Targeting MAPK phosphorylation of Connexin43 provides neuroprotection in stroke.

Connexin43 (Cx43) function is influenced by kinases that phosphorylate specific serine sites located near its C-terminus. Stroke is a powerful inducer of kinase activity, but its effect on Cx43 is unknown. We investigated the impact of wild-type (WT) and knock-in Cx43 with serine to alanine mutations at the protein kinase C (PKC) site Cx43S368A, the casein kinase 1 (CK1) sites Cx43S325A/328Y/330A, and the mitogen-activated protein kinase (MAPK) sites Cx43S255/262/279/282A (MK4) on a permanent middle cerebral artery occlusion (pMCAO) stroke model. We demonstrate that MK4 transgenic animals exhibit a significant decrease in infarct volume that was associated with improvement in behavioral performance. An increase in astrocyte reactivity with a concomitant decrease in microglial reactivity was observed in MK4 mice. In contrast to WT, MK4 astrocytes displayed reduced Cx43 hemichannel activity. Pharmacological blockade of Cx43 hemichannels with TAT-Gap19 also significantly decreased infarct volume in WT animals. This study provides novel molecular insights and charts new avenues for therapeutic intervention associated with Cx43 function.

Matrix-assisted laser desorption/ionization imaging mass spectrometry of intraperitoneally injected danegaptide (ZP1609) for treatment of stroke-reperfusion injury in mice.

RATIONALE: This work focuses on direct matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) detection of intraperitoneally (IP)-injected dipeptide ZP1609 in mouse brain tissue. Direct analysis of drug detection in intact tissue sections provides distribution information that can impact drug development. MALDI-IMS capabilities of uncovering drug transport across the blood-brain barrier are demonstrated. METHODS: Successful peptide detection using MALDI-IMS was achieved using a MALDI TOF/TOF system. Upon optimization of sample preparation procedures for dipeptide ZP1609, an additional tissue acidification procedure was found to greatly enhance signal detection. The imaging data acquired was able to determine successful transport of ZP1609 across the blood-brain barrier. Data obtained from MALDI-IMS can help shape our understanding of biological functions, disease progression, and effects of drug delivery. RESULTS: Direct detection of ZP1609 throughout the brain tissue sections was observed from MALDI-MS images. However, in cases where there was induction of stroke, a peak of lower signal intensity was also detected in the target m/z region. Although distinct differences in signal intensity can be seen between control and experimental groups, fragments and adducts of ZP1609 were investigated using MALDI-IMS to verify detection of the target analyte. CONCLUSIONS: Overall, the data reveals successful penetration of ZP1609 across the blood-brain barrier. The benefits of tissue acidification in the enhancement of detection sensitivity for low-abundance peptides were demonstrated. MALDI-IMS has been shown to be a useful technique in the direct detection of drugs within intact brain tissue sections.

Pannexin1 knockout and blockade reduces ischemic stroke injury in female, but not in male mice.

The membrane channel Pannexin 1 (Panx1) mediates apoptotic and inflammatory signaling cascades in injured neurons, responses previously shown to be sexually dimorphic under ischemic conditions. We tested the hypothesis that Panx1 plays an underlying role in mediating sex differences in stroke outcome responses. Middle-aged, 8-9 month old male and female wild type and Panx1 KO mice were subjected to permanent middle cerebral artery (MCA) occlusion, and infarct size and astrocyte and microglia activation were assessed 4 days later. The sexually dimorphic nature of Panx1 deletion was also explored by testing the effect of probenecid a known Panx1 blocker to alter stroke volume. Panx1 KO females displayed significantly smaller infarct volumes (~ 50 % reduction) compared to their wild-type counterparts, whereas no such KO effect occurred in males. This sex-specific effect of Panx1 KO was recapitulated by significant reductions in peri-infarct inflammation and astrocyte reactivity, as well as smaller infarct volumes in probenecid treated females, but not males. Finally, females showed overall, higher Panx1 protein levels than males under ischemic conditions. These findings unmask a deleterious role for Panx1 in response to permanent MCA occlusion, that is unique to females, and provide several new frameworks for understanding sex differences in stroke outcome.

