Biomedical subjects

K Willecke

Publications and source records attributed to K Willecke.

At least 73 records · Page 4Linked to original sources

PubMed1998

Connexin 32 gap junctions enhance stimulation of glucose output by glucagon and noradrenaline in mouse liver.

Gap junctions connect neighboring cells via intercellular channels composed of connexins (Cx). Connexin 32 (Cx32) is the main connexin in hepatocytes. Gap junctions propagate a signal from periportal to perivenous hepatocytes generated by electrical stimulation of sympathetic liver nerves. Therefore, it was the aim of this study to examine the involvement of hepatocellular gap junctions in hormonal regulation. In perfused livers from wild-type mice and Cx32-deficient mice, the stimulation of glucose release by varying noradrenaline and glucagon concentrations was investigated. At saturating hormone concentrations, glucose release was the same in wild-type and Cx32-deficient livers. However, glucose output was significantly smaller in Cx32-deficient than wild-type livers at half-maximally effective hormone concentrations. Because the two hormones circulate at less than half-saturating concentrations and because they are degraded during passage of blood through the liver, they lose efficiency from the periportal to the perivenous zone. In wild-type livers, this decrease in efficiency can be partially compensated by intercellular signal propagation through gap junctions, resulting in higher hormone actions than in Cx32-deficient livers. It is concluded that gap junctions are not only involved in intercellular propagation of nervous, but also of hormonal signals from periportal to perivenous hepatocytes.

Animals↗

PubMed1998

Downregulation of connexin32 protein and gap-junctional intercellular communication by cytokine-mediated acute-phase response in immortalized mouse hepatocytes.

In the present study, we have analyzed the direct effects of cytokines, which mediate the acute-phase response in liver, on connexin expression and gap-junctional intercellular communication in immortalized MHSV12 mouse hepatocytes. When these cells were stimulated for 24 h with interleukin 1 and interleukin 6, the amount of connexin26 (Cx26) mRNA increased together with beta-fibrinogen mRNA, as expected for this positive acute-phase gene. In contrast, connexin32 (Cx32) mRNA expression was not affected under these conditions. Indirect immunfluorescence revealed a drastic decrease in Cx32 signals, whereas slightly more Cx26 signals were found. Stronger stimulation with interleukin 1 and tumor necrosis factor alpha gave a dose-dependent increase in steady state levels of Cx26 and beta-fibrinogen mRNA, but no further change in Cx32 mRNA level was seen. However, when Cx32 protein was analyzed on immunoblots, we found a 5-fold decrease in expression even at low cytokine doses that did not affect Cx32 mRNA expression. Under these conditions, cell to cell transfer of Lucifer yellow, microinjected into immortalized hepatocytes, was decreased by 70%, suggesting that intercellular communication through Cx32 channels was partially inhibited earlier than other genetic alterations characteristic of the acute-phase response. Thus, the major hepatic gap junction protein was largely downregulated at the beginning of the experimental inflammatory reaction, but about 30% of gap-junctional intercellular communication was maintained. This suggests that, during the acute-phase response, the second hepatic Cx26 protein may compensate in part for the downregulation of the Cx32 protein.

Acute-Phase Reaction↗

PubMed1998

Characterization of the gap junction protein connexin37 in murine endothelium, respiratory epithelium, and after transfection in human HeLa cells.

Affinity-purified antibodies to oligopeptides derived from two different regions of the carboxyterminus and cytoplasmic loop or to the last 103 C-terminal amino acids of mouse connexin37 (Cx37) were used to characterize expression of this gap junctional protein in endothelium of several murine tissues. Cx37 was expressed in endothelium of large blood vessels in brain, liver, kidney, spleen, heart, and lung, but not in capillaries. In addition, weak Cx37 immuno-signals were observed in lung respiratory epithelium of small bronchi and in alveolar epithelial cells of bronchioli. The ratios of Cx37 protein to Cx37 mRNA in adult and embryonic kidney as well as skin were 29-303-fold larger than in lung, suggesting that Cx37 mRNA was translated at different efficiencies in kidney and skin versus lung. Cx37 protein was more abundant in embryonic kidney and lung than in the corresponding adult tissues. After differential centrifugation of plasma membrane fractions in sucrose gradients, we found that Cx37-containing gap junctions in lung were much smaller than Cx32 and Cx26 aggregates from liver. HeLa cells were transfected with mouse Cx37 cDNA. In these cells, mouse Cx37 protein was phosphorylated mainly at serine, less at tyrosine, and very little at threonine residues. Three conductance states were resolved at 110, 240, and 315 pS.

