The role of Connexin 46 promoter in lens and other hypoxic tissues.

Gap junctions are multimeric membrane protein channels that connect the cytoplasm of one cell to another. Much information about connexins regards electrophysiology and channel function but relatively little information is known about non-channel functions of connexins. Lens connexins, Cx43, Cx46 and Cx50, have been extensively studied for their role in lens homeostasis. Connexins allow the movement of small metabolically relevant molecules and ions between cells and this action in the lens prevents cataract formation. Interruption of Cx46 channel function leads to cataract formation due to dysregulation of lens homeostasis. The loss of Cx46 upregulates Cx43 in lens cell culture and suppresses tumor growth in breast and retinoblastoma tumor xenografts. Upregulation of Cx46 in hypoxic tissues has been noted and may be due in part to the effects of hypoxia and HIF activators. Here, we report that the Cx46 promoter is regulated by hypoxia and also offer speculation about the role of Cx46 in lens differentiation and solid tumor growth.

Investigation of the reciprocal relationship between the expression of two gap junction connexin proteins, connexin46 and connexin43.

Connexins are the transmembrane proteins that form gap junctions between adjacent cells. The function of the diverse connexin molecules is related to their tissue-specific expression and highly dynamic turnover. Although multiple connexins have been previously reported to compensate for each other's functions, little is known about how connexins influence their own expression or intracellular regulation. Of the three vertebrate lens connexins, two connexins, connexin43 (Cx43) and connexin46 (Cx46), show reciprocal expression and subsequent function in the lens and in lens cell culture. In this study, we investigate the reciprocal relationship between the expression of Cx43 and Cx46. Forced depletion of Cx43, by tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate, is associated with an up-regulation of Cx46 at both the protein and message level in human lens epithelial cells. An siRNA-mediated down-regulation of Cx43 results in an increase in the level of Cx46 protein, suggesting endogenous Cx43 is involved in the regulation of endogenous Cx46 turnover. Overexpression of Cx46, in turn, induces the depletion of Cx43 in rabbit lens epithelial cells. Cx46-induced Cx43 degradation is likely mediated by the ubiquitin-proteasome pathway, as (i) treatment with proteasome inhibitors restores the Cx43 protein level and (ii) there is an increase in Cx43 ubiquitin conjugation in Cx46-overexpressing cells. We also present data that shows that the C-terminal intracellular tail domain of Cx46 is essential to induce degradation of Cx43. Therefore, our study shows that Cx43 and Cx46 have novel functions in regulating each other's expression and turnover in a reciprocal manner in addition to their conventional roles as gap junction proteins in lens cells.

PKCγ, role in lens differentiation and gap junction coupling.

PURPOSE: To determine the role of PKCγ in the regulation of gap junction coupling in the normal lens, we have compared the properties of coupling in lenses from wild type (WT) and PKC-γ knockout (KO) mice. METHODS: Western blotting, confocal immunofluorescence microscopy, immunoprecipitation, RT-PCR and quantitative real time PCR were used to study gap junction protein and message expression; gap junction coupling conductance and pH gating were measured in intact lenses using impedance studies. RESULTS: There were no gross differences in size, clarity, or expression of full-length Cx46 or Cx50 in lenses from WT and PKCγ KO mice. However, total Cx43 protein expression was ~150% higher in the KO lenses. In WT lenses, Cx43 was found only in epithelial cells whereas in KO lenses, its expression continued into the fiber cells. Gap junction coupling conductance in the differentiating fibers (DF) of PKCγ KO lenses was 34% larger than that of WT. In the mature fiber (MF), the effect was much larger with the KO lenses having an 82% increase in coupling over WT. pH gating of the DF fibers was not altered by the absence of PKCγ. CONCLUSION: PKCγ has a major role in the regulation of gap junction expression and coupling in the normal lens.

Treatment with connexin 46 siRNA suppresses the growth of human Y79 retinoblastoma cell xenografts in vivo.

