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Dale W. Laird - One of the best experts on this subject based on the ideXlab platform.
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Effects of Reduced Connexin43 Function on Mandibular Morphology and Osteogenesis in Mutant Mouse Models of Oculodentodigital Dysplasia
Calcified Tissue International, 2020Co-Authors: Alyssa C. Moore, Dale W. Laird, Elizabeth Jewlal, Kevin Barr, Katherine E. WillmoreAbstract:Mutations in the gene encoding the gap-junctional protein connexin43 (Cx43) are the cause of the human disease Oculodentodigital Dysplasia (ODDD). The mandible is often affected in this disease, with clinical reports describing both mandibular overgrowth and conversely, retrognathia. These seemingly opposing observations underscore our relative lack of understanding of how ODDD affects mandibular morphology. Using two mutant mouse models that mimic the ODDD phenotype (I130T/+ and G60S/+), we sought to uncover how altered Cx43 function may affect mandibular development. Specifically, mandibles of newborn mice were imaged using micro-CT, to enable statistical comparisons of shape. Tissue-level comparisons of key regions of the mandible were conducted using histomorphology, and we quantified the mRNA expression of several cartilage and bone cell differentiation markers. Both G60S/+ and I130T/+ mutant mice had altered mandibular morphology compared to their wildtype counterparts, and the morphological effects were similarly localized for both mutants. Specifically, the biggest phenotypic differences in mutant mice were focused in regions exposed to mechanical forces, such as alveolar bone, muscular attachment sites, and articular surfaces. Histological analyses revealed differences in ossification of the intramembranous bone of the mandibles of both mutant mice compared to their wildtype littermates. However, chondrocyte organization within the secondary cartilages of the mandible was unaffected in the mutant mice. Overall, our results suggest that the morphological differences seen in G60S/+ and I130T/+ mouse mandibles are due to delayed ossification and suggest that mechanical forces may exacerbate the effects of ODDD on the skeleton.
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Effects of reduced connexin43 function on skull development in the Cx43I130T/+ mutant mouse that models Oculodentodigital Dysplasia.
Bone, 2020Co-Authors: Sommer E. Jarvis, Dale W. Laird, Kevin J. Barr, Elizabeth Jewlal, Jae Eun Lee, Gregory M. Kelly, Katherine E. WillmoreAbstract:Oculodentodigital Dysplasia (ODDD) is a disease caused by mutations in the GJA1 gene that encodes the gap-junctional protein connexin43 (Cx43). ODDD affects multiple organs, but craniofacial anomalies are typical. However, details on the timing of phenotypic presentation of these abnormalities and their correspondence with potential cellular changes are incomplete. Here, we perform the first assessment of the development of the ODDD craniofacial phenotype in the Cx43I130T/+ mouse model and show that the phenotypic features commonly found in patients with the disorder arise in mice between E17.5 and birth and become more profound with age. Using mice heterozygous for the I130T mutation of Gja1, we provide a detailed analysis of the craniofacial phenotype in this ODDD model using shape analyses based on micro-CT images. Results show that in addition to differences in facial bone morphology, there are significant shape differences in the cranial base. Mutant mice display delayed ossification at E17.5 and birth, particularly in bones of the face and cranial vault but ossification is normal at three months. Our immunohistochemical analyses of the palatine bone indicate that osteoblast differentiation is delayed in Cx43I130T/+ mice compared to their wildtype littermates, which likely contributes to the phenotypic variations observed in the facial bones. Our histological and immunohistochemical analyses of the synchondroses of the cranial base show no differences in molecular indicators of chondrocyte differentiation in mutant mice, suggesting that the differences to cranial base morphology displayed by Cx43I130T/+ mice are not due to differences in chondrocyte proliferation or differentiation. Together, our findings suggest that Cx43I130T/+ mice represent a surrogate model to not only inform about the craniofacial anomalies found in ODDD patients but also to show that reduced Cx43 function leads to phenotypic changes that are largely due to osteoblast defects.
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Mice harbouring an Oculodentodigital Dysplasia-linked Cx43 G60S mutation have severe hearing loss.
Journal of cell science, 2018Co-Authors: Julia M. Abitbol, Kevin J. Barr, John J. Kelly, Brian L. Allman, Dale W. LairdAbstract:ABSTRACT Given the importance of connexin43 (Cx43, encoded by GJA1 ) function in the central nervous system and sensory organ processing, we proposed that it would also be crucial in auditory function. To that end, hearing was examined in two mouse models of Oculodentodigital Dysplasia that globally express GJA1 mutations resulting in mild or severe loss of Cx43 function. Although Cx43 I130T/+ mutant mice, with ∼50% Cx43 channel function, did not have any hearing loss, Cx43 G60S/+ mutant mice, with ∼20% Cx43 channel function, had severe hearing loss. There was no evidence of inner ear sensory hair cell loss, suggesting that the mechanism for Cx43-linked hearing loss lies downstream in the auditory pathway. Since evidence suggests that Cx26 function is essential for hearing and may be protective against noise-induced hearing loss, we challenged Cx43 I130T/+ mice with a loud noise and found that they had a similar susceptibility to noise-induced hearing loss to that found in controls, suggesting that decreased Cx43 function does not sensitize the mice for environmentally induced hearing loss. Taken together, this study suggests that Cx43 plays an important role in baseline hearing and is essential for auditory processing. This article has an associated First Person interview with the first author of the paper.
