Strain-induced mechanotransduction through primary cilia, extracellular ATP, purinergic calcium signaling, and ERK1/2 transactivates CITED2 and downregulates MMP-1 and MMP-13 gene expression in chondrocytes.

OBJECTIVE: To determine the strain-induced signaling pathways involved in regulating the transactivation of the transcription regulator Cbp/p300 Interacting Transactivator with ED-rich tail 2 (CITED2) and downstream targets in chondrocytes. METHODS: Primary human chondrocytes or C28/I2 chondrocytic cells were subjected to various strain regimes. C57BL/6 mice were subjected to treadmill running. Loss-of-function was carried out using siRNA or inhibitors specific for targeted molecules. mRNA levels were assayed by RT-qPCR, and proteins by western blotting, immunofluorescence, and/or immunohistochemical staining. CITED2 promoter activity was assayed in chondrocytes using wild-type or mutant constructs. RESULTS: Cyclic strain at 5%, 1 Hz induced CITED2 expression and suppressed expression of matrix metalloproteinase (MMP)-1 and -13 at the messenger RNA (mRNA) and protein levels in human chondrocytes. Abolishing primary cilia through knockdown of intraflagellar transport protein (IFT88) attenuated CITED2 gene expression and decreased protein levels. Similar effects were observed with inhibitors of extracellular adenosine triphosphate (ATP) or P2 purinergic receptors, or antagonists of Ca(2+) signaling. Knockdown of IFT88 in articular chondrocytes in vivo diminished treadmill induced-CITED2 expression and upregulated MMPs. Knockdown of hypoxia-inducible factor (HIF)1α, specificity protein 1 (Sp1), or deletion of the shear stress response element (SSRE) in the CITED2 promoter limited cyclic strain-induced transactivation of CITED2. However, the strain induced-transactivation of CITED2 was abolished only on knockdown of HIF1α, Sp1, and SSRE or by loss-of-function of IFT88 or extracellular-signal-regulated kinases (ERK)1/2. CONCLUSIONS: CITED2 transactivation is a critical event in signaling generated by strain and transduced by primary cilia, extracellular ATP, P2 purinergic receptors, and Ca(2+) signaling. Strain-induced CITED2 transactivation requires HIF1α, Sp1, and an intact SSRE and leads to the downregulation of MMPs such as MMP-1 and MMP-13.

Transcriptome profiling of hippocampal CA1 after early-life seizure-induced preconditioning may elucidate new genetic therapies for epilepsy.

Injury of the CA1 subregion induced by a single injection of kainic acid (1 × KA) in juvenile animals (P20) is attenuated in animals with two prior sustained neonatal seizures on P6 and P9. To identify gene candidates involved in the spatially protective effects produced by early-life conditioning seizures we profiled and compared the transcriptomes of CA1 subregions from control, 1 × KA- and 3 × KA-treated animals. More genes were regulated following 3 × KA (9.6%) than after 1 × KA (7.1%). Following 1 × KA, genes supporting oxidative stress, growth, development, inflammation and neurotransmission were upregulated (e.g. Cacng1, Nadsyn1, Kcng1, Aven, S100a4, GFAP, Vim, Hrsp12 and Grik1). After 3 × KA, protective genes were differentially over-expressed [e.g. Cat, Gpx7, Gad1, Hspa12A, Foxn1, adenosine A1 receptor, Ca(2+) adaptor and homeostasis proteins, Cacnb4, Atp2b2, anti-apoptotic Bcl-2 gene members, intracellular trafficking protein, Grasp and suppressor of cytokine signaling (Socs3)]. Distinct anti-inflammatory interleukins (ILs) not observed in adult tissues [e.g. IL-6 transducer, IL-23 and IL-33 or their receptors (IL-F2 )] were also over-expressed. Several transcripts were validated by real-time polymerase chain reaction (QPCR) and immunohistochemistry. QPCR showed that casp 6 was increased after 1 × KA but reduced after 3 × KA; the pro-inflammatory gene Cox1 was either upregulated or unchanged after 1 × KA but reduced by ~70% after 3 × KA. Enhanced GFAP immunostaining following 1 × KA was selectively attenuated in the CA1 subregion after 3 × KA. The observed differential transcriptional responses may contribute to early-life seizure-induced pre-conditioning and neuroprotection by reducing glutamate receptor-mediated Ca(2+) permeability of the hippocampus and redirecting inflammatory and apoptotic pathways. These changes could lead to new genetic therapies for epilepsy.