Tumors with a hypoxic component, including human Y79 retinoblastoma cells, express a specific gap junction protein, Connexin 46 (Cx46), which is usually only found in naturally hypoxic tissues such as the differentiated lens. The aim of this study was to investigate if Cx46 downregulation would suppress Y79 tumor formation in vivo. Five-week old nude mice were subcutaneously implanted with human Y79 retinoblastoma cells and treated with intratumor siRNA injections of 30 µg Cx46 siRNA (n = 6), 30 µg non-silencing siRNA (n = 6), or no siRNA treatment (n = 6) every 2 days for a maximum of 10 treatments. Tumor volume (TV) was calculated from the recorded caliper measurements of length and width. Excised tumors were measured and weighed. Western blot analyses were performed to evaluate Cx46 and Cx43 expression in tumors which received Cx46 siRNA, non-silencing siRNA, or no siRNA treatment. Tumor histopathology was used to assess tumor features. Cx46 siRNA treated Y79 tumors had a reduced TV (287 mm(3) ± 77 mm(3)) when compared to the tumors of mice receiving the negative control siRNA (894 mm(3) ± 218 mm(3); P ≤ 0.03) or no siRNA (1068 mm(3) ± 192 mm(3); P ≤ 0.002). A 6-fold knockdown of Cx46 and a 3-fold rise in Cx43 protein expression was observed from western blots of tumors treated with Cx46 siRNA compared to mice treated with non-silencing siRNA. Knockdown of Cx46 with siRNA had an antitumor effect on human Y79 retinoblastoma tumors in the nude mouse model. The results suggest that anti-Cx46 therapy may be a potential target in the future treatment of retinoblastoma.

Loss of protein kinase Cgamma in knockout mice and increased retinal sensitivity to hyperbaric oxygen.

OBJECTIVE: To determine if loss of protein kinase Cgamma (PKCgamma) results in increased structural damage to the retina by hyperbaric oxygen (HBO), a treatment used for several ocular disorders. METHODS: Six-week-old mice were exposed in vivo to 100% HBO 3 times a week for 8 weeks. Eyes were dissected, fixed, embedded in Epon, sectioned, stained with toluidine blue O, and examined by light microscopy. RESULTS: The thicknesses of the inner nuclear and ganglion cell layers were increased. Destruction of the outer plexiform layer was observed in the retinas of the PKCgamma-knockout mice relative to control mice. Exposure to HBO caused significant degradation of the retina in knockout mice compared with control mice. Damage to the outer segments of the photoreceptor layer and ganglion cell layer was apparent in central retinas of HBO-treated knockout mice. CONCLUSIONS: Protein kinase Cgamma-knockout mice had increased retinal sensitivity to HBO. Results demonstrate that PKCgamma protects retinas from HBO damage. CLINICAL RELEVANCE: Care should be taken in treating patients with HBO, particularly if they have a genetic disease, such as spinocerebellar ataxia type 14, a condition in which the PKCgamma is mutated and nonfunctional.

Loss of Purkinje cells in the PKCgamma H101Y transgenic mouse.

Spinocerebellar ataxia type 14 (SCA14) is an autosomal, dominant neurodegenerative disorder caused by mutations in PKCgamma. The objective of this study was to determine effects of PKCgamma H101Y SCA14 mutation on Purkinje cells in the transgenic mouse. Results demonstrated that wild type PKCgamma-like Purkinje cell localization of HA-tagged PKCgamma H101Y mutant proteins, altered morphology and loss of Purkinje cells were observed in the PKCgamma H101Y SCA14 transgenic mouse at four weeks of age. Failure of stereotypical clasping responses in the hind limbs of transgenic mice was also observed. Further, PKCgamma H101Y SCA14 mutation caused lack of total cellular PKCgamma enzyme activity, loss of connexin 57 phosphorylation on serines, and activation of caspase-12 in the PKCgamma H101Y SCA14 transgenic mouse. Results clearly demonstrate a need for PKCgamma control of gap junctions for maintenance of Purkinje cells. This is the first transgenic mouse to our knowledge which models a human SCA14 mutation.

Hypoxia-regulated activity of PKCepsilon in the lens.