Animals↗

PubMed1998

A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes.

Gap junctions provide direct intercellular communication by linking adjacent cells with aqueous pores permeable to molecules up to 1 kDa in molecular mass and 8-14 A in diameter. The identification of over a dozen connexins in the mammalian gap junction family has stimulated interest in the functional significance of this diversity, including the possibility of selectivity for permeants as seen in other channel classes. Here we present a quantitative comparison of channel permeabilities of different connexins expressed in both HeLa transfectants (rat Cx26, rat Cx32 and mouse Cx45) and Xenopus oocytes (rat Cx26 and rat Cx32). In HeLa cells, we examined permeability to two fluorescent molecules: Lucifer Yellow (LY: anionic, MW 457) and 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI, cationic, MW 350). A comparison of the kinetics of fluorescent dye transfer showed Cx32, Cx26 and Cx45 to have progressively decreasing permeabilities to LY, but increasing permeabilities to DAPI. This pattern was inconsistent with selection based on physical size of the probe, nor could it be accounted for by the differences between clones in the electrical conductance of the monolayers. In Xenopus oocytes, where electrical and dye coupling could be assessed in the same cells, Cx32 coupled oocytes showed an estimated 6-fold greater permeability to LY than those coupled by Cx26, a comparable result to that seen in HeLa cells, where an approximately 9-fold difference was seen. The oocyte system also allowed an examination of Cx32/Cx26 heterotypic gap junction that proved to have a permeability intermediate between the two homotypic forms. Thus, independent of the expression system, it appears that connexins show differential permeabilities that cannot be predicted based on size considerations, but must depend on other features of the probe, such as charge.

Animals↗

PubMed1997

High incidence of spontaneous and chemically induced liver tumors in mice deficient for connexin32.

Connexins are subunits of gap junction channels, which mediate the direct transfer of ions, second messenger molecules and other metabolites between contacting cells. Gap junctions are thought to be involved in tissue homeostasis, embryonic development and the control of cell proliferation [1,2]. It has also been suggested that the loss of intercellular communication via gap junctions may contribute to multistage carcinogenesis [3-5]. We have previously shown that transgenic mice that lack connexin32 (Cx32), the major gap junction protein expressed in hepatocytes, express lower levels of a second hepatic gap junction protein, Cx26, suggesting that Cx32 has a stabilizing effect on Cx26 [6]. Here, we report that male and female one-year-old mice deficient for Cx32 had 25-fold more and 8-fold more spontaneous liver tumors than wild-type mice, respectively. Incorporation of bromodeoxyuridine (BrdU) into the liver was higher for Cx32-deficient mice than for wild-type mice, suggesting that their hepatocyte proliferation rate was higher. Furthermore, intraperitoneal injection, two weeks after birth, of the carcinogen diethylnitrosamine (DEN) led, after one year, both to more liver tumors in Cx32-deficient mice than in controls, and to accelerated tumor growth. Loss of Cx32 protein from hepatic gap junctions is therefore likely to cause enhanced clonal survival and expansion of mutated ('initiated') cells, which results in a higher susceptibility to hepatic tumors. Our results demonstrate that functional gap junctions inhibit the development of spontaneous and chemically induced tumors in mouse liver.

Animals↗

PubMed1997

Structural abnormalities and deficient maintenance of peripheral nerve myelin in mice lacking the gap junction protein connexin 32.

Mutations affecting the connexin 32 (Cx32) gene are associated with the X-linked form of the hereditary peripheral neuropathy Charcot-Marie-Tooth disease (CMTX). We show that Cx32-deficient mice develop a late-onset progressive peripheral neuropathy with abnormalities comparable to those associated with CMTX, thus providing proof of the critical role of Cx32 in the maintenance of peripheral nerve myelin and an animal model for CMTX. Frequently observed features include abnormally thin myelin sheaths, cellular onion bulb formation reflecting myelin degeneration-induced Schwann cell proliferation, and enlarged periaxonal collars while nerve conductance properties are altered only slightly. These observations are consistent with earlier hypotheses suggesting a function of Cx32 as a channel-forming protein that facilitates the communication between the abaxonal and adaxonal aspects of Schwann cell cytoplasm.