Sex-dependent gene regulatory networks of the heart rhythm.

Expression level, control, and intercoordination of 66 selected heart rhythm determinant (HRD) genes were compared in atria and ventricles of four male and four female adult mice. We found that genes encoding various adrenergic receptors, ankyrins, ion channels and transporters, connexins, cadherins, plakophilins, and other components of the intercalated discs form a complex network that is chamber dependent and differs between the two sexes. In addition, most HRD genes in atria had higher expression in males than in females, while in ventricles, expression levels were mostly higher in females than in males. Moreover, significant chamber differences were observed between the sexes, with higher expression in atria than ventricles for males and higher expression in ventricles than atria for females. We have ranked the selected genes according to their prominence (new concept) within the HRD gene web defined as extent of expression coordination with the other web genes and stability of expression. Interestingly, the prominence hierarchy was substantially different between the two sexes. Taken together, these findings indicate that the organizational principles of the heart rhythm transcriptome are sex dependent, with the newly introduced prominence analysis allowing identification of genes that are pivotal for the sexual dichotomy.

Molecular characterization and functional expression of the human cardiac gap junction channel.

Gap junctions permit the passage of ions and chemical mediators from cell to cell. To identify the molecular genetic basis for this coupling in the human heart, we have isolated clones from a human fetal cardiac cDNA library which encode the full-length human cardiac gap junction (HCGJ) mRNA. The predicted amino acid sequence is homologous to the rat cardiac gap junction protein, connexin43 (Beyer, E. D., D. Paul, and D. A. Goodenough. 1987. J. Cell Biol. 105:2621-2629), differing by 9 of 382 amino acids. HCGJ mRNA is detected as early as fetal week 15 and persists in adult human cardiac samples. Genomic DNA analysis suggests the presence of two highly homologous HCGJ loci, only one of which is functional. Stable transfection of the HCGJ cDNA into SKHep1 cells, a human hepatoma line which is communication deficient, leads to the formation of functional channels. Junctional conductance in pairs of transfectants containing 10 copies of the HCGJ sequence is high (approximately 20 nS). Single channel currents are detectable in this expression system and correspond to conductances of approximately 60 pS. These first measurements of the HCGJ channel are similar to the junctional conductance recorded between pairs of rat or guinea pig cardiocytes.

Electrophysiological properties of gap junctions between dissociated pairs of rat hepatocytes.

Physiological properties of isolated pairs of rat hepatocytes were examined within 5 h after dissociation. These cells become round when separated, but cell pairs still display membrane specializations. Most notably, canaliculi are often present at appositional membranes which are flanked by abundant gap and tight junctions. These cell pairs are strongly dye-coupled; Lucifer Yellow CH injected into one cell rapidly diffuses to the other. Pairs of hepatocytes are closely coupled electrically. Conductance of the junctional membrane is not voltage sensitive: voltage clamp studies demonstrate that gj is constant in response to long (5 s) transjunctional voltage steps of either polarity (to greater than +/- 40 mV from rest). Junctional conductance (gj) between hepatocyte pairs is reduced by exposure to octanol (0.1 mM) and by intracellular acidification. Normal intracellular pH (pHi), measured with a liquid ion exchange microelectrode, was generally 7.1-7.4, and superfusion with saline equilibrated with 100% CO2 reduced pHi to 6.0-6.5. In the pHi range 7.5-6.6, gj was constant. Below pH 6.6, gj steeply decreased and at 6.1 coupling was undetectable. pHi recovered when cells were rinsed with normal saline; in most cases gj recovered in parallel so that gj values were similar for pHs obtained during acidification or recovery. The low apparent pK and very steep pHi-gj relation of the liver gap junction contrast with higher pKs and more gradually rising curves in other tissues. If H+ ions act directly on the junctional molecules, the channels that are presumably homologous in different tissues must differ with respect to reactive sites or their environment.

Junctional communication is induced in migrating capillary endothelial cells.