PURPOSE: To show that hypoxia is necessary to prevent opacification of the lens. Protein kinase C (PKC)-epsilon serves a role that is distinct from PKC-gamma when both PKC isoforms are expressed in the lens. PKCepsilon serves a very important role in hypoxic conditions, helping to prevent opacification of the lens. METHODS: Digital image analysis, confocal microscopy, dye transfer assay, coimmunoprecipitation, Western blot analysis, and enzyme activity assays were used, respectively, to study opacification of the lens, intercellular communications, cellular localization of connexin-43 (Cx43), and the interactions between PKCepsilon, PKCgamma, and Cx43 in the lens epithelial cells. RESULTS: Hypoxic conditions (1%-5% of oxygen) were very important in maintaining clarity of the lenses of wild-type (WT) mice. Normoxic conditions induced opacification of the WT lens. Lenses from the PKCepsilon-knockout mice underwent rapid opacification, even in hypoxic conditions. Hypoxia did not induce apoptosis in the lens epithelial cells, judging by the absence of active caspase-3, and it did not change intercellular communication and did not affect the number and localization of junctional Cx43 plaques in the lens epithelial cell culture. Hypoxia activated PKCepsilon, whereas phorbol ester (TPA), oxidation (H(2)O(2)), and insulin-like growth factor-1 (IGF-1) activated PKCgamma and decreased the activity of PKCepsilon. Hypoxia did not induce the phosphorylation of the Cx43. CONCLUSIONS: Hypoxia-induced activation of PKCepsilon is very important in surviving hypoxia and maintaining the clarity of the lens. However, PKCgamma is utilized in the control of Cx43 gap junctions.

Protection of retinal cells from ischemia by a novel gap junction inhibitor.

Retinal cells which become ischemic will pass apoptotic signal to adjacent cells, resulting in the spread of damage. This occurs through open gap junctions. A class of novel drugs, based on primaquine (PQ), was tested for binding to connexin 43 using simulated docking studies. A novel drug has been synthesized and tested for inhibition of gap junction activity using R28 neuro-retinal cells in culture. Four drugs were initially compared to mefloquine, a known gap junction inhibitor. The drug with optimal inhibitory activity, PQ1, was tested for inhibition and was found to inhibit dye transfer by 70% at 10 microM. Retinal ischemia was produced in R28 cells using cobalt chloride as a chemical agent. This resulted in activation of caspase-3 which was prevented by PQ1, the gap junction inhibitor. Results demonstrate that novel gap junction inhibitors may provide a means to prevent retinal damage during ischemia.

Synthesis and anti-breast cancer activities of substituted quinolines.

Promising anti-breast cancer agents derived from substituted quinolines were discovered. The quinolines were readily synthesized in a large scale from a sequence of reactions starting from 4-acetamidoanisole. The Michael addition product was isolated as the reaction intermediate in the ring closing reaction of 4-amino-5-nitro-2-(3-trifluoromethylphenyloxy)anisole with methyl vinyl ketone leading to 6-methoxy-4-methyl-8-nitro-5-(3-trifluoromethylphenyloxy)quinoline (14). The amino function of 8-amino-6-methoxy-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, prepared from 14, was connected to various side chains via alkylation with N-(3-iodopropyl)phthalimide, Michael addition with acrylonitrile, and reductive amination with various heterocycle carboxaldehydes, such as imidazole-4-carboxaldehyde, thiophene-2-carboxaldehyde, and 2-furaldehyde. Effects of the substituted quinolines on cell viability of T47D breast cancer cells using trypan blue exclusion assay were examined. The results showed that the IC(50) value of 6-methoxy-8-[(2-furanylmethyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline is 16+/-3nM, the lowest IC(50) out of all the quinolines tested. IC(50) values of three other quinolines are in the nanomolar range, a desirable range for pharmacological testing.

Protection from ataxia-linked apoptosis by gap junction inhibitors.

Mutations in the protein kinase C gamma (PKCgamma) gene cause spinocerebellar ataxia type 14 (SCA14), a heterogeneous neurodegenerative disorder. Synthetic peptides (C1B1) serve as gap junction inhibitors through activation of PKCgamma control of gap junctions. We investigated the neuroprotective potential of these peptides against SCA14 mutation-induced cell death using neuronal HT22 cells. The C1B1 synthetic peptides completely restored PKCgamma enzyme activity and subsequent control of gap junctions. PKCgamma SCA14 mutant proteins were shown to cause aggregation which initially resulted in endoplasmic reticulum (ER) stress and cell apoptosis as demonstrated by phosphorylation of PERK on Thr981, activation of caspase-12, increases in BiP/GRP78 protein levels, and consequent activation of caspase-3. Pre-incubation with C1B1 peptides completely abolished these SCA14 effects on ER stress and caspase-3 activation, suggesting that C1B1 peptides protect cells from apoptosis through inhibition of gap junctions by restoration of PKCgamma control of gap junctions, which may result in neuroprotection in SCA14.