Afferent Pathways↗

PubMed1997

Advances in Charcot-Marie-Tooth disease research: cellular function of CMT-related proteins, transgenic animal models, and pathomechanisms. The European CMT Consortium.

The First Workshop of the European Consortium on Charcot-Marie-Tooth (CMT) disease brought together neuroscientists, molecular and cell biologists, neuropathologists, neurologists, and geneticists with a common interest in the understanding of the fundamental mechanisms that underlie the pathogenesis of CMT. The interdisciplinary group of 25 expert scientists discussed recent advances in (i) molecular genetics and histopathology of CMT, (ii) development of suitable animal models, (iii) understanding of the cellular function of CMT-related proteins, and (iv) studies using nerve biopsies from CMT patients. In this minireview, we summarize the key findings presented and discuss their impact on CMT research.

Animals↗

PubMed1997

Coexpression of connexin45 and -32 in oligodendrocytes of rat brain.

Connexin proteins are the subunits of gap junction channels, and are encoded by a gene family. Although several connexin mRNAs were detected in brain, only a few connexin-proteins have been localized to specific cell types in this tissue. Here we describe expression of connexin45 protein in oligodendrocytes in rat hippocampus. Double immunofluorescent staining using specific antibodies to connexin45 and connexin32 paired with cell-type specific marker proteins revealed that connexin45 and connexin32 were co-expressed and colocalized in oligodendrocytes. Each of the connexin antibodies gave rise to the same pattern of punctate fluorescence in the plasma membrane of cell bodies and proximal processes of oligodendrocytes. Connexins in the plasma membrane of oligodendrocytes may form gap junctions between oligodendrocytes, or between oligodendrocytes and astrocytes. Expression of connexin45 in oligodendrocytes may prevent dysmyelinating effects of connexin32 mutations in the central nervous system of Charcot-Marie-Tooth (X-type) patients.

Animals↗

PubMed1997

Expression pattern of connexin gene products at the early developmental stages of the mouse cardiovascular system.

The synchronized contraction of myocytes in cardiac muscle requires the structural and functional integrity of the gap junctions present between these cells. Gap junctions are clusters of intercellular channels formed by transmembrane proteins of the connexin (Cx) family. Products of several Cx genes have been identified in the mammalian heart (eg, Cx45, Cx43, Cx40, and Cx37), and their expression was shown to be regulated during the development of the myocardium. Cx43, Cx40, and Cx45 are components of myocyte gap junctions, and it has also been demonstrated that Cx40 was expressed in the endothelial cells of the blood vessels. The aim of the present work was to investigate the expression and regulation of Cx40, Cx43, and Cx37 during the early stages of mouse heart maturation, between 8.5 days post coitum (dpc), when the first rhythmic contractions appear, and 14.5 dpc, when the four-chambered heart is almost completed. At 8.5 dpc, only the reverse-transcriptase polymerase chain reaction technique has allowed identification of Cx43, Cx40, and Cx37 gene transcripts in mouse heart, suggesting a very low activity level of these genes. From 9.5 dpc, all three transcripts became detectable in whole-mount in situ-hybridized embryos, and the most obvious result was the labeling of the vascular system with Cx40 and Cx37 anti-sense riboprobes. Cx40 and Cx37 gene products (transcript and/or protein) were demonstrated to be expressed in the vascular endothelial cells at all stages examined. By contrast, only Cx37 gene products were found in the endothelial cells of the endocardium. In heart, Cx37 was expressed exclusively in these cells, which rules out any direct involvement of this Cx in the propagation of electrical activity between myocytes and the synchronization of contractions. Between 9.5 and 11.5 dpc, Cx40 gene activation in myocytes was demonstrated to proceed according to a caudorostral gradient involving first the primitive atrium and the common ventricular chamber (9.5 dpc) and then the right ventricle (11.5 dpc). During this period of heart morphogenesis, there is clearly a temporary and asymmetrical regionalization of the Cx40 gene expression that is superimposed on the functional regionalization. In addition, comparison of Cx40 and Cx43 distribution at the above developmental stages has shown that these Cxs have overlapping (left ventricle) or complementary (atrial tissue and right ventricle) expression patterns.

