Identification of two regulatory elements within the promoter region of the mouse connexin 43 gene.
ABSTRACT To define the minimal sequences required for expression of the connexin 43 gene (cx43) in myometrial cells, we generated 5' deletion constructs of a fragment extending 1686 base pairs upstream and 162 base pairs downstream of the transcription start site and determined their ability to drive expression of the chloramphenicol acetyltransferase reporter gene in transfected myometrial cell lines. Our investigation revealed two cis-acting regulatory elements within this fragment. Deletion of a region extending from -102 to -92 led to an increase of the promoter activity by greater than 10-fold, indicating a presence of a repressor element. Deletion of a region extending from -72 to -62 caused a decrease of the promoter activity of a similar extent, implying the existence of a positive element. Electrophoretic mobility shift assays demonstrated that synthetic oligonucleotides derived from these two small regions can each bind with a nuclear protein(s) prepared from myometrial cells, and an introduction of three and two base substitutions into each of these oligomers was sufficient to abolish their protein binding capability. These same mutations, when incorporated in the chloramphenicol acetyltransferase constructs, diminished regulatory functions of the negative and positive elements, and the protein(s) that bind to these functional elements was found in several tissues known to express cx43 gene.
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ABSTRACT: Members of the connexin gene family are integral membrane proteins that form hexamers called connexons. Most cells express two or more connexins. Open connexons found at the nonjunctional plasma membrane connect the cell interior with the extracellular milieu. They have been implicated in physiological functions including paracrine intercellular signaling and in induction of cell death under pathological conditions. Gap junction channels are formed by docking of two connexons and are found at cell-cell appositions. Gap junction channels are responsible for direct intercellular transfer of ions and small molecules including propagation of inositol trisphosphate-dependent calcium waves. They are involved in coordinating the electrical and metabolic responses of heterogeneous cells. New approaches have expanded our knowledge of channel structure and connexin biochemistry (e.g., protein trafficking/assembly, phosphorylation, and interactions with other connexins or other proteins). The physiological role of gap junctions in several tissues has been elucidated by the discovery of mutant connexins associated with genetic diseases and by the generation of mice with targeted ablation of specific connexin genes. The observed phenotypes range from specific tissue dysfunction to embryonic lethality.Physiological Reviews 11/2003; 83(4):1359-400. · 26.87 Impact Factor
Article: Progressive atrioventricular conduction defects and heart failure in mice expressing a mutant Csx/Nkx2.5 homeoprotein.[show abstract] [hide abstract]
ABSTRACT: A DNA nonbinding mutant of the NK2 class homeoprotein Nkx2.5 dominantly inhibits cardiogenesis in Xenopus embryos, causing a small heart to develop or blocking heart formation entirely. Recently, ten heterozygous CSX/NKX2.5 homeoprotein mutations were identified in patients with congenital atrioventricular (AV) conduction defects. All four missense mutations identified in the human homeodomain led to markedly reduced DNA binding. To examine the effect of a DNA binding-impaired mutant of mouse Csx/Nkx2.5 in the embryonic heart, we generated transgenic mice expressing one such allele, I183P, under the beta-myosin heavy chain promoter. Unexpectedly, transgenic mice were born apparently normal, but the accumulation of Csx/Nkx2.5(I183P) mutant protein in the embryo, neonate, and adult myocardium resulted in progressive and profound cardiac conduction defects and heart failure. P-R prolongation observed at 2 weeks of age rapidly progressed into complete AV block as early as 4 weeks of age. Expression of connexins 40 and 43 was dramatically decreased in the transgenic heart, which may contribute to the conduction defects in the transgenic mice. This transgenic mouse model may be useful in the study of the pathogenesis of cardiac dysfunction associated with CSX/NKX2.5 mutations in humans.Journal of Clinical Investigation 08/2001; 108(2):189-201. · 15.39 Impact Factor
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ABSTRACT: We have examined factors concerned with the maintenance of uterine quiescence during pregnancy and the onset of uterine activity at term in an animal model, the sheep, and in primate species. We suggest that in both species the fetus exerts a critical role in the processes leading to birth, and that activation of the fetal hypothalamic-pituitary-adrenal axis is a central mechanism by which the fetal influence on gestation length is exerted. Increased cortisol output from the fetal adrenal gland is a common characteristic across animal species. In primates, there is, in addition, increased output of estrogen precursor from the adrenal in late gestation. The end result, however, in primates and in sheep is similar: an increase in estrogen production from the placenta and intrauterine tissues. We have revised the pathway by which endocrine events associated with parturition in the sheep come about and suggest that fetal cortisol directly affects placental PGHS expression. In human pregnancy we suggest that cortisol increases PGHS expression, activity, and PG output in human fetal membranes in a similar manner. Simultaneously, cortisol contributes to decreases in PG metabolism and to a feed-forward loop involving elevation of CRH production from intrauterine tissues. In human pregnancy, there is no systemic withdrawal of progesterone in late gestation. We have argued that high circulating progesterone concentrations are required to effect regionalization of uterine activity, with predominantly relaxation in the lower uterine segment, allowing contractions in the fundal region to precipitate delivery. This new information, arising from basic and clinical studies, should further the development of new methods of diagnosing the patient at risk of preterm labor, and the use of scientifically based strategies specifically for the management of this condition, which will improve the health of the newborn.Endocrine Reviews 11/2000; 21(5):514-50. · 19.93 Impact Factor