Protein kinase C as a stress sensor.

While there are many reviews which examine the group of proteins known as protein kinase C (PKC), the focus of this article is to examine the cellular roles of two PKCs that are important for stress responses in neurological tissues (PKC gamma and epsilon) and in cardiac tissues (PKC epsilon). These two kinases, in particular, seem to have overlapping functions and interact with an identical target, connexin 43 (Cx43), a gap junction protein which is central to proper control of signals in both tissues. While PKC gamma and PKC epsilon both help protect neural tissue from ischemia, PKC epsilon is the primary PKC isoform responsible for responding to decreased oxygen, or ischemia, in the heart. Both do this through Cx43. It is clear that both PKC gamma and PKC epsilon are necessary for protection from ischemia. However, the importance of these kinases has been inferred from preconditioning experiments which demonstrate that brief periods of hypoxia protect neurological and cardiac tissues from future insults, and that this depends on the activation, translocation, or ability for PKC gamma and/or PKC epsilon to interact with distinct cellular targets, especially Cx43. This review summarizes the recent findings which define the roles of PKC gamma and PKC epsilon in cardiac and neurological functions and their relationships to ischemia/reperfusion injury. In addition, a biochemical comparison of PKC gamma and PKC epsilon and a proposed argument for why both forms are present in neurological tissue while only PKC epsilon is present in heart, are discussed. Finally, the biochemistry of PKCs and future directions for the field are discussed, in light of this new information.

Protein kinase C-gamma activation in the early streptozotocin diabetic rat lens.

PURPOSE: The purpose of this study is to demonstrate the early activation of the protein kinase C-gamma (PKC-gamma) pathway in the streptozotocin (STZ)-induced diabetic rat lens. METHODS: Twelve-week-old male and female Sprague-Dawley rats were injected with 80 mg/kg (body weight) of STZ (N-[methylnitrosocarbamoyl]-D-glucosamine) intraperitoneally. Very high glucose (VHG) diabetes was defined as a nonfasting blood glucose level of at least 450 mg/dl, confirmed by daily monitoring with Accu-Check Advantage test strips, and occurred about 2 weeks after STZ administration. All assayed lenses were from VHG or age-matched control rats, harvested within 24 hr of VHG detection. PKC-gamma activation was measured by enzyme activity assay and by Western blotting to show autophosphorylation on Thr514. Cellular insulin-like growth factor-1 (IGF-1), PKC-gamma phosphorylation of Cx43 on Ser368, and activation of phospholipase C-gamma 1 (PLC-gamma 1), extracellular signal-regulated kinase (ERK1/2), and caspase-3 were determined by Western blotting. Endogenous diacylglycerol (DAG) levels were measured with a DAG assay kit. Lens gap junction activity was determined by the microinjection/Lucifer yellow dye transfer assay. Electron microscopy was applied to affirm fiber cell damage in the VHG diabetic lenses. RESULTS: In the lenses of VHG diabetic rats, PKC-gamma enzyme was activated. PKC-gamma could be further activated by 400 nM phorbol-12-myristate-13-acetate (PMA), but the PKC-gamma protein levels remained constant. No elevation of IGF-1 level was observed. Western blots showed that activation of PKC-gamma may be due to activation of PLC-gamma 1, which synthesized endogenous DAG, a native PKC activator. The level of PKC-gamma -catalyzed phosphorylation of Cx43 on Ser368 and resulting inhibition of lens gap junction dye transfer activity was increased in the VHG diabetic lenses. At this early time period, the diabetic lens showed no activation of either caspase-3 or ERK1/2. Only a single fiber cell layer deep within the cortex (approximately 90 cell layers from capsule surface) showed vacuoles and damaged cell connections. CONCLUSIONS: Early activation of PLC-gamma 1 and elevated DAG were observed within VHG diabetic lenses. These were correlated with activation of PKC-gamma, phosphorylation of Cx43 on Ser368, and inhibition of dye transfer. Abnormal signaling from PKC-gamma to Cx43 in the epithelial cells/early fiber cells, observed within VHG diabetic lenses, may be responsible for fiber cell damage deeper in the lens cortex.