The C-terminal domain of connexin43 modulates cartilage structure via chondrocyte phenotypic changes.

Chondrocytes in cartilage and bone cells population express connexin43 (Cx43) and gap junction intercellular communication (GJIC) is essential to synchronize cells for coordinated electrical, mechanical, metabolic and chemical communication in both tissues. Reduced Cx43 connectivity decreases chondrocyte differentiation and defective Cx43 causes skeletal defects. The carboxy terminal domain (CTD) of Cx43 is located in the cytoplasmic side and is key for protein functions. Here we demonstrated that chondrocytes from the CTD-deficient mice, K258stop/Cx43KO and K258stop/K258stop, have reduced GJIC, increased rates of proliferation and reduced expression of collagen type II and proteoglycans. We observed that CTD-truncated mice were significantly smaller in size. Together these results demonstrated that the deletion of the CTD negatively impacts cartilage structure and normal chondrocyte phenotype. These findings suggest that the proteolytic cleavage of the CTD under pathological conditions, such as under the activation of metalloproteinases during tissue injury or inflammation, may account for the deleterious effects of Cx43 in cartilage and bone disorders such as osteoarthritis.

Cx26 knockout predisposes the mammary gland to primary mammary tumors in a DMBA-induced mouse model of breast cancer.

Down-regulation of the gap junction protein connexin26 (Cx26) is an early event following breast cancer onset and has led to Cx26 being classically described as a tumor suppressor. Interestingly, mutations in theCx26 gene (GJB2) reduce or ablate Cx26 gap junction channel function and are the most common cause of genetic deafness. It is unknown if patients with loss-of-function GJB2 mutations have a greater susceptibility to breast tumorigenesis or aggressive breast cancer progression. To investigate these possibilities, 7, 12-dimethylbenz[α]anthracene (DMBA)-induced tumor development was evaluated in BLG-Cre; Cx26fl/fl mice expressing Cre under the ß-Lactoglobulin promoter (Cre+) compared to Cx26fl/fl controlmice (Cre-) following pituitary isograft driven Cx26 knockout. A significantly increased number of DMBA-treated Cre+ mice developed primary mammary tumors, as well as developed multiple tumors, compared to Cre- mice. Primary tumors of Cre+ mice were of multiple histological subtypes and had similar palpable tumour onset and growth rate compared to tumors from Cre- mice. Lungs were evaluated for evidence of metastases revealing a similar percentage of lung metastases in Cre+ and Cre- mice. Together, our results suggest that loss of Cx26 predisposes the mammary gland to chemically induced mammary tumour formation which may have important implications to patients with GJB2 mutations.

Podoplanin: a marker for reactive gliosis in gliomas and brain injury.

Reactive astrogliosis is associated with many pathologic processes in the central nervous system, including gliomas. The glycoprotein podoplanin (PDPN) is upregulated in malignant gliomas. Using a syngeneic intracranial glioma mouse model, we show that PDPN is highly expressed in a subset of glial fibrillary acidic protein-positive astrocytes within and adjacent to gliomas. The expression of PDPN in tumor-associated reactive astrocytes was confirmed by its colocalization with the astrocytic marker S100ß and with connexin43, a major astrocytic gap junction protein. To determine whether the increase in PDPN is a general feature of gliosis, we used 2 mouse models in which astrogliosis was induced either by a needle injury or ischemia and observed similar upregulation of PDPN in reactive astrocytes in both models. Astrocytic PDPN was also found to be coexpressed with nestin, an intermediate filament marker for neural stem/progenitor cells. Our findings confirm that expression of PDPN is part of the normal host response to brain injury and gliomas, and suggest that it may be a novel cell surface marker for a specific population of reactive astrocytes in the vicinity of gliomas and nonneoplastic brain lesions. The findings also highlight the heterogeneity of glial fibrillary acidic protein-positive astrocytes in reactive gliosis.

Pannexin 2 protein expression is not restricted to the CNS.