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Specific functional pathologies of Cx43 mutations associated with Oculodentodigital Dysplasia.
Molecular biology of the cell, 2016Co-Authors: John J. Kelly, Qing Shao, Donglin Bai, Jessica L. Esseltine, Ethylin Wang Jabs, Jacinda B. Sampson, Mari Auranen, Dale W. LairdAbstract:Oculodentodigital Dysplasia (ODDD) is a rare genetic disease that affects the development of multiple organs in the human body. More than 70 mutations in the gap junction connexin43 (Cx43) gene, GJA1, are associated with ODDD, most of which are inherited in an autosomal dominant manner. Many patients exhibit similar clinical presentations. However, there is high intrafamilial and interfamilial phenotypic variability. To better understand this variability, we established primary human dermal fibroblast cultures from several ODDD patients and unaffected controls. In the present study, we characterized three fibroblast lines expressing heterozygous p.L7V, p.G138R, and p.G143S Cx43 variants. All ODDD fibroblasts exhibited slower growth, reduced migration, and defective cell polarization, traits common to all ODDD fibroblasts studied so far. However, we found striking differences in overall expression levels, with p.L7V down-regulated at the mRNA and protein level. Although all of the Cx43 variants could traffic to the cell surface, there were stark differences in gap junction plaque formation, gap junctional intercellular communication, Cx43 phosphorylation, and hemichannel activity among Cx43 variants, as well as subtle differences in myofibroblast differentiation. Together these findings enabled us to discover mutation-specific pathologies that may help to predict future clinical outcomes.
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Manipulating Cx43 expression triggers gene reprogramming events in dermal fibroblasts from Oculodentodigital Dysplasia patients.
The Biochemical journal, 2015Co-Authors: Jessica L. Esseltine, Qing Shao, John J. Kelly, Jacinda B. Sampson, Tao Huang, Dale W. LairdAbstract:Oculodentodigital Dysplasia (ODDD) is primarily an autosomal dominant disorder linked to over 70 GJA1 gene [connexin43 (Cx43)] mutations. For nearly a decade, our laboratory has been investigating the relationship between Cx43 and ODDD by expressing disease-linked mutants in reference cells, tissue-relevant cell lines, 3D organ cultures and by using genetically modified mouse models of human disease. Although salient features of Cx43 mutants have been revealed, these models do not necessarily reflect the complexity of the human context. To further overcome these limitations, we have acquired dermal fibroblasts from two ODDD-affected individuals harbouring D3N and V216L mutations in Cx43, along with familial controls. Using these ODDD patient dermal fibroblasts, which naturally produce less GJA1 gene product, along with RNAi and RNA activation (RNAa) approaches, we show that manipulating Cx43 expression triggers cellular gene reprogramming. Quantitative RT-PCR, Western blot and immunofluorescent analysis of ODDD patient fibroblasts show unusually high levels of extracellular matrix (ECM)-interacting proteins, including integrin α5β1, matrix metalloproteinases as well as secreted ECM proteins collagen-I and laminin. Cx43 knockdown in familial control cells produces similar effects on ECM expression, whereas Cx43 transcriptional up-regulation using RNAa decreases production of collagen-I. Interestingly, the enhanced levels of ECM-associated proteins in ODDD V216L fibroblasts is not only a consequence of increased ECM gene expression, but also due to an apparent deficit in collagen-I secretion which may further contribute to impaired collagen gel contraction in ODDD fibroblasts. These findings further illuminate the altered function of Cx43 in ODDD-affected individuals and highlight the impact of manipulating Cx43 expression in human cells.
William A Paznekas - One of the best experts on this subject based on the ideXlab platform.
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tyrosine dependent basolateral targeting of human connexin43 eyfp in madin darby canine kidney cells can be disrupted by the Oculodentodigital Dysplasia mutation l90v
FEBS Journal, 2009Co-Authors: Jana Chtchetinin, William A Paznekas, Ethylin Wang Jabs, Wes D. Gifford, Albert LaiAbstract:Polarized membrane sorting of connexin 43 (Cx43) has not been well-characterized. Based on the presence of a putative sorting signal, YKLV(286–289), within its C-terminal cytoplasmic domain, we hypothesized that Cx43 is selectively expressed on the basolateral surface of Madin–Darby canine kidney (MDCK) cells in a tyrosine-dependent manner. We generated stable MDCK cell lines expressing human wild-type and mutant Cx43–eYFP, and analyzed the membrane localization of Cx43–eYFP within polarized monolayers using confocal microscopy and selective surface biotinylation. We found that wild-type Cx43–eYFP was selectively targeted to the basolateral membrane domain of MDCK cells. Substitution of alanine for Y286 disrupted basolateral targeting of Cx43–eYFP. Additionally, substitution of a sequence containing the transferrin receptor internalization signal, LSYTRF, for PGYKLV(284–289) also disrupted basolateral targeting. Taken together, these results indicate that Y286 in its native amino acid sequence is necessary for targeting Cx43–eYFP to the basolateral membrane domain of MDCK cells. To determine whether the F52dup or L90V Oculodentodigital Dysplasia -associated mutations could affect polarized sorting of Cx43–eYFP, we analyzed the expression of these Cx43–eYFP mutant constructs and found that the L90V mutation disrupted basolateral expression. These findings raise the possibility that some oculodentodigitial Dysplasia-associated mutations contribute to disease by altering polarized targeting of Cx43.