Amino Acid Sequence↗

PubMed1996

Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice.

The gap junctional protein connexin32 is expressed in hepatocytes, exocrine pancreatic cells, Schwann cells, and other cell types. We have inactivated the connexin32 gene by homologous recombination in the mouse genome and have generated homozygous connexin32-deficient mice that were viable and fertile but weighed on the average approximately 17% less than wild-type controls. Electrical stimulation of sympathetic nerves in connexin32-deficient liver triggered a 78% lower amount of glucose mobilization from glycogen stores, when compared with wild-type liver. Thus, connexin32-containing gap junctions are essential in mouse liver for maximal intercellular propagation of the noradrenaline signal from the periportal (upstream) area, where it is received from sympathetic nerve endings, to perivenous (downstream) hepatocytes. In connexin32-defective liver, the amount of connexin26 protein expressed was found to be lower than in wild-type liver, and the total area of gap junction plaques was approximately 1000-fold smaller than in wild-type liver. In contrast to patients with connexin32 defects suffering from X chromosome-linked Charcot-Marie-Tooth disease (CMTX) due to demyelination in Schwann cells of peripheral nerves, connexin32-deficient mice did not show neurological abnormalities when analyzed at 3 months of age. It is possible, however, that they may develop neurodegenerative symptoms at older age.

Animals↗

PubMed1996

Molecular cloning and functional expression of mouse connexin-30,a gap junction gene highly expressed in adult brain and skin.

A new gap junction gene isolated from the mouse genome codes for a connexin protein of 261 amino acids. Because of its theoretical molecular mass of 30.366 kDa, it is named connexin-30. Within the connexin gene family, this protein is most closely related to connexin-26 (77% amino acid sequence identity). The coding region of mouse connexin-30 is uninterrupted by introns and is detected in the mouse genome as a single copy gene that is assigned to mouse chromosome 14 by analysis of mouse x hamster somatic cell hybrids. Abundant amounts of connexin-30 mRNA (two transcripts of 2.0 and 2.3 kilobase pairs) were found after 4 weeks of postnatal development in mouse brain and skin. Microinjection of connexin-30 cRNA into Xenopus oocytes induced formation of functional gap junction channels that gated somewhat asymmetrically in response to transjunctional voltage and at significantly lower voltage (Vo = +38 and -46 mV) than the closely homologous connexin-26 channels (Vo = 89 mV). Heterotypic pairings of connexin-30 with connexin-26 and connexin-32 produced channels with highly asymmetric and rectifying voltage gating, respectively. This suggests that the polarity of voltage gating and the cationic selectivity of connexin-30 are similar to those of its closest homologue, connexin-26.

Amino Acid Sequence↗

PubMed1996

Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.

In gene therapy to treat cancer, typically only a fraction of the tumor cells can be successfully transfected with a gene. However, in the case of brain tumor therapy with the thymidine kinase gene from herpes simplex virus (HSV-tk), not only the cells transfected with the gene but also neighboring others can be killed in the presence of ganciclovir. Such a "bystander" effect is reminiscent of our previous observation that the effect of certain therapeutic agents may be enhanced by their diffusion through gap junctional intercellular communication (GJIC). Herein, we present the evidence, from in vitro studies, that gap junctions could indeed be responsible for such a gene therapy bystander effect. We used HeLa cells for this purpose, since they show very little, if any, ability to communicate through gap junctions. When HeLa cells were transfected with HSV-tk gene and cocultured with nontransfected cells, only HSV-tk-transfected HeLa cells (tk+) were killed by ganciclovir. However, when HeLa cells transfected with a gene encoding for the gap junction protein, connexin 43 (Cx43), were used, not only tk+ cells, but also tk- cells were killed, presumably due to the transfer, via Cx43-mediated GJIC, of toxic ganciclovir molecules phosphorylated by HSV-tk to the tk- cells. Such bystander effect was not observed when tk+ and tk- cells were cocultured without direct cell-cell contact between those two types of cells. Thus, our results give strong evidence that the bystander effect seen in HSV-tk gene therapy may be due to Cx-mediated GJIC.