Using an in vitro model in which a confluent monolayer of capillary endothelial cells is mechanically wounded, gap junction-mediated intercellular communication has been studied by loading the cells with the fluorescent dye, Lucifer Yellow. Approximately 40-50% of the cells in a nonwounded confluent monolayer were coupled in groups of four to five cells (basal level). Basal levels of communication were also observed in sparse and preconfluent cultures, but were reduced in postconfluent monolayers. 30 min after wounding, coupling was markedly reduced between cells lining the wound. Communication at the wound was partially reestablished by 2 h, exceeded basal levels after 6 h and reached a maximum after 24 h, at which stage approximately 90% of the cells were coupled in groups of six to seven cells. When the wound had closed (after 8 d), the increase in communication was no longer observed. Induction of wound-associated communication was unaffected by exposure of the cells to the DNA synthesis inhibitor mitomycin C, but was prevented by the protein synthesis inhibitor, cycloheximide. The induction of wound-associated communication was also inhibited when migration was prevented by placing the cells immediately after wounding at 22 degrees C or after exposure to cytochalasin D, suggesting that the increase in communication is dependent on cells migrating into the wound area. In contrast, migration was not prevented when coupling was blocked by exposure of the cells to retinoic acid, although this agent did disrupt the characteristic sheet-like pattern of migration typically seen during endothelial repair. These results suggest that junctional communication may play an important role in wound repair, possibly by coordinating capillary endothelial cell migration.

Proteoglycans and glycosaminoglycans induce gap junction synthesis and function in primary liver cultures.

Intercellular communication via gap junctions, as measured by dye and electrical coupling, disappears within 12 h in primary rat hepatocytes cultured in serum-supplemented media or within 24 h in cells in a serum-free, hormonally defined medium (HDM) designed for hepatocytes. Glucagon and linoleic acid/BSA were the primary factors in the HDM responsible for the extended life span of the electrical coupling. After 24 h of culture, no hormone or growth factor tested could restore the expression of gap junctions. After 4-5 d of culture, the incidence of coupling was undetectable in a serum-supplemented medium and was only 4-5% in HDM alone. However, treatment with glycosaminoglycans or proteoglycans of 24-h cultures, having no detectable gap junction protein, resulted in synthesis of gap junction protein and of reexpression of electrical and dye coupling within 48 h. Most glycosaminoglycans were inactive (heparan sulfates, chondroitin-6 sulfates) or only weakly active (dermatan sulfates, chondroitin 4-sulfates, hyaluronates), the weakly active group increasing the incidence of coupling to 10-30% with the addition of 50-100 micrograms/ml of the factor. Treatment of the cells with 50-100 micrograms/ml of heparins derived from lung or intestine resulted in cells with intermediate levels of coupling (30-50%). By contrast, 10-20 micrograms/ml of chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, or liver-derived heparin resulted in dye coupling in 80-100% of the cells, with numerous cells showing dye spread from a single injected cell. Sulfated polysaccharides of glucose (dextran sulfates) or of galactose (carrageenans) were inactive or only weakly active except for lambda-carrageenan, which induced up to 70% coupling (albeit no multiple coupling in the cultures). The abundance of mRNA (Northern blots) encoding gap junction protein and the amounts of the 27-kD gap junction polypeptide (Western blots) correlated with the degree of electrical and dye coupling indicating that the active glycosaminoglycans and proteoglycans are inducing synthesis and expression of gap junctions. Thus, proteoglycans and glycosaminoglycans, especially those found in abundance in the extracellular matrix of liver cells, are important in the regulation of expression of gap junctions and, thereby, in the regulation of intercellular communication in the liver. The relative potencies of heparins from different tissue sources at inducing gap junction expression are suggestive of functional tissue specificity for these glycosaminoglycans.

Substituted benzyl acetates: a new class of compounds that reduce gap junctional conductance by cytoplasmic acidification.