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Tyrosine-dependent basolateral targeting of human connexin43–eYFP in Madin–Darby canine kidney cells can be disrupted by the Oculodentodigital Dysplasia mutation L90V
The FEBS journal, 2009Co-Authors: Jana Chtchetinin, William A Paznekas, Ethylin Wang Jabs, Wes D. Gifford, Albert LaiAbstract:Polarized membrane sorting of connexin 43 (Cx43) has not been well-characterized. Based on the presence of a putative sorting signal, YKLV(286–289), within its C-terminal cytoplasmic domain, we hypothesized that Cx43 is selectively expressed on the basolateral surface of Madin–Darby canine kidney (MDCK) cells in a tyrosine-dependent manner. We generated stable MDCK cell lines expressing human wild-type and mutant Cx43–eYFP, and analyzed the membrane localization of Cx43–eYFP within polarized monolayers using confocal microscopy and selective surface biotinylation. We found that wild-type Cx43–eYFP was selectively targeted to the basolateral membrane domain of MDCK cells. Substitution of alanine for Y286 disrupted basolateral targeting of Cx43–eYFP. Additionally, substitution of a sequence containing the transferrin receptor internalization signal, LSYTRF, for PGYKLV(284–289) also disrupted basolateral targeting. Taken together, these results indicate that Y286 in its native amino acid sequence is necessary for targeting Cx43–eYFP to the basolateral membrane domain of MDCK cells. To determine whether the F52dup or L90V Oculodentodigital Dysplasia -associated mutations could affect polarized sorting of Cx43–eYFP, we analyzed the expression of these Cx43–eYFP mutant constructs and found that the L90V mutation disrupted basolateral expression. These findings raise the possibility that some oculodentodigitial Dysplasia-associated mutations contribute to disease by altering polarized targeting of Cx43.
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GJA1 mutations, variants, and connexin 43 dysfunction as it relates to the Oculodentodigital Dysplasia phenotype.
Human mutation, 2009Co-Authors: William A Paznekas, Simeon A Boyadjiev, Barbara A. Karczeski, Sascha Vermeer, R. Brian Lowry, Martin B. Delatycki, Faivre Laurence, Pasi A. Koivisto, Lionel Van Maldergem, Joann N BodurthaAbstract:The predominantly autosomal dominant disorder, Oculodentodigital Dysplasia (ODDD) has high penetrance with intra- and interfamilial phenotypic variability. Abnormalities observed in ODDD affect the eye, dentition, and digits of the hands and feet. Patients present with a characteristic facial appearance, narrow nose, and hypoplastic alae nasi. Neurological problems, including dysarthria, neurogenic bladder disturbances, spastic paraparesis, ataxia, anterior tibial muscle weakness, and seizures, are known to occur as well as conductive hearing loss, cardiac defects, and anomalies of the skin, hair, and nails. In 2003, our analysis of 17 ODDD families revealed that each had a different mutation within the human gap junction alpha 1 (GJA1) gene which encodes the protein connexin 43 (Cx43). Since then at least 17 publications have identified an additional 26 GJA1 mutations and in this study, we present 28 new cases with 18 novel GJA1 mutations. We include tables summarizing the 62 known GJA1 nucleotide changes leading to Cx43 protein alterations and the phenotypic information available on 177 affected individuals from 54 genotyped families. Mutations resulting in ODDD occur in each of the nine domains of the Cx43 protein, and we review our functional experiments and those in the literature, examining the effects of 13 different Cx43 mutations upon gap junction activity.
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Oculodentodigital Dysplasia connexin43 mutations result in non-functional connexin hemichannels and gap junctions in C6 glioma cells
Journal of Cell Science, 2006Co-Authors: Albert Lai, William A Paznekas, Ethylin Wang Jabs, Wes D. Gifford, Andrew CharlesAbstract:Oculodentodigital Dysplasia (ODDD) is a rare developmental disorder characterized by craniofacial and limb abnormalities. Over 35 separate mutations in human connexin43 (Cx43) causing ODDD have been identified. Several mutations are also associated with central nervous system involvement, including white-matter changes detected by magnetic resonance imaging. As Cx43 is abundantly expressed in astrocytes, we hypothesized that the mutant Cx43 proteins that produce neurological dysfunction have abnormal functional characteristics in astrocytes. To understand how ODDD-associated mutations affect Cx43 signaling in cells of glial origin, we conducted studies in rat C6 glioma cells, a communication-deficient glial cell line that expresses low levels of Cx43. We generated stable cell lines expressing enhanced yellow fluorescent protein (eYFP)-tagged human Cx43 constructs encoding wild-type and six eYFP-tagged mutant Cx43 mutants: Y17S, G21R, A40V, F52dup, L90V and I130T. Of these, Y17S, L90V and I130T are associated with neurological abnormalities. We found that all mutants could be detected on the cell surface. Y17S, G21R, A40V, L90V and I130T formed triton-resistant plaques representing gap junctions, although the relative ability to form plaques was decreased in these mutants compared with the wild type. F52dup formed dramatically reduced numbers of plaques. Propidium iodide uptake experiments demonstrated that all mutants were associated with reduced connexin hemichannel function compared with wild type. Scrape-loading experiments performed on the same stable cell lines showed reduced gap junctional dye transfer in all mutants compared with the wild type. These studies demonstrated that ODDD-associated Cx43 mutations result in non-functional connexin hemichannels and gap junction functions in a glial cell line regardless of whether the particular mutant is associated with neurological dysfunction.