Cell Communication↗

PubMed1996

Connexin expression systems: to what extent do they reflect the situation in the animal?

Intercellular communication is mediated by specialized cell-cell contact areas known as gap junctions. Connexins are the constitutive proteins of gap junction intercellular channels. Various cell expression systems are used to express connexins and, in turn, these expression systems can then be tested for their ability to form functional cell-cell channels. In this review, expression of murine endogenous connexins in primary cells and established cell lines is compared with results obtained by expression of exogenous connexins in Xenopus oocytes and cultured mammalian cells. In addition, first reports on characterization of connexin-deficient mice are discussed.

Animals↗

PubMed1996

Effect of tumor-promoting and anti-promoting chemicals on the viability and junctional coupling of human HeLa cells transfected with DNAs coding for various murine connexin proteins.

Gap-junctional intercellular communication is thought to be essential for maintaining cellular homeostasis and growth control. Its perturbation entails toxicological implications and it has been correlated with the in vivo tumor-promoting potential of chemicals. Little is known about the mechanism(s) responsible for the tumor promoters interference with the cellular coupling. Moreover, nongenotoxic carcinogens, as well as connexins (gap-junctional protein subunits), are known to be organ-/tissue-specific; this implies that the effect of different agents should be evaluated on their specific target, that is, connexin. To investigate the role of different connexins in regulating gap-junctional gating and to compare the properties of homotypic junctional channels, we evaluated the effects of tissue-specific tumor promoters and anti-promoters on the viability and intercellular coupling (dye-transfer) of HeLa cells stably transfected with cDNAs coding for connexin(cx)43, cx40, cx26 and cx32. The results demonstrate that the transfectants possess individual junctional permeabilities, differentially affected by the chemicals, they also show different sensitivities to the cytotoxic effect of the compounds. These findings confirm that connexin diversity may be responsible for the different gating properties of gap-junctional channels, being also suggestive for their separate functions and independent regulatory mechanisms.

Animals↗

PubMed1996

Incompatibility of connexin 40 and 43 Hemichannels in gap junctions between mammalian cells is determined by intracellular domains.

Murine connexin 40 (Cx40) and connexin 43 (Cx43) do not form functional heterotypic gap junction channels. This property may contribute to the preferential propagation of action potentials in murine conductive myocardium (expressing Cx40) which is surrounded by working myocardium, expressing Cx43. When mouse Cx40 and Cx43 were individually expressed in cocultured human HeLa cells, no punctate immunofluorescent signals were detected on apposed plasma membranes between different transfectants, using antibodies specific for each connexin, suggesting that Cx40 and Cx43 hemichannels do not dock to each other. We wanted to identify domains in these connexin proteins which are responsible for the incompatibility. Thus, we expressed in HeLa cells several chimeric gene constructs in which different extracellular and intracellular domains of Cx43 had been spliced into the corresponding regions of Cx40. We found that exchange of both extracellular loops (E1 and E2) in this system (Cx40*43E1,2) was required for formation of homotypic and heterotypic conductive channels, although the electrical properties differed from those of Cx40 or Cx43 channels. Thus, the extracellular domains of Cx43 can be directed to form functional homo- and heterotypic channels. Another chimeric construct in which both extracellular domains and the central cytoplasmic loop (E1, E2, and C2) of Cx43 were spliced into Cx40 (Cx40*43E1,2,C2) led to heterotypic coupling only with Cx43 and not with Cx40 transfectants. Thus, the central cytoplasmic loop of Cx43 contributed to selectivity. A third construct, in which only the C-terminal domain (C3) of Cx43 was spliced into Cx40, i.e., Cx40*43C3, showed neither homotypic nor heterotypic coupling with Cx40 and Cx43 transfectants, suggesting that the C-terminal region of Cx43 determined incompatibility.

Animals↗

PubMed1996

Expression of the gap junction proteins connexin31 and connexin43 correlates with communication compartments in extraembryonic tissues and in the gastrulating mouse embryo, respectively.