Conductance of gap junctions in many preparations has been shown to be sensitive to cytoplasmic pH, decreasing as pH decreases below 7.5 in fish and amphibian embryos and below 7.1 in crayfish septate axon. We have found a new class of compounds, benzyl acetate derivatives, that reversibly decrease junctional conductance, gj, when applied in low concentration (approximately 1 mM). Simultaneous intracellular pH (pHi) measurements show that the ester effects are attributable to reduction in pHi. The sensitivity of gj to these compounds and the relative lack of side effects make these agents attractive for studies of the role played by gap junctions in normal tissue function. In addition, the finding of cytoplasmic acidification in response to cell exposure to esters suggests caution in interpretation of results obtained using esterified compounds for intracellular loading.

Carbon tetrachloride at hepatotoxic levels blocks reversibly gap junctions between rat hepatocytes.

Electrical coupling and dye coupling between pairs of rat hepatocytes were reversibly reduced by brief exposure to halogenated methanes (CBrCl3, CCl4, and CHCl3). The potency of different halomethanes in uncoupling hepatocytes was comparable to their hepatotoxicity in vivo, and the rank order was the same as that of their tendency to form free radicals. The effect of carbon tetrachloride (CCl4) on hepatocytes was substantially reduced by prior treatment with SKF 525A, an inhibitor of cytochrome P-450, and by exposure to the reducing reagent beta-mercaptoethanol. Halomethane uncoupling occurred with or without extracellular calcium and did not change intracellular concentrations of calcium and hydrogen ions or the phosphorylation state of the main gap-junctional protein. Thus the uncoupling appears to depend on cytochrome P-450 oxidative metabolism in which free radicals are generated and may result from oxidation of the gap-junctional protein or of a regulatory molecule that leads to closure of gap-junctional channels. Decreases in junctional conductance may be a rapid cellular response to injury that protects healthy cells by uncoupling them from unhealthy ones.

Voltage dependence of junctional conductance in early amphibian embryos.

Isolated pairs of blastomeres from early amphibian embryos (Ambystoma, Rana, Xenopus) are electrontonically coupled. Junctional conductance and permeability to the dye Lucifer Yellow are markeldy and reversibly decreased by moderate transjunctional polarization in either direction. The relationship between junctional conductance and transjunctional voltage is sufficiently steep that a physiological role in regulation of intercellular communication is plausible.

Electrotonic coupling: effective sign reversal by inhibitory neurons.

Neurons in the buccal ganglia of Navanax inermis which control circumferential muscles of the pharynx showed typical electrotonic coupling when there was little synaptic activity in them. When there was much inhibitory activity, the effective sign of coupling was reversed; that is, hyperpolarization and depolarization of one cell caused depolarization and hyperpolarization of the others. A neural circuit explaining these results involes inhibitory neurons electronically coupled to and also inhibitory to the circumferential neurons that are themselves coupled. This circuit offers considerable flexibility for mediation of different activity patterns in this simple neuronal system.

Gap junctional conductance is a simple and sensitive function of intracellular pH.

The pH of the cytoplasm (pHt) measured with pH-sensitive microelectrodes in cleavage-stage blastomeres of amphibian (Ambystoma) and teleost (Fundulus) embryos is about 7.7. In electrotonically coupled cell pairs, junctional conductance is rapidly and reversibly reduced by acidification of the cytoplasm. The relation between junctional conductance and pHi is the same for increasing and decreasing pH and is independent of the rate of change over a wide range. The relation is well fitted by a Hill curve with K = 50 nM (pK = 7.3) and n = 4 to 5. The closure of gap junction channels at low pHi appears to be a cooperative process involving several charged sites. The absence of hysteresis and identity of effects for fast and slow pHi changes implies that protons act directly on the channel macromolecules and not through an intermediate in the cytoplasm.

Gap junctional conductance and permeability are linearly related.

The permeability of gap junctions to tetraethylammonium ions was measured in isolated pairs of blastomeres from Rana pipiens L. and compared to the junctional conductance. In this system, the junctional conductance is voltage-dependent and decreases with moderate transjunctional voltage of either sign. The permeability to tetraethylammonium ions was determined by injecting one cell of a pair with tetraethylammonium and monitoring its changing concentration in the prejunctional and postjunctional cells with ion-selective electrodes. Junctional conductance was determined by current-clamp and voltage-clamp techniques. For different cell pairs in which the transjunctional voltage was small and the junctional conductance at its maximum value, the permeability to tetraethylammonium ions was proportional to the junctional conductance. In individual cell pairs, a reduction in the junctional conductance induced by voltage was accompanied by a proportional reduction in the permeability of the gap junction over a wide range. The diameter of the tetraethylammonium ion (8.0 to 8.5 A, unhydrated) is larger than that of the potassium ion (4.6 A, hydrated), the predominant current-carrying species. The proportionality between the permeability to tetraethylammonium ions and the junctional conductance, measured here with exceptionally fine time resolution, indicates that a common gap junctional pathway mediates both electrical and chemical fluxes between cells, and that closure of single gap junction channels by voltage is all or none.