Protein kinase C gamma mutations in the C1B domain cause caspase-3-linked apoptosis in lens epithelial cells through gap junctions.

Failure to control oxidative stress is closely related to aging and to a diverse range of human diseases. We have reported that protein kinase C gamma (PKCgamma) acts as a primary oxidative stress sensor in the lens. PKCgamma has a Zn-finger C1B stress switch domain, residues 101-150. Mutation, H101Y, in the C1B domain of PKCgamma proteins causes a failure of the PKCgamma oxidative stress response [Lin, D., Takemoto, D.J., 2005. Oxidative activation of protein kinase Cgamma through the C1 domain. Effects on gap junctions. J. Biol. Chem. 280, 13682-13693]. Some human neurodegenerative spinocerebellar ataxia type 14 are caused by mutations in the PKCgamma C1B domain. In the current study we have investigated the effects of these mutations on lens epithelial cell responses to oxidative stress. The results demonstrate that PKCgamma C1B mutants had lower basal enzyme activities and were not activated by H(2)O(2). Furthermore, the PKCgamma mutations caused a failure of endogenous wild type PKCgamma to be activated by H(2)O(2). These PKCgamma mutations abolished the effect of H(2)O(2) on phosphorylation of Cx43 and Cx50 by H(2)O(2) activation of PKCgamma. The cells with PKCgamma C1B mutations had more Cx43 and/or Cx50 gap junction plaques which were not decreased by H(2)O(2). Since open gap junctions could have a bystander effect this could cause apoptosis to occur. H(2)O(2) (100 microM, 3 h) activated a caspase-3 apoptotic pathway in the lens epithelial cells but was more severe in cells expressing PKCgamma mutations. The presence of 18alpha-glycyrrhetinic acid (AGA), an inhibitor of gap junctions, decreased Cx43 and Cx50 protein levels and gap junction plaque number. This reduction in gap junctions by AGA resulted in inhibition of H(2)O(2)-induced apoptosis. Our results demonstrate that there is a dominant negative effect of PKCgamma C1B mutations on endogenous PKCgamma which results in loss of control of gap junctions. Modeled structures suggest that the severity of C1B mutation effects may be related to the extent of loss of C1B structure. Mutations in the C1B domain of PKCgamma result in increased apoptosis in lens epithelial cells. This can be prevented by a gap junction inhibitor. Thus, propagation of apoptosis from cell-to-cell in lens epithelial cells may be through open gap junctions. The control of gap junctions requires PKCgamma.

ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43.

We have previously reported that protein kinase C gamma (PKC-gamma) is activated by phorbol-12-myristate-13-acetate (TPA) and that this causes PKC-gamma translocation to membranes and phosphorylation of the gap junction protein, connexin 43 (Cx43). This phosphorylation, on S368 of Cx43, causes disassembly of Cx43 out of cell junctional plaques resulting in the inhibition of dye transfer. The purpose of this study is to identify the specific role of zonula occludens protein-1 (ZO-1), a tight junction protein with recently established effects on gap junctions, in this PKC-gamma-driven Cx43 disassembly. For this purpose, ZO-1 levels in lens epithelial cells in culture were decreased by up to 70% using specific siRNA. The down-regulation of ZO-1 caused a stable interaction of PKC-gamma with Cx43 even without normal enzyme activation by TPA. However, after TPA activation of the PKC-gamma, the Cx43 did not disassemble out of plaques even though the PKC-gamma enzyme was activated and the Cx43 was phosphorylated on S368. Confocal microscopy demonstrated that the siRNA treatment caused a loss of ZO-1 from borders of large junctional Cx43 cell-to-cell plaques and resulted in the accumulation of Cx43 aggregates inside of cells. Loss of the specific "plaquetosome" arrangement of large Cx43 plaques surrounded by ZO-1 was accompanied by a complete loss of functional dye transfer. These results suggest that ZO-1 is required for Cx43 control, both for dye transfer, and, for the PKC-gamma-driven disassembly response.