We have characterized the pattern of connexin expression in embryonic and extraembryonic tissues during early mouse development. In the preimplantation blastocyst, at 3.5 days post coitum (dpc), immunofluorescent signals specific for connexin31 and connexin43 proteins were present in both the inner cell mass and the trophectoderm, as shown by confocal laser scan microscopy. Immediately after implantation at 6.5 dpc, however, we find complete compartmentation of these two connexins: connexin31 mRNA and protein are expressed exclusively in cells derived from the trophectoderm lineage, whereas connexin43 mRNA and protein are detected in cells derived from the inner cell mass. This expression pattern of connexin31 and connexin43 is maintained at 7.5 dpc when the axial polarity of the mouse embryo is established. It correlates with the communication compartments in extraembryonic tissues and the gastrulating mouse embryo, respectively. The communication boundary between those compartments may be due to incompatibility of connexin31 and connexin43 hemichannels, which do not communicate with each other in cell culture.

Animals↗

PubMed1996

A second alternative transcript of the gap junction gene connexin32 is expressed in murine Schwann cells and modulated in injured sciatic nerve.

Four connexin32 (Cx32) cDNA clones isolated from a rat sciatic nerve cDNA library differ in the nucleotide sequence of their 5' untranslated region (UTR) from the corresponding Cx32 cDNA clones previously characterized from liver. The new Cx32 5'UTR sequence detected in the sciatic nerve cDNA clones is identical to one previously found in the 6.5 kb intron of the murine Cx32 gene. Using primer extension and S1 nuclease protection analysis, we determined the transcriptional starting point of this new alternative Cx32 transcript expressed in the sciatic nerve. This starting point is located 444 bp (409 bp) upstream of exon2 in a region previously described as an intron of the Cx32 gene in the rat (and mouse) genome, respectively. The alternative exon1B comprises 99 bp in rat, but 97 bp in the mouse genome, and is spliced to the same exon2 acceptor site also used for splicing of exon1 in liver. Both transcripts are likely to code for the same Cx32 protein whose reading frame is located in exon2. The putative promoter region, upstream of the alternative exon1B, contains a TATAAA motif and has been sequenced and noticed before by Miller et al. (Biosci. Rep. 8, 455-464, (1988)). The alternative exon1B transcript is highly expressed in the sciatic nerve, (i.e. Schwann cells) and very low in liver (i.e. hepatocytes). Its expression is regulated after sciatic nerve injury. The time course of expression was similar to previously established myelin genes and, therefore, we suggest that the expression of the alternative exon1B Cx32 transcript is related to the process of myelination. Very recently, we have characterized another alternative Cx32 exon1A which is transcribed in mouse embryonic stem cells but not in the sciatic nerve (Dahl et al., submitted for publication, 1995). Thus, the murine Cx32 gene is likely to be regulated by three alternative promoters that appear to be activated in a cell type-specific manner.

Animals↗

PubMed1995

Negative growth control of HeLa cells by connexin genes: connexin species specificity.

In order to examine whether different connexin gene species exert different degrees of tumor-suppressing activity, we characterized growth characteristics of a gap junction-deficient human cancer cell line, HeLa cells, before and after transfection with cDNA for three different connexins, connexin (cx) 26, cx 40, and cx 43. All transfected cell lines (3 clones transfected with the cx 26 gene, 2 clones with cx 40, and 1 with cx 43) showed establishment of gap junctional intercellular communication (GJIC). Two of the cx 26-transfected clones showed significantly slower growth compared with the parental HeLa cells. When transfectants were grown in soft agar, the three cx 26-transfected clones grew much less than the other transfectants and parent HeLa cells. When injected into nude mice, the two cx 26 clones which exhibited the highest amount of cx 26 transcript induced almost no tumors, whereas other transfectants, including the cx 26 clone which exhibited the lowest amount of cx 26 transcript, were tumorigenic. Among transfectants of various connexin genes, there was no good inverse correlation between their GJIC and tumorigenicity. GJIC levels were significantly higher in tumors induced in nude mice by clone cx 26 A and E transfectants. These results suggest that all of the connexin genes examined could induce recovery of GJIC of HeLa cells, but only the cx 26 gene exerts strong negative growth control on HeLa cells; thus, this connexin gene may have different functions from other connexin genes.

Animals↗