Cytokine-induced programmed death of cultured sympathetic neurons.

Programmed cell death (PCD) of sympathetic neurons is inhibited by nerve growth factor. However, factors that induce PCD of these cells are unknown. Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor, neuropoietic cytokines known to regulate sympathetic neuron gene expression, were examined for effects on survival of cultured sympathetic neurons. Treatment with LIF or ciliary neurotrophic factor caused neuronal death in a dose-dependent fashion. Inhibition of RNA or protein synthesis, or treatment with potassium, all of which prevent PCD after nerve growth factor deprivation, prevented LIF-induced death. The morphologic and ultrastructural characteristics of the neuronal death induced by LIF and by nerve growth factor deprivation were similar. Furthermore, LIF treatment resulted in DNA fragmentation with a characteristic "ladder" on Southern blot analysis. These observations suggest that neuron numbers may be regulated by factors which initiate PCD, as well as by factors which prevent it.

Gap junctions remain open during cytochrome c-induced cell death: relationship of conductance to 'bystander' cell killing.

Previous reports have shown that gap junctions relay cell death in many cell types. However, changes in electrical coupling and their dynamics during cell death are poorly understood. We performed comprehensive studies of electrical coupling following induction of cell death by single-cell cytochrome c (cyC) injection in paired Xenopus oocytes. Cell death was rapidly induced by cyC in injected cells, and cell death was also observed in uninjected bystander cells electrically coupled to the cyC-injected oocytes. Gap junction currents either remained at pre-cyC injection levels or increased dramatically as the injected cell died. Nonjunctional currents increased in injected cells immediately following cyC injection; nonjunctional currents increased slowly in uninjected bystander cells. Bystander cell death occurred only when junctional conductance was approximately 6 muS. Both 1,2-bis-(o-aminophenoxy)-ethane-N,N,-N',N'-tetraacetic acid tetraacetoxy-methyl ester and Xestospongin C inhibited bystander cell death in pairs that had reached the death conductance threshold, suggesting that Ca(2+) and inositol 1,4,5 triphosphate are involved in the process.

Wnt-1 regulation of connexin43 in cardiac myocytes.

Gap junction channels composed of connexin43 (Cx43) are essential for normal heart formation and function. We studied the potential role of the Wnt family of secreted polypeptides as regulators of Cx43 expression and gap junction channel function in dissociated myocytes and intact hearts. Neonatal rat cardiomyocytes responded to Li(+), which mimics Wnt signaling, by accumulating the effector protein beta-catenin and by inducing Cx43 mRNA and protein markedly. Induction of Cx43 expression was also observed in cardiomyocytes cocultured with Rat-2 fibroblasts or N2A neuroblastoma cells programmed to secrete bioactive Wnt-1. By transfecting a Cx43 promoter-reporter gene construct into cardiomyocytes, we demonstrated that the inductive effect of Wnt signaling was transcriptionally mediated. Enhanced expression of Cx43 increased cardiomyocyte cell coupling, as determined by Lucifer Yellow dye transfer and by calcium wave propagation. Conversely, in a transgenic cardiomyopathic mouse model that exhibits ventricular arrhythmias and gap junctional remodeling, beta-catenin and Cx43 expression were downregulated concordantly. In response to Wnt signaling, the accumulating Cx43 colocalized with beta-catenin in the junctional membrane; moreover, forced expression of Cx43 in cardiomyocytes reduced the transactivation potential of beta-catenin. These findings demonstrate that Wnt signaling is an important modulator of Cx43-dependent intercellular coupling in the heart, and they support the hypothesis that dysregulated signaling contributes to altered impulse propagation and arrhythmia in the myopathic heart.