Pannexins (Panx) are proteins homologous to the invertebrate gap junction proteins called innexins (Inx) and are traditionally described as transmembrane channels connecting the intracellular and extracellular compartments. Three distinct Panx paralogs (Panx1, Panx2 and Panx3) have been identified in vertebrates but previous reports on Panx expression and functionality focused primarily on Panx1 and Panx3 proteins. Several gene expression studies reported that Panx2 transcript is largely restricted to the central nervous system (CNS) hence suggesting that Panx2 might serve an important role in the CNS. However, the lack of suitable antibodies prevented the creation of a comprehensive map of Panx2 protein expression and Panx2 protein localization profile is currently mostly inferred from the distribution of its transcript. In this study, we characterized novel commercial monoclonal antibodies and surveyed Panx2 expression and distribution at the mRNA and protein level by real-time qPCR, Western blotting and immunofluorescence. Panx2 protein levels were readily detected in every tissue examined, even when transcriptional analysis predicted very low Panx2 protein expression. Furthermore, our results indicate that Panx2 transcriptional activity is a poor predictor of Panx2 protein abundance and does not correlate with Panx2 protein levels. Despite showing disproportionately high transcript levels, the CNS expressed less Panx2 protein than any other tissues analyzed. Additionally, we showed that Panx2 protein does not localize at the plasma membrane like other gap junction proteins but remains confined within cytoplasmic compartments. Overall, our results demonstrate that the endogenous expression of Panx2 protein is not restricted to the CNS and is more ubiquitous than initially predicted.

Mammary gland specific knockdown of the physiological surge in Cx26 during lactation retains normal mammary gland development and function.

Connexin26 (Cx26) is the major Cx protein expressed in the human mammary gland and is up-regulated during pregnancy while remaining elevated throughout lactation. It is currently unknown if patients with loss-of-function Cx26 mutations that result in hearing loss and skin diseases have a greater susceptibility to impaired breast development. To investigate if Cx26 plays a critical role in mammary gland development and differentiation, a novel Cx26 conditional knockout mouse model was generated by crossing Cx26fl/fl mice with mice expressing Cre under the ß-Lactoglobulin promoter. Conditional knockdown of Cx26 from the mammary gland resulted in a dramatic reduction in detectable gap junction plaques confirmed by a significant ∼65-70% reduction in Cx26 mRNA and protein throughout parturition and lactation. Interestingly, this reduction was accompanied by a decrease in mammary gland Cx30 gap junction plaques at parturition, while no change was observed for Cx32 or Cx43. Whole mount, histological and immunofluorescent assessment of breast tissue revealed comparatively normal lobuloalveolar development following pregnancy in the conditionally knockdown mice compared to control mice. In addition, glands from genetically-modified mice were capable of producing milk proteins that were evident in the lumen of alveoli and ducts at similar levels as controls, suggesting normal gland function. Together, our results suggest that low levels of Cx26 expression throughout pregnancy and lactation, and not the physiological surge in Cx26, is sufficient for normal gland development and function.

Comorbid Aß toxicity and stroke: hippocampal atrophy, pathology, and cognitive deficit.

Numerous clinical and epidemiological reports indicate that patients with history of vascular illness such as stroke are more likely to develop dementia as the clinical manifestation of Alzheimer's disease. However, there are little data regarding the pathologic mechanisms that link vascular risk factors to the factors associated with dementia onset. We provide evidence that suggests intriguing detrimental interactions between stroke and ß-amyloid (Aß) toxicity in the hippocampus. Stroke was induced by unilateral striatal injection of endothelin-1, the potent vasoconstrictor. Aß toxicity was modeled by bilateral intracerebroventricular injections of the toxic fragment Aß. Gross morphologic changes in comorbid Aß and stroke rats were enlargement of the lateral ventricles with concomitant shrinkage of the hippocampus. The hippocampus displayed a series of synergistic biochemical alterations, including microgliosis, deposition of Aß precursor protein fragments, and cellular degeneration. In addition, there was bilateral induction of connexin43, reduced neuronal survival, and impaired dendritic development of adult-born immature neurons in the dentate gyrus of these rats compared with either rats alone. Behaviorally, there was impairment in the hippocampal-based discriminative fear-conditioning to context task indicating learning and memory deficit. These results suggest an insight into the relationship between hippocampal atrophy, pathology, and functional impairment. Our work not only highlights the exacerbated pathology that emerges when Aß toxicity and stroke occur comorbidly but also demonstrates that this comorbid rat model exhibits physiopathology that is highly characteristic of the human condition.

