New strategies for the treatment of colic: modifying the parent/infant interaction

Cystic fibrosis (CF) is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Its product is a cyclic AMP-dependent Cl- channel, that is defective in CF. Since cAMP regulates the expression of many genes and since the 5'-flanking region of the CFTR gene contains cAMP response elements, we hypothesized that intracellular cAMP might modulate not only the cAMP-dependent Cl- channel CFTR, but also CFTR gene expression in epithelial cells. To accomplish this, we investigated Cl- secretion and CFTR-mRNA levels in HT-29 and T84 colon carcinoma epithelial cells before and after exposure to forskolin and 8-bromo-cAMP for 12 hr. While resting T84 cells increased Cl- secretion in response to forskolin strongly and immediately, HT-29 cells did not, although both cell lines showed highly increased Cl- efflux in response to A23187, a calcium ionophore. Interestingly, prolonged exposure to forskolin (12 hr) induced a clear decrease of CFTR-mRNA levels in T84 cells, but an increase of CFTR-mRNA levels in HT-29 cells, thus demonstrating different behaviour of CFTR gene regulation in different epithelial cells in response to intracellular cAMP. These results suggest that cells with an effective cAMP-dependent Cl- channel (CFTR) respond to prolonged stimulation of this channel with down-regulation of CFTR gene expression, while cells with no effective cAMP-dependent Cl--secretion respond with an up-regulation of CFTR gene expression.     

Characterization of a potent and specific class of antisense oligonucleotide inhibitor of human protein kinase C-alpha expression

The use of antisense oligonucleotides to inhibit the expression of targeted mRNA sequences is becoming increasingly commonplace. Although effective, the most widely used oligonucleotide modification (phosphorothioate) has some limitations. In previous studies we have described a 20-mer phosphorothioate oligodeoxynucleotide inhibitor of human protein kinase C-alpha expression. In an effort to identify improved antisense inhibitors of protein kinase C expression, a series of 2' modifications have been incorporated into the protein kinase C-alpha targeting oligonucleotide, and the effects on oligonucleotide biophysical characteristics and pharmacology evaluated. The incorporation of 2'-O-(2-methoxy)ethyl chemistry resulted in a number of significant improvements in oligonucleotide characteristics. These include an increase in hybridization affinity toward a complementary RNA (1.5 degrees C per modification) and an increase in resistance toward both 3'-exonuclease and intracellular nucleases. These improvements result in a substantial increase in oligonucleotide potency (>20-fold after 72 h). The most active compound identified was used to examine the role played by protein kinase C-alpha in mediating the phorbol ester-induced changes in c-fos, c-jun, and junB expression in A549 lung epithelial cells. Depletion of protein kinase C-alpha protein expression by this oligonucleotide lead to a reduction in c-jun expression but not c-fos or junB. These results demonstrate that 2'-O-(2-methoxy)ethyl-modified antisense oligonucleotides are 1) effective inhibitors of protein kinase C-alpha expression, and 2) represent a class of antisense oligonucleotide which are much more effective inhibitors of gene expression than the widely used phosphorothioate antisense oligodeoxynucleotides.     

Hepatitis C viral genome in a subset of primary hepatic lymphomas

Formoterol, a beta2-adrenergic agonist has been shown in ovariectomized rat models to have anabolic effects on bone. However, those studies did not determine whether the effect of formoterol was by a direct action on bone cells themselves or indirectly via anabolic action on muscle. To address the question of whether formoterol could directly affect osteoblast function we investigated the expression patterns of beta3-adrenergic receptors (betaARs) in human osteoblast-like cells and functional coupling to gene expression. Northern blot analysis showed that betaAR subtypes are expressed at different levels in the osteoblast-like cell lines TE-85, SaOS-2, MG-63, and OHS-4. beta1AR expression was found in SaOS-2, OHS-4, and TE-85, but not MG-63 cells. beta2ARs are expressed at higher levels in MG-63 cells than in TE-85 and SaOS-2 cells, but were not detected in OHS-4 cells. PCR analysis paralleled the northern blot analysis except that beta3AR expression was found in one of three human primary osteoblast cDNAs tested. beta3AR expression was not found in any of the osteoblast-like cell lines. The nonspecific betaAR agonist, isoproterenol, and the beta2AR-specific agonist, formoterol, induced c-fos gene expression in cultured SaOS-2 cells in an immediate early fashion. This effect was inhibited by the beta2AR-specific antagonist, ICI 118551, but not by the beta1AR-specific antagonist, CGP 20712, indicating that induction of c-fos gene expression is specifically mediated by beta2ARs. c-fos gene expression was induced by both isoproterenol and formoterol via increases in cAMP, which in turn activated the cAMP/PKA pathway; the PKA inhibitor, H89, inhibited c-fos gene expression. Thus, betaARs are expressed in osteoblast-like cells and are coupled to c-fos gene expression via the beta2AR, increases in cAMP levels and activation of a PKA-dependent pathway.     