Gap junctions in the brain: where, what type, how many and why?

Gap junctions represent well-documented means of intercellular communication in various tissues, including the brain, where they function as portals allowing the exchange of electrolytes, second messengers and metabolites between cells. In view of the enormous recent surge of information dealing with the cellular and molecular biology of gap junctions in non-nervous tissue, as well as current interest in the cell biology of glia, this review is intended to provide an overview of the molecular and functional implications of gap-junction-mediated intercellular communication in the nervous system.

X-linked dominant Charcot-Marie-Tooth disease and other potential gap-junction diseases of the nervous system.

Gap junctions play important roles in the exchange of information and metabolites in the nervous system. These roles are highlighted by peripheral neuropathy (X-linked dominant Charcot-Marie-Tooth disease) that is associated with mutations in a gap-junction protein (connexin32), resulting in loss of function, and by somatic dysfunctions where changes in expression, organization or function of gap junctions are associated with neuronal hyper- or hypoexcitability. In this review, the causes and consequences of this gap-junction-related peripheral neuropathy and other pathological conditions of the nervous system, where dysfunctions of junctional communication are considered to play a casual role, are considered.

Calmodulin kinase pathway mediates the K+-induced increase in Gap junctional communication between mouse spinal cord astrocytes.

Astrocytes are coupled to one another by gap junction channels that allow the diffusion of ions and small molecules throughout the interconnected syncytium. In astrocytes, gap junctions are believed to participate in spatial buffering removing the focal excess of potassium resultant from intense neuronal activity by current loops through the syncytium and are also implicated in the propagation of astrocytic calcium waves, a form of extraneuronal signaling. Gap junctions can be modulated by several factors, including elevation of extracellular potassium concentration. Because K(+) elevation is a component of spinal cord injury, we evaluated the extent to which cultured spinal cord astrocytes is affected by K(+) levels and obtained evidence suggesting that a Ca(2+)-calmodulin (CaM) protein kinase is involved in the K(+)-induced increased coupling. Exposure of astrocytes to high K(+) solutions induced a dose-dependent increase in dye coupling; such increased coupling was greatly attenuated by reducing extracellular Ca(2+) concentration, prevented by nifedipine, and potentiated by Bay-K-8644. These results indicate that K(+)-induced increased coupling is mediated by a signaling pathway that is dependent on the influx of Ca(2+) through L-type Ca(2+) channels. Evidence supporting the participation of the CaM kinase pathway on K(+)-induced increased coupling was obtained in experiments showing that calmidazolium and KN-93 totally prevented the increase in dye and electrical coupling induced by high K(+) solutions. Because no changes in connexin43 expression levels or distribution were observed in astrocytes exposed to high K(+) solutions, we propose that the increased junctional communication is related to an increased number of active channels within gap junction plaques.

Reciprocal regulation of the junctional proteins claudin-1 and connexin43 by interleukin-1beta in primary human fetal astrocytes.

Vertebrate tissues use multiple junctional types to establish and maintain tissue architecture, including gap junctions for cytoplasmic connectivity and tight junctions (TJs) for paracellular and/or cell polarity barriers. The integral membrane proteins of gap junctions are connexins, whereas TJs are a complex between occludin and members of a recently characterized multigene family, the claudins. In normal brain, astrocytes are coupled by gap junctions composed primarily of connexin43 (Cx43), whereas TJs have not been detected in these cells. We now show that treatment of primary human astrocytes with the cytokine interleukin-1beta (IL-1beta) causes rapid induction of claudin-1, with an expression pattern reciprocal to loss of Cx43. Treatment also led to protracted downregulation of occludin but no change in expression of zonula occludens proteins ZO-1 and -2. Immunofluorescence staining localized claudin-1 to cell membranes in IL-1beta-treated astrocytes, whereas freeze-fracture replicas showed strand-like arrays of intramembranous particles in treated cells resembling rudimentary TJ assemblies. We conclude that in human astrocytes, IL-1beta regulates expression of the claudin multigene family and that gap and tight junction proteins are inversely regulated by this proinflammatory cytokine. We suggest that in pathological conditions of the human CNS, elevated IL-1beta expression fundamentally alters astrocyte-to-astrocyte connectivity.