Cerebral ischemic injury is enhanced in a model of oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is a rare autosomal dominant disease that results in visible developmental anomalies of the limbs, face, eyes and teeth. Recently analysis of human connexin43 (Cx43) DNA sequences has revealed a number of different missense, duplication and frame shift mutations resulting in this phenotype. A mouse model of this disorder has been created with a missense point mutation of the glycine amino acid at position 60 to serine (G60S). Heterozygote +/G60S mice exhibit a similar ODDD phenotype as observed in humans. In addition to the malformations listed above, ODDD patients often have neurological findings. In the brain, Cx43 is highly expressed in astrocytes and has been shown to play a role in neuroprotection. We were interested in determining the effect of the +/G60S mutation following stroke. Four days after middle cerebral artery occlusion the volume of infarct was larger in mice with the +/G60S mutation. In astrocyte-neuron co-cultures, exposure to glutamate also resulted in greater cellular death in the +/G60S mutants. Protein levels of Cx43 in the mutant mouse were found to be reduced when compared to the normal tissue. Cx43 protein was observed as a continual line of small punctate aggregates in the plasma membrane with increased intracellular localization, which is distinct from the larger plaques seen in the normal mouse astrocytes. Functionally, primary +/G60S astrocytes exhibited reduced gap junctional coupling and increased hemichannel activity, which may underlie the mechanism of increased damage during stroke. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

Role of gap junction protein connexin43 in astrogliosis induced by brain injury.

Astrogliosis is a process that involves morphological and biochemical changes associated with astrocyte activation in response to cell damage in the brain. The upregulation of intermediate filament proteins including glial fibrillary acidic protein (GFAP), nestin and vimentin are often used as indicators for astrogliosis. Although connexin43 (Cx43), a channel protein widely expressed in adult astrocytes, exhibits enhanced immunoreactivity in the peri-lesion region, its role in astrogliosis is still unclear. Here, we correlated the temporal and spatial expression of Cx43 to the activation of astrocytes and microglia in response to an acute needle stab wound in vivo. We found large numbers of microglia devoid of Cx43 in the needle wound at 3 days post injury (dpi) while reactive astrocytes expressing Cx43 were present in the peripheral zone surrounding the injury site. A redistribution of Cx43 to the needle site, corresponding to the increased presence of GFAP-positive reactive astrocytes in the region, was only apparent from 6 dpi and sustained until at least 15 dpi. Interestingly, the extent of microglial activation and subsequent astrogliosis in the brain of Cx43 knockout mice was significantly larger than those of wild type, suggesting that Cx43 expression limits the degree of microgliosis. Although Cx43 is not essential for astrogliosis and microglial activation induced by a needle injury, our results demonstrate that Cx43 is a useful marker for injury induced astrogliosis due to its enhanced expression specifically within a small region of the lesion for an extended period. As a channel protein, Cx43 is a potential in vivo diagnostic tool of asymptomatic brain injury.

The connexin43 C-terminal region mediates neuroprotection during stroke.

Connexin43 plays an important role in neuroprotection in experimental stroke models; reducing the expression of this gap junction protein in astrocytes enhances injury upon middle cerebral artery occlusion (MCAO). Because the C-terminal region of connexin43 isimportant for channel activity, we carried out MCAO stroke experiments in mice expressing a truncated form of connexin43 (Cx43DeltaCT mice). Brain sections were analyzed for infarct volume, astrogliosis, and inflammatory cell invasion 4 days after MCAO. Adult cortices and astrocyte cultures were examined for connexin43 (Cx43) expression by immunohistochemistry and Western blot. Cultured astrocytes were also examined for dye coupling, channel conductance, hemichannel activity, and Ca wave propagation. The Cx43DeltaCT mice exhibit enhanced cerebral injury after stroke. Astrogliosis was reduced and inflammatory cell invasion was increased inthe peri-infarct region in these mice compared with controls; Cx43 expression was also altered. Lastly, cultured astrocytes from Cx43DeltaCT mice were less coupled and displayed alterations in channel gating, hemichannel activity, and Ca wave properties. These results suggest that astrocytic Cx43 contributed to the regulation of cell death after stroke and support the view that the Cx43 C-terminal region is important in protection in cerebral ischemia.