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Functional Characterization of Connexin43 Mutations Found in Patients With Oculodentodigital Dysplasia
Circulation research, 2005Co-Authors: Junko Shibayama, William A Paznekas, Ethylin Wang Jabs, Akiko Seki, Steven M. Taffet, Mario Delmar, Hassan Hussein MusaAbstract:Specific mutations in GJA1, the gene encoding the gap junction protein connexin43 (Cx43), cause an autosomal dominant disorder called Oculodentodigital Dysplasia (ODDD). Here, we characterize the effects of 8 of these mutations on Cx43 function. Immunochemical studies have shown that most of the mutant proteins formed gap junction plaques at the sites of cell-cell apposition. However, 2 of the mutations (a codon duplication in the first extracellular loop, F52dup, and a missense mutation in the second extracellular loop, R202H, produced full-length connexins that failed to properly form gap junction plaques. Cx43 proteins containing ODDD mutations found in the N-terminus (Y17S), first transmembrane domain (G21R, A40V), second transmembrane domain (L90V), and cytoplasmic loop (I130T, K134E) do form gap junction plaques but show compromised channel function. L90V, I130T, and K134E demonstrated a significant decrease in junctional conductance relative to Cx43WT. Mutations Y17S, G21R, and A40V demonstrated a complete lack of functional electrical coupling even in the presence of significant plaque formation between paired cells. Heterologous channels formed by coexpression of Cx43WT and mutation R202H resulted in electrically functional gap junctions that were not permeable to Lucifer yellow. Therefore, the mutations found in ODDD not only cause phenotypic variability, but also result in various functional consequences. Overall, our data show an extensive range of molecular phenotypes, consistent with the pleiotropic nature of the clinical syndrome as a whole.
Ethylin Wang Jabs - One of the best experts on this subject based on the ideXlab platform.
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Specific functional pathologies of Cx43 mutations associated with Oculodentodigital Dysplasia.
Molecular biology of the cell, 2016Co-Authors: John J. Kelly, Qing Shao, Donglin Bai, Jessica L. Esseltine, Ethylin Wang Jabs, Jacinda B. Sampson, Mari Auranen, Dale W. LairdAbstract:Oculodentodigital Dysplasia (ODDD) is a rare genetic disease that affects the development of multiple organs in the human body. More than 70 mutations in the gap junction connexin43 (Cx43) gene, GJA1, are associated with ODDD, most of which are inherited in an autosomal dominant manner. Many patients exhibit similar clinical presentations. However, there is high intrafamilial and interfamilial phenotypic variability. To better understand this variability, we established primary human dermal fibroblast cultures from several ODDD patients and unaffected controls. In the present study, we characterized three fibroblast lines expressing heterozygous p.L7V, p.G138R, and p.G143S Cx43 variants. All ODDD fibroblasts exhibited slower growth, reduced migration, and defective cell polarization, traits common to all ODDD fibroblasts studied so far. However, we found striking differences in overall expression levels, with p.L7V down-regulated at the mRNA and protein level. Although all of the Cx43 variants could traffic to the cell surface, there were stark differences in gap junction plaque formation, gap junctional intercellular communication, Cx43 phosphorylation, and hemichannel activity among Cx43 variants, as well as subtle differences in myofibroblast differentiation. Together these findings enabled us to discover mutation-specific pathologies that may help to predict future clinical outcomes.
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tyrosine dependent basolateral targeting of human connexin43 eyfp in madin darby canine kidney cells can be disrupted by the Oculodentodigital Dysplasia mutation l90v
FEBS Journal, 2009Co-Authors: Jana Chtchetinin, William A Paznekas, Ethylin Wang Jabs, Wes D. Gifford, Albert LaiAbstract:Polarized membrane sorting of connexin 43 (Cx43) has not been well-characterized. Based on the presence of a putative sorting signal, YKLV(286–289), within its C-terminal cytoplasmic domain, we hypothesized that Cx43 is selectively expressed on the basolateral surface of Madin–Darby canine kidney (MDCK) cells in a tyrosine-dependent manner. We generated stable MDCK cell lines expressing human wild-type and mutant Cx43–eYFP, and analyzed the membrane localization of Cx43–eYFP within polarized monolayers using confocal microscopy and selective surface biotinylation. We found that wild-type Cx43–eYFP was selectively targeted to the basolateral membrane domain of MDCK cells. Substitution of alanine for Y286 disrupted basolateral targeting of Cx43–eYFP. Additionally, substitution of a sequence containing the transferrin receptor internalization signal, LSYTRF, for PGYKLV(284–289) also disrupted basolateral targeting. Taken together, these results indicate that Y286 in its native amino acid sequence is necessary for targeting Cx43–eYFP to the basolateral membrane domain of MDCK cells. To determine whether the F52dup or L90V Oculodentodigital Dysplasia -associated mutations could affect polarized sorting of Cx43–eYFP, we analyzed the expression of these Cx43–eYFP mutant constructs and found that the L90V mutation disrupted basolateral expression. These findings raise the possibility that some oculodentodigitial Dysplasia-associated mutations contribute to disease by altering polarized targeting of Cx43.
