The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects.

Oculodentodigital dysplasia (ODDD) is a rare condition characterized by a typical facial appearance and variable findings of the eyes, teeth, and fingers. ODDD is caused by mutations in the GJA1 gene in chromosome 6q22 and inherited in an autosomal dominant manner in the majority of the patients. However, in recent clinical reports, autosomal recessive ODDD cases due to by GJA1 mutations were also described. Here, we report on a 14-year-old boy with microphthalmia, microcornea, narrow nasal bridge, hypoplastic alae nasi, prominent columnella, hypodontia, dental caries, and partial syndactyly of the 2nd and 3rd toes. These clinical findings were concordant with the diagnosis of ODDD, and a novel homozygous mutation (c.442C>T, p.Arg148Ter) was determined in the GJA1 gene leading to a premature stop codon. His phenotypically normal parents were found to be carriers of the same mutation. This is the third family in the literature in which ODDD segregates in an autosomal recessive manner.

Oculodentodigital dysplasia (ODDD) is a clinically variable genetic disorder caused by mutations of the GJA1 gene, predominantly inherited in an autosomal dominant fashion. In rare cases ODDD can also exhibit autosomal recessive mode of inheritance. The phenotype of ODDD comprises craniofacial (short and narrow palpebral fissure, thin, narrow nose with hypoplastic alae nasi), dental (oligodontia, hypoplastic enamel), and digital abnormalities (syndactyly of finger 4/5, hypoplastic phalanges). Ocular manifestation is typical and involves microphthalmia, microcornea, glaucoma, congenital malformations of iris or vitreous, ectopic pupils or strabismus. To date, only 67 GJA1 mutations have been described to underlie ODDD and most of them (i.e. 97%) represent missense substitutions. In this report, we describe three (two familial and one sporadic) non-consanguineous cases presenting with ODDD features in whom we identified novel missense heterozygous mutations of the GJA1 gene: c.317T>G (p. L106R), c.G139C (p.D47H), and c.C257A (p.S86Y). The first two mutations were inherited from an affected parent, whereas the latter one occurred de novo. The mutations affect highly conserved amino acid residues located in the different portions of the GJA1 protein. Our report broadens the spectrum of probably pathogenic mutations associated with ODDD phenotype and demonstrates that the amino acid substitutions at highly conserved positions 47, 86, 106 may affect protein functioning and lead to the development of this syndrome. Together with molecular data, we provide a brief clinical description of the affected individuals.

In this study, hypoxia inducible factor-1α (HIF-1α) and hypoxia inducible factor-1β (HIF-1β) from small abalone Haliotis diversicolor were cloned. The cDNA of H. diversicolor HIF-1α (HdHIF-1α) is 2,833 bp encoding a protein of 711aa and H. diversicolor HIF-1β (HdHIF-1β) is 1919 bp encoding a protein of 590aa. Similar to other species\' HIF-1, HdHIF-1 has one basic helix-loop-helix (bHLH) domain and two Per-Arnt-Sim (PAS) domains, and HdHIF-1α has a oxygen-dependent degradation domain (ODDD) with two proline hydroxylation motifs and a C-terminal transactivation domain (C-TAD) with an asparagine hydroxylation motif. Under normoxic conditions, HdHIF-1α and HdHIF-1β mRNAs were constitutively present in all examined tissues. Under hypoxia (2.0mg/L DO at 25°C) stress, HdHIF-1α expression was up-regulated in gills at 4h, 24h and 96 h, and in hemocytes at 24h and 96 h, while HdHIF-1β remained relatively constant. Under thermal stress (31°C), HdHIF-1α expression was significantly increased in gills at 4h, and hemocytes at 0 h and 4 h, while HdHIF-1β expression still remained relatively constant. These results suggested that HIF-1α may play an important role in adaption to poor environment in H. diversicolor.

Connexin43 (CX43), encoded by Gja1 in the mouse, is highly expressed in decidual cells and is known to be important for the transformation of stromal cells into the compact decidua and for neoangiogenesis. Here we investigated if the dominant Gja1(Jrt) mutation encoding CX43(G60S) in mice, which results in a phenotype resembling oculodentodigital dysplasia in humans, has an impact on decidualization, angiogenesis, and implantation. We found a reduced mean weight of fetuses at Gestational Day 17.5 in dams carrying this mutation, with the growth deficiency being independent of fetal genotype. Although the mutant implantation sites exhibited a reduction in CX43 protein, with most immunoreactivity being cytoplasmic, the decidua was morphologically intact at Embryonic Days 5.5 to 7.5. However, the mutation resulted in enhanced and irregular angiogenesis and an increased level of expression of the angiogenic factor-encoding genes Vegfa, Flt1, Kdr, and Fgf2 as well as the prolactin-related gene Prl6a. Moreover, immunolocalization of VEGFA, FLT1, and KDR revealed a homogeneous distribution pattern in the mesometrial as well as antimesometrial decidua of the mutants. Most obviously, uterine NK cells are drastically diminished in the mesometrial decidua of the mutant mice. Invasion of ectoplacental cone cells was disoriented, and placentation was established more laterally in the implantation chambers. It was concluded that the CX43(G60S) mutant impairs control of decidual angiogenesis, leading to dysmorphic placentation and fetal growth restriction. This phenomenon could contribute to the reduced fetal weights and viability of pups born of Gja1(Jrt)/+ dams.

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\'.

Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel-Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers.