furthermore, overexpression of the mutant protein impeded IR-induced G2 arrest. In conclusion, we have shown that S1219 phosphorylation by ATM is required for proper execution of DNA damage response.

Effects of lactate dehydrogenase suppression and glycerol-3-phosphate dehydrogenase overexpression on cellular metabolism.

In order to conduct a physiological functional study of lactate dehydrogenase (LDH) and glycerol-3-phosphate dehydrogenase (GPDH), we engineered a CHO dhfr(-) cell, by overexpressing either the anti-sense LDH-A RNA (anti-LDH cells) or GPDH (GP3 cells), or both (GP3/anti-LDH cells). LDH activity in the cell cytosol, and lactate content and pHe change in the growth media were found to decrease according to the order: cell lines GP3/anti-LDH > anti-LDH > GP3 > CHO. Intracellular ATP contents, representing the extent of respiration rate, also decreased, according to a rank order as follows: GP3 > CHO > GP3/anti-LDH > anti-LDH. We also attempted to identify and characterize any physiological changes occurring in the cells which harbored diverse metabolic pathways. First, anti-LDH cells with heightened respiration rates were found to display a higher degree of sensitivity to the prooxidant tert-butyl hydroperoxide (tBOOH), and the mitochondrial complex III inhibitor, antimycin A, than the GPDH-expressing cells (GP3 and GP3/anti-LDH), which have a lower respiration rate. Second, the anti-sense LDH-A RNA-expressing cells (anti-LDH and GP3/anti-LDH) evidenced a higher degree of resistance to apoptosis by cell-cell contact inhibition, and a faster doubling time ( approximately 19 h compared with approximately 26 h) than the CHO and GP3 cells. Additionally, cell growth in an extended culture under HCO(3) (-)-free conditions to induce a steep acidification could be maintained with the anti-sense LDH-A RNA-expressing cells, but could not be maintained with the CHO and GP3 cells. Third, we observed that the most appropriate cell line for the optical production of a certain therapeutic protein (Tissue-Plasminogen Activator) was the GP3/anti-LDH cells. Collectively, our data indicate a variety of physiological roles for LDH and GPDH, including cellular acidosis, oxidoresistance, apoptosis by both acidosis and cell-cell contact inhibition, cell growth, and the generation of recombinant proteins.

Modification of glycolysis affects cell sensitivity to apoptosis induced by oxidative stress and mediated by mitochondria.

The effect of alteration of the glycolytic pathway on cell damage induced by oxidative stress was investigated with dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cells that either overexpress cytosolic glycerol-3-phosphate dehydrogenase (CHO/cGPDH cells) or are depleted of the A subunit of lactate dehydrogenase as a result of anti-sense RNA expression (CHO/anti-LDH cells). The extent of oxidative phosphorylation in CHO/anti-LDH and CHO/cGPDH cells was increased and decreased, respectively, relative to that in parental CHO cells, as revealed by measurement of the intracellular content of ATP, the rate of cellular O(2) consumption, the mitochondrial membrane potential (DeltaPsi(m)), and the generation of reactive oxygen species. The sensitivity of these cell lines to cell death induced by the exogenous oxidant tert-butyl hydroperoxide decreased according to the rank order CHO/anti-LDH>CHO>CHO/cGPDH. Exogenous pyruvate markedly increased the sensitivity of CHO/cGPDH cells to oxidant-induced death. The differences among the three cell lines in susceptibility to oxidant-induced death were reflected in the proportion of oxidant-treated cells with a subdiploid DNA content, with a collapsed DeltaPsi(m), and with cytochrome c in the cytosol, indicating that death was mediated by apoptosis. These results demonstrate that the influx of respiratory substrate into mitochondria is an important determinant of cell sensitivity to oxidant-induced apoptosis.