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Tyrosine-dependent basolateral targeting of human connexin43–eYFP in Madin–Darby canine kidney cells can be disrupted by the Oculodentodigital Dysplasia mutation L90V
The FEBS journal, 2009Co-Authors: Jana Chtchetinin, William A Paznekas, Ethylin Wang Jabs, Wes D. Gifford, Albert LaiAbstract:Polarized membrane sorting of connexin 43 (Cx43) has not been well-characterized. Based on the presence of a putative sorting signal, YKLV(286–289), within its C-terminal cytoplasmic domain, we hypothesized that Cx43 is selectively expressed on the basolateral surface of Madin–Darby canine kidney (MDCK) cells in a tyrosine-dependent manner. We generated stable MDCK cell lines expressing human wild-type and mutant Cx43–eYFP, and analyzed the membrane localization of Cx43–eYFP within polarized monolayers using confocal microscopy and selective surface biotinylation. We found that wild-type Cx43–eYFP was selectively targeted to the basolateral membrane domain of MDCK cells. Substitution of alanine for Y286 disrupted basolateral targeting of Cx43–eYFP. Additionally, substitution of a sequence containing the transferrin receptor internalization signal, LSYTRF, for PGYKLV(284–289) also disrupted basolateral targeting. Taken together, these results indicate that Y286 in its native amino acid sequence is necessary for targeting Cx43–eYFP to the basolateral membrane domain of MDCK cells. To determine whether the F52dup or L90V Oculodentodigital Dysplasia -associated mutations could affect polarized sorting of Cx43–eYFP, we analyzed the expression of these Cx43–eYFP mutant constructs and found that the L90V mutation disrupted basolateral expression. These findings raise the possibility that some oculodentodigitial Dysplasia-associated mutations contribute to disease by altering polarized targeting of Cx43.
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Oculodentodigital Dysplasia connexin43 mutations result in non-functional connexin hemichannels and gap junctions in C6 glioma cells
Journal of Cell Science, 2006Co-Authors: Albert Lai, William A Paznekas, Ethylin Wang Jabs, Wes D. Gifford, Andrew CharlesAbstract:Oculodentodigital Dysplasia (ODDD) is a rare developmental disorder characterized by craniofacial and limb abnormalities. Over 35 separate mutations in human connexin43 (Cx43) causing ODDD have been identified. Several mutations are also associated with central nervous system involvement, including white-matter changes detected by magnetic resonance imaging. As Cx43 is abundantly expressed in astrocytes, we hypothesized that the mutant Cx43 proteins that produce neurological dysfunction have abnormal functional characteristics in astrocytes. To understand how ODDD-associated mutations affect Cx43 signaling in cells of glial origin, we conducted studies in rat C6 glioma cells, a communication-deficient glial cell line that expresses low levels of Cx43. We generated stable cell lines expressing enhanced yellow fluorescent protein (eYFP)-tagged human Cx43 constructs encoding wild-type and six eYFP-tagged mutant Cx43 mutants: Y17S, G21R, A40V, F52dup, L90V and I130T. Of these, Y17S, L90V and I130T are associated with neurological abnormalities. We found that all mutants could be detected on the cell surface. Y17S, G21R, A40V, L90V and I130T formed triton-resistant plaques representing gap junctions, although the relative ability to form plaques was decreased in these mutants compared with the wild type. F52dup formed dramatically reduced numbers of plaques. Propidium iodide uptake experiments demonstrated that all mutants were associated with reduced connexin hemichannel function compared with wild type. Scrape-loading experiments performed on the same stable cell lines showed reduced gap junctional dye transfer in all mutants compared with the wild type. These studies demonstrated that ODDD-associated Cx43 mutations result in non-functional connexin hemichannels and gap junction functions in a glial cell line regardless of whether the particular mutant is associated with neurological dysfunction.