Involvement of the cytoplasmic C-terminal domain of connexin43 in neuronal migration.

During brain development, young neurons closely associate with radial glial while migrating from the ventricular zone (VZ) to the cortical plate (CP) of the neocortex. It has been shown previously that gap junctions are needed for this migration to occur properly, but the precise mechanism responsible is still in question. Here, we used Cre recombinase, driven by the nestin promoter, to conditionally knock-out a floxed coding DNA of the connexin43 (Cx43) gene in mice. Radial glia in the VZ normally express connexin43. They undergo divisions that produce neurons and astrocytes and serve as migratory guides for the daughter cells that they produce. Based on histological analysis, we suggest that removing Cx43 from radial glia alters the normal lamination of the mouse neocortex. To monitor newborn neurons during development, we introduced a plasmid containing green fluorescent protein driven by a neuronal (Talpha1 tubulin) promoter into the embryonic neocortex using in utero electroporation. The transfected migrating neurons remain in the VZ/intermediate zone (IZ) of the Cx43 conditional knock-out (Cx43cKO) animals, whereas in Cx43(fl/fl) mice, neurons migrate through the IZ into the CP, indicating that deletion of Cx43 from nestin-positive cells disrupts neuronal migration. We were able to rescue migration of Cx43cKO neurons by electroporating a cytomegalovirus-Cx43 expression plasmid into the embryonic cortex. In contrast, a C-terminal truncated form of Cx43 failed to rescue neuronal migration. In addition, Cx43(K258stop) mice, in which Cx43 lacks the last 125 amino acid residues of the cytoplasmic C-terminal domain, gave results similar to those seen with the Cx43cKO mice. This study illustrates that deletion of the C-terminal domain of Cx43 alters neuronal migration in the neocortex.

Dose-dependent differential upregulation of CCN1/Cyr61 and CCN3/NOV by the gap junction protein Connexin43 in glioma cells.

Gap junctions form channels that allow exchange of materials between cells and are composed of transmembrane protein subunits called connexins. While connexins are believed to mediate cellular signaling by permitting intercellular communication to occur, there is also increasing evidence that suggest connexins may mediate growth control via a junction-independent mechanism. Connexin43 (Cx43) is the most abundant gap junction protein found in astrocytes, and gliomas exhibit reduced Cx43 expression. We have previously observed that restoration of Cx43 levels in glioma cells led to increased expression of CCN3 (NOV) proteins. We now report that overexpression of Cx43 in C6-glioma cells (C6-Cx43) also upregulates the expression of CCN1 (Cyr61). Both CCN1 and CCN3 belong to the Cyr61/Connective tissue growth factor/Nephroblastoma-overexpressed (CCN) family of secretory proteins. The CCN proteins are tightly associated with the extracellular matrix and have important roles in cell proliferation and migration. CCN1 promotes growth in glioma cells, as shown by the increased proliferation rate of CCN1-overexpressing C6 cells. In addition to its effect on cell growth, CCN1 also increased the motility of glioma cells in the presence of extracellular substrates such as fibronectin. Gliomas expressing high levels of Cx43 preferentially upregulated CCN3 which resulted in reduced growth rate. CCN3 could also be observed in Cx43 gap junction plaques in confluent C6-Cx43H culture at the stationary phase of their growth. Our results suggest that the dissimilar growth characteristics between high and low Cx43 expressors may be due to differential regulation of CCN3 by varying levels of Cx43.

Expression of connexins in embryonic mouse neocortical development.