Endothelins inhibit cyclic-AMP induced renin gene expression in cultured mouse juxtaglomerular cells

We have recently described that endothelins-1 to -3 equipotently inhibit cAMP stimulated renin secretion from cultured mouse juxtaglomerular cells by a process involving phospholipase C activation. This study examined the influence of endothelin-2 on renin gene expression in renal juxtaglomerular cells. To this end we semiquantitated renin mRNA levels by competitive RT-PCR in primary cultures of mouse renal juxtaglomerular cells after 20 hours of incubation. We found that endothelin-2 (0.1 to 100 nmol/liter) did not change basal renin gene expression. The adenylate cyclase activator forskolin (3 mumol/ liter) increased renin mRNA levels to 400% of the controls and this stimulation was dose-dependently attenuated by ET-2 to 250% of the control value. The effect of ET-2 was mimicked by the ETB-receptor agonist sarafotoxin S6c. The kinase inhibitor staurosporine (100 nmol/ liter) increased renin secretion and renin mRNA levels. Combination of staurosporine with forskolin produced the same effects on renin secretion and renin mRNA levels as did staurosporine alone. In the presence of both forskolin and staurosporine ET-2 had no significant effect on renin secretion and renin gene expression. The phorbol ester PMA (30 nmol/ liter), which was used to stimulate protein kinase C activity, attenuated cAMP stimulated renin secretion and renin mRNA levels. Lowering the extracellular concentration of calcium by the addition of 1 mmol/liter EGTA did not inhibit the effect of ET-2 on cAMP induced renin secretion and renin gene expression. These findings suggest that endothelins inhibit cAMP stimulated renin gene expression by an event that is mediated via ETB receptors. This inhibitory effect may in part involve protein kinase C activation.     

Activation of protein kinase A prevents the ethanol-induced up-regulation of delta-opioid receptor mRNA in NG108-15 cells

We have used a sensitive solution hybridization assay with a riboprobe transcribed from the coding sequence of the delta-opioid receptor gene (DOR) to study the up-regulation of the DOR mRNA by ethanol in NG108-15 cells. Exposure of the cells to compounds that increase cAMP levels (forskolin, forskolin + IBMX, or dibutyryl cAMP) resulted in the attenuation of ethanol-induced up-regulation of DOR mRNA. The inactive analogue of forskolin, 1,9-dideoxy forskolin had no effect. Northern blot analysis of RNA extracts from ethanol-, forskolin- or ethanol + forskolin-treated cells showed proportional changes in each of the multiple DOR mRNA bands, so that no difference was observed in the fraction of the total hybridization signal produced by each band of the DOR mRNA. In the absence of ethanol, forskolin or dibutyryl cAMP reduced the basal levels of DOR mRNA. The cAMP analogue (Rp)-cAMPS, a protein kinase A (PKA) inhibitor, increased DOR mRNA levels. However, the combination of (Rp)-cAMPS and ethanol did not further increase DOR mRNA levels compared to ethanol or (Rp)-cAMPS alone. Signaling through cAMP and PKA down-regulates DOR mRNA levels. The ethanol-induced increase in DOR mRNA levels in NG108-15 cells appears to be mediated via a reduction of PKA.