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Functional Characterization of Connexin43 Mutations Found in Patients With Oculodentodigital Dysplasia
Circulation research, 2005Co-Authors: Junko Shibayama, William A Paznekas, Ethylin Wang Jabs, Akiko Seki, Steven M. Taffet, Mario Delmar, Hassan Hussein MusaAbstract:Specific mutations in GJA1, the gene encoding the gap junction protein connexin43 (Cx43), cause an autosomal dominant disorder called Oculodentodigital Dysplasia (ODDD). Here, we characterize the effects of 8 of these mutations on Cx43 function. Immunochemical studies have shown that most of the mutant proteins formed gap junction plaques at the sites of cell-cell apposition. However, 2 of the mutations (a codon duplication in the first extracellular loop, F52dup, and a missense mutation in the second extracellular loop, R202H, produced full-length connexins that failed to properly form gap junction plaques. Cx43 proteins containing ODDD mutations found in the N-terminus (Y17S), first transmembrane domain (G21R, A40V), second transmembrane domain (L90V), and cytoplasmic loop (I130T, K134E) do form gap junction plaques but show compromised channel function. L90V, I130T, and K134E demonstrated a significant decrease in junctional conductance relative to Cx43WT. Mutations Y17S, G21R, and A40V demonstrated a complete lack of functional electrical coupling even in the presence of significant plaque formation between paired cells. Heterologous channels formed by coexpression of Cx43WT and mutation R202H resulted in electrically functional gap junctions that were not permeable to Lucifer yellow. Therefore, the mutations found in ODDD not only cause phenotypic variability, but also result in various functional consequences. Overall, our data show an extensive range of molecular phenotypes, consistent with the pleiotropic nature of the clinical syndrome as a whole.
Albert Lai - One of the best experts on this subject based on the ideXlab platform.
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tyrosine dependent basolateral targeting of human connexin43 eyfp in madin darby canine kidney cells can be disrupted by the Oculodentodigital Dysplasia mutation l90v
FEBS Journal, 2009Co-Authors: Jana Chtchetinin, William A Paznekas, Ethylin Wang Jabs, Wes D. Gifford, Albert LaiAbstract:Polarized membrane sorting of connexin 43 (Cx43) has not been well-characterized. Based on the presence of a putative sorting signal, YKLV(286–289), within its C-terminal cytoplasmic domain, we hypothesized that Cx43 is selectively expressed on the basolateral surface of Madin–Darby canine kidney (MDCK) cells in a tyrosine-dependent manner. We generated stable MDCK cell lines expressing human wild-type and mutant Cx43–eYFP, and analyzed the membrane localization of Cx43–eYFP within polarized monolayers using confocal microscopy and selective surface biotinylation. We found that wild-type Cx43–eYFP was selectively targeted to the basolateral membrane domain of MDCK cells. Substitution of alanine for Y286 disrupted basolateral targeting of Cx43–eYFP. Additionally, substitution of a sequence containing the transferrin receptor internalization signal, LSYTRF, for PGYKLV(284–289) also disrupted basolateral targeting. Taken together, these results indicate that Y286 in its native amino acid sequence is necessary for targeting Cx43–eYFP to the basolateral membrane domain of MDCK cells. To determine whether the F52dup or L90V Oculodentodigital Dysplasia -associated mutations could affect polarized sorting of Cx43–eYFP, we analyzed the expression of these Cx43–eYFP mutant constructs and found that the L90V mutation disrupted basolateral expression. These findings raise the possibility that some oculodentodigitial Dysplasia-associated mutations contribute to disease by altering polarized targeting of Cx43.
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Tyrosine-dependent basolateral targeting of human connexin43–eYFP in Madin–Darby canine kidney cells can be disrupted by the Oculodentodigital Dysplasia mutation L90V
The FEBS journal, 2009Co-Authors: Jana Chtchetinin, William A Paznekas, Ethylin Wang Jabs, Wes D. Gifford, Albert LaiAbstract:Polarized membrane sorting of connexin 43 (Cx43) has not been well-characterized. Based on the presence of a putative sorting signal, YKLV(286–289), within its C-terminal cytoplasmic domain, we hypothesized that Cx43 is selectively expressed on the basolateral surface of Madin–Darby canine kidney (MDCK) cells in a tyrosine-dependent manner. We generated stable MDCK cell lines expressing human wild-type and mutant Cx43–eYFP, and analyzed the membrane localization of Cx43–eYFP within polarized monolayers using confocal microscopy and selective surface biotinylation. We found that wild-type Cx43–eYFP was selectively targeted to the basolateral membrane domain of MDCK cells. Substitution of alanine for Y286 disrupted basolateral targeting of Cx43–eYFP. Additionally, substitution of a sequence containing the transferrin receptor internalization signal, LSYTRF, for PGYKLV(284–289) also disrupted basolateral targeting. Taken together, these results indicate that Y286 in its native amino acid sequence is necessary for targeting Cx43–eYFP to the basolateral membrane domain of MDCK cells. To determine whether the F52dup or L90V Oculodentodigital Dysplasia -associated mutations could affect polarized sorting of Cx43–eYFP, we analyzed the expression of these Cx43–eYFP mutant constructs and found that the L90V mutation disrupted basolateral expression. These findings raise the possibility that some oculodentodigitial Dysplasia-associated mutations contribute to disease by altering polarized targeting of Cx43.