During embryonic development, young neurons migrate from the ventricular zone to the cortical plate of the cerebral cortex. Disturbances in this neuronal migration have been associated with numerous diseases such as mental retardation, double cortex, Down syndrome, and epilepsy. One possible cause of these neuropathologies is an aberration in normal gap junctional communication. At least 20 connexin (Cx) genes encode gap junction proteins in mice and humans. A proper understanding of the role of specific connexins in the developing brain requires the characterization of their spatial and temporal pattern of expression. In the current study we performed all the experiments on mouse developing cortex at embryonic days (E) 14, 16, and 18, timepoints that are highly active with regard to cortical development. Using reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemistry, we found that among the family of gap junction proteins, Cx26, Cx36, Cx37, Cx43, and Cx45 were expressed in the developing cortex of mice, Cx30 and Cx32 were absent, while Cx40 was expressed at a very low level. Our results demonstrate that Cx26 and Cx37 were evenly distributed in the cortical layers of developing brain, while Cx36 and Cx43 were more abundant in the ventricular zone and cortical plate. Cx45 distribution appeared to be more abundant at E18 compared to the other timepoints (E14 and E16). Thus, the present study provides identification and the distribution pattern for Cxs associated with cortical development during normal neuronal migration.

Tissue microarray analysis of connexin expression and its prognostic significance in human breast cancer.

Breast cancer accounts for approximately 15% of all cancer deaths. Currently, axillary nodal status is the most reliable prognostic indicator for breast cancer. Tumor size and histological grade are used to stage breast cancer. Estrogen receptor/progesterone receptor (ER/PR) and HER-2/neu status are useful in predicting patient survival and relapse. Ki67, an indicator of proliferative activity, also correlates well with prognosis. Connexin proteins form gap junction channels, permitting intercellular exchange of ions and small molecules. Reduced connexin protein levels and impaired gap junctional intercellular communication are associated with tumor phenotypes. This study investigated the prognostic value of connexin proteins as breast cancer markers. Tissue microarrays, containing 438 cases of invasive breast carcinoma, were stained with Cx26, Cx32, and Cx43 antibodies. The degree of connexin immunoreactivity was determined and then correlated with patient outcome, tumor grade, tumor size, lymph node status, and immunohistochemical markers, such as p53, ER/PR status, Ki67 and c-erbB-2 expression. Cx26, Cx32, or Cx43 did not correlate well with tumor grade, tumor size, p53 or c-erbB-2 status. There was an inverse correlation between Cx32 and lymph node status (P <0.05) and a positive correlation between Cx43 and PR status (P <0.01). Cx32 and Cx43 correlated positively with ER status (P <0.01). Cx43 correlated negatively with Ki67 expression (P <0.01). Cx26, Cx32, and Cx43 did not correlate with patient outcome. Based on our observations in this study, connexin proteins do not appear to be reliable indicators of breast cancer prognosis.

Tumor-suppressive effects of pannexin 1 in C6 glioma cells.

Mammalian gap junction proteins, connexins, have long been implicated in tumor suppression. Recently, a novel family of proteins named pannexins has been identified as the mammalian counterpart of the invertebrate gap junction proteins, innexins. To date, pannexin 1 (Panx1) and pannexin 2 (Panx2) mRNAs are reported to be expressed in the brain. Most neoplastic cells, including rat C6 gliomas, exhibit reduced connexin expression, aberrant gap junctional intercellular communication (GJIC), and an increased proliferation rate. When gap junctions are up-regulated by transfecting C6 cells with connexin43, GJIC is restored and the proliferation is reduced. In this study, we examined the tumor-suppressive effects of Panx1 expression in C6 cells. Reverse transcription-PCR analysis revealed that C6 cells do not express any of the pannexin transcripts, whereas its nontumorigenic counterpart, rat primary astrocytes, exhibited mRNAs for all three pannexins. On generation of stable C6 transfectants with tagged Panx1 [myc or enhanced green fluorescent protein (EGFP)], a localization of Panx1 expression to the Golgi apparatus and plasma membrane was observed. In addition, Panx1 transfectants exhibited a flattened morphology, which differs greatly from the spindle-shaped control cells (EGFP only). Moreover, Panx1 expression increased gap junctional coupling as shown by the passage of sulforhodamine 101. Finally, we showed that stable expression of Panx1 in C6 cells significantly reduced cell proliferation in monolayers, cell motility, anchorage-independent growth, and in vivo tumor growth in athymic nude mice. Altogether, we conclude that the loss of pannexin expression may participate in the development of C6 gliomas, whereas restoration of Panx1 plays a tumor-suppressive role.

