Evidence for modulation of cell-to-cell electrical coupling by cAMP in mouse islets of Langerhans

The effects of forskolin on electrical coupling among pancreatic β-cells were studied. Two microelectrodes were used to measure membrane potentials simultaneously in pairs of islet β-cells. Intracellular injection of a current pulse (ΔI) elicited a membrane response ΔV₁ in the injected cell and also a response ΔV₂ in a nearby β-cell confirming the existence of cell-to-cell electrical coupling among islet β-cells. In the presence of glucose (7 mM), application of forskolin evoked a transient depolarization of the membrane and electrical activity suggesting that the drug induced a partial inhibition of the β-cell membrane K⁺ conductance. Concomitant with this depolarization of the membrane there was a marked decrease in β-cell input resistance (ΔV₂/ΔI) suggesting that exposure to forskolin enhanced intercellular coupling. Direct measurements of the coupling ratio ΔV₂/ΔV₁ provided further support to the idea that forskolin enhances electrical coupling among islet cells. Indeed, application of forskolin reversibly increased the coupling ratio. These results suggest that cAMP might be involved in the modulation of electrical coupling among islet β-cells.     

Promoter elements of the mouse 21-hydroxylase (Cyp-21) gene involved in cell-selective and cAMP-dependent gene expression

Cyp-21 (the mouse steroid 21-hydroxylase gene) is expressed exclusively in cells of the adrenal cortex, is induced by ACTH and cAMP, and is required for corticosteroid synthesis. This review examines the molecular basis for the regulated expression of Cyp-21 in the ACTH-responsive, mouse adrenocortical tumor cell line, Y1. We demonstrate that 330 bp of 5′-flanking DNA from the Cyp-21 gene are sufficient for cell-selective and ACTH-induced expression of Cyp-21, and that this promoter region comprises multiple, closely spaced enhancer elements each of which is required for promoter function. Within this promoter, we define three related elements that contain variations of an AGGTCA motif and that contribute to the cell-selective expression of Cyp-21. Variations of these same AGGTCA-bearing elements are also involved in the expression of Cyp 11a and Cyp 11b in Y1 adrenocortical cells. These elements interact with the same or closely related nuclear proteins found only in steroidogenic cell lines. Taken together, these results suggest that shared elements contribute to the adrenal cell-selective expression of at least three steroidogenic cytochrome P450 genes.

The element at −170 and the related elements at −65, −140 and −210 in the Cyp-21 promoter are not active as enhancers in the mutant Y1 cell line, Kin-8. Kin-8 cells contain a mutation in the regulatory subunit of the type 1 cAMP-dependent protein kinase that renders the enzyme resistant to activation by cAMP. Therefore, these elements appear to be selectively dependent upon an intact cAMP-dependent protein kinase for enhancer function. Individually, none of these elements confer cAMP-dependence to a reporter gene driven by a heterologous promoter. On the basis of these observations, we suggest that ACTH- and cAMP-dependent expression of Cyp-21 requires the combined actions of the element at −170, and the related elements at −140, −210 and −65.     

Phosphoinositide 3-kinase is involved in Xenopus and Labrus melanophore aggregation

Melanophores are pigmented cells capable of quick colour changes through coordinated transport of their intracellular pigment granules. We demonstrate the involvement of phosphoinositide 3-kinase (PI3-K) in Xenopus and Labrus aggregation by the use of the PI3-K inhibitor, LY-294002. In Xenopus, wortmannin-insensitive PI3-K was found to be essential for the aggregation, mitogen-activated protein kinase (MAPK) activation and tyrosine phosphorylation of a 280-kDa protein, and for the maintenance of low cyclic adenosine 3′:5′-monophosphate (cAMP) during the aggregated state. Pre-aggregated cells disperse completely to LY-294002 at 50–100 μM, involving a transient elevation in cAMP due to adenylate cyclase (AC) stimulation or to inhibition of cyclic nucleotide phosphodiesterase (PDE). The inactive analogue LY-303511 did not induce dispersion at the same concentrations. PDE4 and/or PDE2 was found to be involved in melanosome aggregation. The similar kinetics of LY-294002 and various PDE inhibitors indicates that the elevation of cAMP might be due to inhibition of PDE. In Labrus melanophores, LY-294002 had a less dramatic effect, probably due to less dependence on PDE in regulation of cAMP levels. In Xenopus aggregation, we suggest that melatonin stimulation of the Mel1c receptor via G_βγ activates PI3-K that, directly or indirectly via MAPK, activates PDE.     