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Oculodentodigital Dysplasia connexin43 mutations result in non-functional connexin hemichannels and gap junctions in C6 glioma cells
Journal of Cell Science, 2006Co-Authors: Albert Lai, William A Paznekas, Ethylin Wang Jabs, Wes D. Gifford, Andrew CharlesAbstract:Oculodentodigital Dysplasia (ODDD) is a rare developmental disorder characterized by craniofacial and limb abnormalities. Over 35 separate mutations in human connexin43 (Cx43) causing ODDD have been identified. Several mutations are also associated with central nervous system involvement, including white-matter changes detected by magnetic resonance imaging. As Cx43 is abundantly expressed in astrocytes, we hypothesized that the mutant Cx43 proteins that produce neurological dysfunction have abnormal functional characteristics in astrocytes. To understand how ODDD-associated mutations affect Cx43 signaling in cells of glial origin, we conducted studies in rat C6 glioma cells, a communication-deficient glial cell line that expresses low levels of Cx43. We generated stable cell lines expressing enhanced yellow fluorescent protein (eYFP)-tagged human Cx43 constructs encoding wild-type and six eYFP-tagged mutant Cx43 mutants: Y17S, G21R, A40V, F52dup, L90V and I130T. Of these, Y17S, L90V and I130T are associated with neurological abnormalities. We found that all mutants could be detected on the cell surface. Y17S, G21R, A40V, L90V and I130T formed triton-resistant plaques representing gap junctions, although the relative ability to form plaques was decreased in these mutants compared with the wild type. F52dup formed dramatically reduced numbers of plaques. Propidium iodide uptake experiments demonstrated that all mutants were associated with reduced connexin hemichannel function compared with wild type. Scrape-loading experiments performed on the same stable cell lines showed reduced gap junctional dye transfer in all mutants compared with the wild type. These studies demonstrated that ODDD-associated Cx43 mutations result in non-functional connexin hemichannels and gap junction functions in a glial cell line regardless of whether the particular mutant is associated with neurological dysfunction.
Qing Shao - One of the best experts on this subject based on the ideXlab platform.
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Specific functional pathologies of Cx43 mutations associated with Oculodentodigital Dysplasia.
Molecular biology of the cell, 2016Co-Authors: John J. Kelly, Qing Shao, Donglin Bai, Jessica L. Esseltine, Ethylin Wang Jabs, Jacinda B. Sampson, Mari Auranen, Dale W. LairdAbstract:Oculodentodigital Dysplasia (ODDD) is a rare genetic disease that affects the development of multiple organs in the human body. More than 70 mutations in the gap junction connexin43 (Cx43) gene, GJA1, are associated with ODDD, most of which are inherited in an autosomal dominant manner. Many patients exhibit similar clinical presentations. However, there is high intrafamilial and interfamilial phenotypic variability. To better understand this variability, we established primary human dermal fibroblast cultures from several ODDD patients and unaffected controls. In the present study, we characterized three fibroblast lines expressing heterozygous p.L7V, p.G138R, and p.G143S Cx43 variants. All ODDD fibroblasts exhibited slower growth, reduced migration, and defective cell polarization, traits common to all ODDD fibroblasts studied so far. However, we found striking differences in overall expression levels, with p.L7V down-regulated at the mRNA and protein level. Although all of the Cx43 variants could traffic to the cell surface, there were stark differences in gap junction plaque formation, gap junctional intercellular communication, Cx43 phosphorylation, and hemichannel activity among Cx43 variants, as well as subtle differences in myofibroblast differentiation. Together these findings enabled us to discover mutation-specific pathologies that may help to predict future clinical outcomes.
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Manipulating Cx43 expression triggers gene reprogramming events in dermal fibroblasts from Oculodentodigital Dysplasia patients.
The Biochemical journal, 2015Co-Authors: Jessica L. Esseltine, Qing Shao, John J. Kelly, Jacinda B. Sampson, Tao Huang, Dale W. LairdAbstract:Oculodentodigital Dysplasia (ODDD) is primarily an autosomal dominant disorder linked to over 70 GJA1 gene [connexin43 (Cx43)] mutations. For nearly a decade, our laboratory has been investigating the relationship between Cx43 and ODDD by expressing disease-linked mutants in reference cells, tissue-relevant cell lines, 3D organ cultures and by using genetically modified mouse models of human disease. Although salient features of Cx43 mutants have been revealed, these models do not necessarily reflect the complexity of the human context. To further overcome these limitations, we have acquired dermal fibroblasts from two ODDD-affected individuals harbouring D3N and V216L mutations in Cx43, along with familial controls. Using these ODDD patient dermal fibroblasts, which naturally produce less GJA1 gene product, along with RNAi and RNA activation (RNAa) approaches, we show that manipulating Cx43 expression triggers cellular gene reprogramming. Quantitative RT-PCR, Western blot and immunofluorescent analysis of ODDD patient fibroblasts show unusually high levels of extracellular matrix (ECM)-interacting proteins, including integrin α5β1, matrix metalloproteinases as well as secreted ECM proteins collagen-I and laminin. Cx43 knockdown in familial control cells produces similar effects on ECM expression, whereas Cx43 transcriptional up-regulation using RNAa decreases production of collagen-I. Interestingly, the enhanced levels of ECM-associated proteins in ODDD V216L fibroblasts is not only a consequence of increased ECM gene expression, but also due to an apparent deficit in collagen-I secretion which may further contribute to impaired collagen gel contraction in ODDD fibroblasts. These findings further illuminate the altered function of Cx43 in ODDD-affected individuals and highlight the impact of manipulating Cx43 expression in human cells.