Genistein and quercetin increase connexin43 and suppress growth of breast cancer cells.

Connexin proteins form gap junctions, which permit direct exchange of cytoplasmic contents between neighboring cells. Evidence indicates that gap junctional intercellular communication (GJIC) is important for maintaining homeostasis and preventing cell transformation. Furthermore, connexins may have independent functions including tumor growth suppression. Most tumors express less connexins, have reduced GJIC and have increased growth rates compared with non-tumorigenic cells. The purpose of this study was to determine whether common flavonoids, genistein and quercetin, increase connexin43 (Cx43) levels, improve GJIC and suppress growth of a metastatic human breast tumor cell line (MDA-MB-231). Quercetin (2.5, 5 microg/ml) and genistein (0.5, 2.5, 15 microg/ml) upregulated Cx43 but failed to increase GJIC. Cx43 localized to the plasma membrane following genistein treatment (2.5, 15 mug/ml). In contrast, Cx43 aggregated in the perinuclear region following quercetin treatment (0.5, 2.5, 5, 15 microg/ml). Both genistein (15 microg/ml) and quercetin (2.5, 5, 15 microg/ml) significantly reduced MDA-MB-231 cell proliferation. In summary, genistein and quercetin increase Cx43 and suppress MDA-MB-231 cell proliferation at physiologically relevant concentrations. These results demonstrate that genistein and quercetin are potential anti-breast cancer agents.

Nontransformed cells can normalize gap junctional communication with transformed cells.

We demonstrate that the Src kinase can augment gap junctional communication between cells derived from homozygous null Cx43 knockout mice. The total conductance between Src transformed cells was nearly twice that of nontransformed cells. In addition, the unitary conductance of the majority of single channel events between transformed cells was about 35% greater than that of nontransformed cells. Analysis showed that both nontransformed and transformed cells expressed at least two populations of channels, suggesting that Src increased junctional conductance by up-regulating one population and/or by increasing the unitary conductance of another population of channels. Interestingly, the conductance displayed by heterologous pairs of transformed and nontransformed cells resembled that of nontransformed cells. The majority of single channel events between heterologous pairs shifted back to lower conductances that were exhibited by nontransformed cells. Thus, nontransformed cells can effectively "normalize" the conductance of gap junction channels expressed by adjacent tumor cells.

CCN3 (NOV) interacts with connexin43 in C6 glioma cells: possible mechanism of connexin-mediated growth suppression.

Many tumor cells exhibit aberrant gap junctional intercellular communication, which can be restored by transfection with connexin genes. We have previously discovered that overexpression of connexin43 (Cx43) in C6 glioma cells not only reduces proliferation but also leads to production of soluble growth-inhibitory factors. We identified that several members of the CCN (Cyr61/connective tissue growth factor/nephroblastoma-overexpressed) family are up-regulated following Cx43 expression, including CCN3 (NOV). We now report evidence for an association between CCN3 and Cx43. Western blot analysis demonstrated that the 48-kDa full-length CCN3 protein was present in the lysate and conditioned medium of growth-suppressed C6-Cx43 cells, as well as primary astrocytes, but not in C6 parental and human glioma cells. Immunocytochemical examination of CCN3 revealed diffuse localization in parental C6 cells, whereas transfection of C6 cells with Cx43 (C6-Cx43) or with a modified Cx43 tagged to green fluorescent protein on its C terminus (Cx43-GFP) resulted in punctate staining, suggesting that CCN3 co-localizes with Cx43 in plaques at the plasma membrane. In cells expressing a C-terminal truncation of Cx43 (Cx43Delta244-382), this co-localization was lost. Glutathione S-transferase pull-down assay and co-immunoprecipitation demonstrated that CCN3 was able to physically interact with Cx43. In contrast, CCN3 was not found to associate with Cx43Delta244-382. Similar experiments revealed that CCN3 did not co-localize or associate with other connexins, including Cx40 or Cx32. Taken together, these data support an interaction of CCN3 with the C terminus of Cx43, which could play an important role in mediating growth control induced by specific gap junction proteins.