PKCgamma knockout mouse lenses are more susceptible to oxidative stress damage.

Cataracts, or lens opacities, are the leading cause of blindness worldwide. Cataracts increase with age and environmental insults, e.g. oxidative stress. Lens homeostasis depends on functional gap junctions. Knockout or missense mutations of lens gap junction proteins, Cx46 or Cx50, result in cataractogenesis in mice. We have previously demonstrated that protein kinase Cgamma (PKCgamma) regulates gap junctions in the lens epithelium and cortex. In the current study, we further determined whether PKCgamma control of gap junctions protects the lens from cataractogenesis induced by oxidative stress in vitro, using PKCgamma knockout and control mice as our models. The results demonstrate that PKCgamma knockout lenses are normal at 2 days post-natal when compared to control. However, cell damage, but not obvious cataract, was observed in the lenses of 6-week-old PKCgamma knockout mice, suggesting that the deletion of PKCgamma causes lenses to be more susceptible to damage. Furthermore, in vitro incubation or lens oxidative stress treatment by H(2)O(2) significantly induced lens opacification (cataract) in the PKCgamma knockout mice when compared to controls. Biochemical and structural results also demonstrated that H(2)O(2) activation of endogenous PKCgamma resulted in phosphorylation of Cx50 and subsequent inhibition of gap junctions in the lenses of control mice, but not in the knockout. Deletion of PKCgamma altered the arrangement of gap junctions on the cortical fiber cell surface, and completely abolished the inhibitory effect of H(2)O(2) on lens gap junctions. Data suggest that activation of PKCgamma is an important mechanism regulating the closure of the communicating pathway mediated by gap junction channels in lens fiber cells. The absence of this regulatory mechanism in the PKCgamma knockout mice may cause those lenses to have increased susceptibility to oxidative damage.

Expression of superoxide dismutase in whole lens prevents cataract formation.

PURPOSE: Oxidative damage is a major factor causing cataracts, which account for almost half of human blindness cases worldwide. In this study, we wished to determine if overexpression of superoxide dismutase (SOD) in intact lenses could prevent cataract formation induced by oxidative stress. METHODS: Fresh, intact lenses from 6-week-old male/female Sprague Dawley rats were incubated with plasmid DNA encoding the human SOD1 (Cu/Zn-SOD) gene at 37 degrees C in a CO2 cell culture chamber with 95% air and 5% CO2. SOD1 expression was determined by western blotting and SOD enzyme activity. Lenses with or without overexpression of SOD1 were treated with H2O2 and cataract formation was examined. SOD1 regulation of protein kinase Cgamma (PKCgamma) was determined by PKCgamma enzyme activity assay. Intact lens gap junctions were determined by dye transfer assay. RESULTS: In the lens overexpression system, SOD1 cDNA was fused to EYFP to generate EYFP:SOD1 fusion proteins which allow detection from endogenous SOD1. Incubation of intact lenses with plasmid DNA produced EYFP:SOD1 fusion proteins as determined by western blot using anti-GFP or anti-SOD1 antibodies. This caused significant increases in SOD enzyme activity. Data indicated that SOD1 plasmid DNA can be expressed as a functional enzyme in intact lenses in culture. Lenses overexpressing SOD1 remained clear after H2O2 treatment at 100 muM for 24 h, similar to control. Overexpression of SOD1 diminished the effect of H2O2 on PKCgamma activation and subsequent inhibition of gap junctions, indicating that overexpression of SOD1 may reduce reactive oxygen species (ROS) production, and this would prevent the normal H2O2 effect on cataract formation. CONCLUSIONS: Overexpression of SOD1 in whole lens prevents H2O2-induced oxidative damage (cataract formation) to the lens and subsequent control of gap junctions by protein kinase Cgamma.

Lignans are involved in the antitumor activity of wheat bran in colon cancer SW480 cells.