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Myogenic bladder defects in mouse models of human Oculodentodigital Dysplasia.
The Biochemical journal, 2014Co-Authors: Tao Huang, Qing Shao, Kevin J. Barr, Jamie Simek, Glenn I. Fishman, Dale W. LairdAbstract:To date, over 65 mutations in the gene encoding Cx43 (connexin43) have been linked to the autosomal-dominant disease ODDD (Oculodentodigital Dysplasia). A subset of these patients experience bladder incontinence which could be due to underlying neurogenic deterioration or aberrant myogenic regulation. BSMCs (bladder smooth muscle cells) from wild-type and two Cx43 mutant lines (Cx43(G60S) and Cx43(I130T)) that mimic ODDD exhibit a significant reduction in total Cx43. Dye transfer studies revealed that the G60S mutant was a potent dominant-negative inhibitor of co-expressed Cx43, a property not equally shared by the I130T mutant. BSMCs from both mutant mouse strains were defective in their ability to contract, which is indicative of phenotype changes due to harbouring the Cx43 mutants. Upon stretching, Cx43 levels were significantly elevated in controls and mutants containing BSMCs, but the non-muscle myosin heavy chain A levels were only reduced in cells from control mice. Although the Cx43(G60S) mutant mice showed no difference in voided urine volume or frequency, the Cx43(I130T) mice voided less frequently. Thus, similar to the diversity of morbidities seen in ODDD patients, genetically modified mice also display mutation-specific changes in bladder function. Furthermore, although mutant mice have compromised smooth muscle contraction and response to stretch, overriding bladder defects in Cx43(I130T) mice are likely to be complemented by neurogenic changes.
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Autosomal recessive GJA1 (Cx43) gene mutations cause Oculodentodigital Dysplasia by distinct mechanisms.
Journal of Cell Science, 2013Co-Authors: Tao Huang, Qing Shao, Donglin Bai, Robert Lorentz, Andrew Macdonald, Li Xin, Dale W. LairdAbstract:Oculodentodigital Dysplasia (ODDD) is mainly an autosomal dominant human disease caused by mutations in the GJA1 gene, which encodes the gap junction protein connexin43 (Cx43). Surprisingly, there have been two autosomal recessive mutations reported that cause ODDD: a single amino acid substitution (R76H) and a premature truncation mutation (R33X). When expressed in either gap junctional intercellular communication (GJIC)-deficient HeLa cells or Cx43-expressing NRK cells, the R76H mutant trafficked to the plasma membrane to form gap junction-like plaques, whereas the R33X mutant remained diffusely localized throughout the cell, including the nucleus. As expected, the R33X mutant failed to form functional channels. In the case of the R76H mutant, dye transfer studies in HeLa cells and electrical conductance analysis in GJIC-deficient N2a cells revealed that this mutant could form functional gap junction channels, albeit with reduced macroscopic and single channel conductance. Alexa 350 dye transfer studies further revealed that the R76H mutant had no detectable negative effect on the function of co-expressed Cx26, Cx32, Cx37 or Cx40, whereas the R33X mutant exhibited significant dominant or trans-dominant effects on Cx43 and Cx40 as manifested by a reduction in wild-type connexin gap junction plaques. Taken together, our results suggest that the trans-dominant effect of R33X together with its complete inability to form a functional channel may explain why patients harboring this autosomal recessive R33X mutant exhibit greater disease burden than patients harboring the R76H mutant.
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Structure and functional studies of N-terminal Cx43 mutants linked to Oculodentodigital Dysplasia
Molecular biology of the cell, 2012Co-Authors: Qing Shao, Xiang-qun Gong, Donglin Bai, Qin Liu, Robert Lorentz, Gary S. Shaw, Dale W. LairdAbstract:Mutations in the gene encoding connexin-43 (Cx43) cause the human development disorder known as Oculodentodigital Dysplasia (ODDD). In this study, ODDD-linked Cx43 N-terminal mutants formed nonfunctional gap junction–like plaques and exhibited dominant-negative effects on the coupling conductance of coexpressed endogenous Cx43 in reference cell models. Nuclear magnetic resonance (NMR) protein structure determination of an N-terminal 23–amino acid polypeptide of wild-type Cx43 revealed that it folded in to a kinked α-helical structure. This finding predicted that W4 might be critically important in intramolecular and intermolecular interactions. Thus we engineered and characterized a W4A mutant and found that this mutant formed a regular, nonkinked α-helix but did not form functional gap junctions. Furthermore, a G2V variant peptide of Cx43 showed a kinked helix that now included V2 interactions with W4, resulting in the G2V mutant forming nonfunctional gap junctions. Also predicted from the NMR structures, a G2S mutant was found to relieve these interactions and allowed the protein to form functional gap junctions. Collectively, these studies suggest that the nature of the mutation conveys loss of Cx43 function by distinctly different mechanisms that are rooted in the structure of the N-terminal region.