Binding of the C6-zinc cluster protein, AFLR, to the promoters of aflatoxin pathway biosynthesis genes in Aspergillus parasiticus

AFLR is a Zn₂Cys₆-type sequence-specific DNA-binding protein that is thought to be necessary for expression of most of the genes in the aflatoxin pathway gene cluster in Aspergillus parasiticus and A. flavus, and the sterigmatocystin gene cluster in A. nidulans. However, it was not known whether AFLR bound to the promoter regions of each of the genes in the cluster. Recently, A. nidulans AFLR was shown to bind to the motif 5′-TCGN₅CGA-3′. In the present study, we examined the binding of AFLR to promoter regions of 11 genes in the A. parasiticus cluster. Based on electrophoretic mobility shift assays, the genes nor1, pksA, adhA, norA, ver1, omtA, ordA, and, vbs, had at least one 5′-TCGN₅CGA-3′ binding site within 200 bp of the translation start site, and pksA and ver1 had an additional binding site further upstream. Although the promoter region of avnA lacked this motif, AFLR bound weakly to the sequence 5′-TCGCAGCCCGG-3′ at −110 bp. One region in the promoter of the divergently transcribed genes aflR/aflJ bound weakly to AFLR even though it contained a site with at most only 7 bp of the 5′-TCGN₅CGA-3′ motif. This partial site may be recognized by a monomeric form of AFLR. Based on a comparison of 16 possible sites, the preferred binding sequence was 5′-TCGSWNNSCGR-3′.     

Expression pattern of 5''-nucleotidase in Dictyostelium.

In order to analyze the expression pattern of the 5′-nucleotidase (5nt) gene in Dictyostelium, we made a fusion construct in which the 5nt promoter directed the expression of β-galactosidase gene. The reporter gene was not active in vegetative amoebae but was expressed during the aggregation stage. At the slug stage, 5nt was highly expressed in pstAB cells. As the slug moved along the substratum, high activity of β-galactosidase was detected in cells that were left behind in the slime trail. In the completed fruiting body, 5nt was expressed in the lower cup, the anterior like cells (ALC) and the basal disc.     

Identification of a 3.2 kb 5'-flanking region of the murine keratocan gene that directs beta-galactosidase expression in the adult corneal stroma of transgenic mice

The mouse keratocan gene (Ktcn) expression tracks the corneal morphogenesis during eye development and becomes restricted to keratocytes of the adult, implicating a cornea-specific gene regulation of the mouse Ktcn [J. Biol. Chem., 273 (1998) 22 584–22 588]. To examine the functionality of the mouse Ktcn promoter, we have cloned and sequenced a 3.2 kb genomic DNA fragment 5′ of the mouse Ktcn gene, which was used to prepare a reporter gene construct that contained the 3.2 kb 5′ flanking sequence, exon 1 and 0.4 kb of intron 1 of Ktcn, and β-geo hybrid reporter gene. The β-galactosidase (βGal) activity was assayed in tissues of two of five transgenic mouse lines obtained via microinjection. In adult transgenic mice, βGal activity was detected only in cornea, not in other tissues (e.g. lens, retina, sclera, lung, heart, liver, diaphragm, kidney, and brain). During ocular development, the spatial–temporal expression patterns of the βGal recapitulated that of endogenous Ktcn in transgenic mice. Using XGal staining, strong βGal activity was first detected in periocular tissues of E13.5 embryos, and restricted to corneal keratocytes at E14.5 and thereafter. Interestingly, in addition to cornea, βGal activity was transiently found in some non-ocular tissues, i.e. ears, snout, and limbs of embryos of E13.5 and E14.5 but was no longer detected in those tissues of E16.5 embryos. The transient expression of endogenous keratocan in non-ocular tissues during embryonic development was confirmed by in situ hybridization. Taken together, our results suggest that the 3.2 kb Ktcn promoter contains sufficient cis-regulatory elements to drive heterologous minigene expression in cells expressing keratocan. The identification of keratocyte-specific expression of βGal reporter gene in the adult transgenic mice is an important first step in characterizing the Ktcn promoter in order to use it to drive a foreign gene expression in corneal stroma.     

Specific expression in mouse mesoderm- and neural crest-derived tissues of a human PDGFRA promoter/lacZ transgene

The platelet-derived growth factor -receptor (PDGFR-) displays a lineage-specific expression pattern in the mouse embryo and is required for normal development of mesoderm and cephalic neural crest derivatives. The purpose of the present study was to demonstrate the in vivo promoter function of genomic DNA fragments representing the 5′-flanking part of the human PDGFRA gene. 2.2, 0.9 and 0.4 kb PDGFRA promoter fragments, ligated to a lacZ reporter gene, were microinjected into fertilized mouse eggs and transgenic mouse lines were established. The expression patterns were basically similar in the 2.2 and 0.9 kb lines and overlapped grossly the endogenous Pdgfra gene expression pattern. The transgenic line with the highest expression level was chosen for detailed analysis. Expression was, as expected, mainly confined to tissues of mesodermal and neural crest origin. No expression was found in epithelial tissues of endo- or ectodermal origin. The promoter fragments were also active in neuroepithelium and in certain neuronal cell types that did not faithfully express PDGFR- mRNA, while they failed to specify reporter expression in PDGFR- expressing O-2A progenitor cells and other glial elements of the central nervous system. Thus, the isolated human PDGFRA promoter contains most but not all of the regulatory elements that are necessary to establish tissue specific gene expression during development.