Wheat bran was shown to provide protection against colorectal cancer in human intervention and animal studies. Our recent study showed, however, that antitumor activities of wheat bran from various wheat cultivars differed significantly even when wheat fiber was equal in diets. We hypothesized that phytochemical lignans in wheat bran may account for the differences among wheat cultivars in cancer prevention. The concentration of a major lignan, secoisolariciresinol diglycoside, was determined by HPLC in 4 selected wheat cultivars (i.e., Madison, Ernie, Betty, and Arapahoe). The lignan concentrations and their antitumor activities, previously determined in APC-Min mice, were correlated (r = 0.73, P < 0.02). The cancer preventive mechanisms of 2 prominent lignan metabolites (enterolactone and enterodiol) were further studied in human colonic cancer SW480 cells. Treatment with enterolactone and enterodiol, alone or in combination, at 0-40 micromol/L resulted in dose- and time-dependent decreases in cell numbers. Although the cytotoxicity as measured by trypan blue staining in adherent cells was not affected, DNA flow cytometric analysis indicated that the treatments induced cell cycle arrest at the S-phase. Western blot analysis for cyclin A, a required protein for S/G2 transition, showed that the cyclin A protein levels decreased after treatment with enterodiol or the combination of enterolactone and enterodiol at 40 micromol/L for 72 h. Apoptosis analysis by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay showed an increased percentage of apoptotic cells in the floating cells after enterodiol alone or combined treatments. These results suggest for the first time that lignans may contribute, at least in part, to the cancer prevention by wheat bran observed in APC-Min mice. Inhibition of cancer cell growth by lignan metabolites seems to be mediated by cytostatic and apoptotic mechanisms.

Oxidative activation of protein kinase Cgamma through the C1 domain. Effects on gap junctions.

The accumulation of reactive oxygen species (ROS, for example H2O2) is linked to several chronic pathologies, including cancer and cardiovascular and neurodegenerative diseases (Gate, L., Paul, J., Ba, G. N., Tew, K. D., and Tapiero, H. (1999) Biomed. Pharmacother. 53, 169-180). Protein kinase C (PKC) gamma is a unique isoform of PKC that is found in neuronal cells and eye tissues. This isoform is activated by ROS such as H2O2. Mutations (H101Y, G118D, S119P, and G128D) in the PKCgamma Cys-rich C1B domain caused a form of dominant non-episodic cerebellar ataxia in humans (Chen, D.-H., Brkanac, Z., Verlinde, C. L. M. J., Tan, X.-J., Bylenok, L., Nochli, D., Matsushita, M., Lipe, H., Wolff, J., Fernandez, M., Cimino, P. J., Bird, T. D., and Raskind, W. H. (2003) Am. J. Hum. Genet. 72, 839-849; van de Warrenburg, B. P. C., Verbeek, D. S., Piersma, S. J., Hennekam, F. A. M., Pearson, P. L., Knoers, N. V. A. M., Kremer, H. P. H., and Sinke, R. J. (2003) Neurology 61, 1760-1765). This could be due to a failure of the mutant PKCgamma proteins to be activated by ROS and to subsequently inhibit gap junctions. The purpose of this study was to demonstrate the cellular mechanism of activation of PKCgamma by H2O2 and the resultant effects on gap junction activity. H2O2 stimulated PKCgamma enzyme activity independently of elevations in cellular diacylglycerol, the natural PKC activator. Okadaic acid, a phosphatase inhibitor, did not affect H2O2-stimulated PKCgamma activity, indicating that dephosphorylation was not involved. The reductant, dithiothreitol, abolished the effects of H2O2, suggesting a direct oxidation of PKCgamma at the Cys-rich C1 domain. H2O2 induced the C1 domain of PKCgamma to translocate to plasma membranes, whereas the C2 domain did not. Direct effects of H2O2 on PKCgamma were demonstrated using two-dimensional SDS-PAGE. Results demonstrated that PKCgamma formed disulfide bonds in response to H2O2. H2O2-activated PKCgamma was targeted into caveolin-1- and connexin 43-containing lipid rafts, and the PKCgamma phosphorylated the connexin 43 gap junction proteins on Ser-368. This resulted in disassembly of connexin 43 gap junction plaques and decreased gap junction activity. Results suggested that H2O2 caused oxidation of the C1 domain, activation of the PKCgamma, and inhibition of gap junctions. This inhibition of gap junctions could provide a protection to cells against oxidative stress.

Differential phosphorylation of connexin46 and connexin50 by H2O2 activation of protein kinase Cgamma.

PURPOSE: Fiber cell gap junction proteins connexin 46 (Cx46) and connexin 50 (Cx50) play distinct roles in the avascular lens. The purpose of this study was to determine how protein kinase Cgamma (PKCgamma) differentially regulates phosphorylation of Cx46 and Cx50 in oxidatively stressed lenses. METHODS: Sprague Dawley rats (six week old) were used in the experiments. PKCgamma enzyme activity was analyzed by use of the PepTag assay kit. Phosphorylation of caveolin-1, Cx46, and Cx50 was determined by immunoblotting. Lipid rafts were isolated by continuous sucrose gradient centrifugation. Lipid raft-localization of PKCgamma, Cx46, or Cx50 was demonstrated by immunoblotting. Association of caveolin-1 with PKCgamma, Cx46, or Cx50 was revealed by co-immunoprecipitation. RESULTS: H2O2 (100 microM) stimulated PKCgammaactivation in rat whole lens. Activated PKCgamma was recruited into caveolin-1 (Cav-1) containing lipid rafts and this activation enhanced the coimmunoprecipitation of Cav-1, Cx46, and Cx50 with PKCgamma. Both Cx50 and Cx46 were associated with Cav-1 in lipid rafts. H2O2 significantly induced threonine (Thr) phosphorylation of Cx46 and Cx50, and serine (Ser) phosphorylation of Cx50. However, There was only a small stimulation of Cx46 phosphorylation at Ser by H2O2, as Cx46 was already phosphorylated. CONCLUSIONS: Activation of PKCgamma by H2O2 stimulated differential Ser phosphorylation of Cx50 versus Cx46, within lipid rafts. This suggests that Cx50 and Cx46 may have different functions in lens.

Inhibition of gap junction activity through the release of the C1B domain of protein kinase Cgamma (PKCgamma) from 14-3-3: identification of PKCgamma-binding sites.

We have shown previously that insulin-like growth factor-I or lens epithelium-derived growth factor increases the translocation of protein kinase Cgamma (PKCgamma)to the membrane and the phosphorylation of Cx43 by PKCgamma and causes a subsequent decrease of gap junction activity (Nguyen, T. A., Boyle, D. L., Wagner, L. M., Shinohara, T., and Takemoto, D. J. (2003) Exp. Eye Res. 76, 565-572; Lin, D., Boyle, D. L., and Takemoto, D. J. (2003) Investig. Ophthalmol. Vis. Sci. 44, 1160-1168). Gap junction activity in lens epithelial cells is regulated by PKCgamma-mediated phosphorylation of Cx43. PKCgamma activity is stimulated by growth factor-regulated increases in the synthesis of diacylglycerol but is inhibited by cytosolic docking proteins such as 14-3-3. Here we have identified two sites on the PKCgamma-C1B domain that are responsible for its interaction with 14-3-3epsilon. Two sites, C1B1 (residues 101-112) and C1B5 (residues 141-151), are located within the C1 domain of PKCgamma. C1B1 and/or C1B5 synthetic peptides can directly compete for the binding of 14-3-3epsilon, resulting in the release of endogenous cellular PKCgamma from 14-3-3epsilon, in vivo or in vitro, in activation of PKCgamma enzyme activity, phosphorylation of PKCgamma, in the subsequent translocation of PKCgamma to the membrane, and in inhibition of gap junction activity. Gap junction activity was decreased by at least 5-fold in cells treated with C1B1 or C1B5 peptides when compared with a control. 100 microM of C1B1 or C1B5 peptides also caused a 10- or 4-fold decrease of Cx43 plaque formation compared with control cells. The uptake of these synthetic peptides into cells was verified by using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry. We have demonstrated that the activity and localization of PKCgamma are regulated by its binding to 14-3-3epsilon at the C1B domain of PKCgamma. Synthetic peptides corresponding to these regions of PKCgamma successfully competed for the binding of 14-3-3epsilon to endogenous PKCgamma, resulting in inhibition of gap junction activity. This demonstrates that synthetic peptides can be used to exogenously regulate gap junctions.