Influence of retinoic acid and of cyclic AMP on the expression of choline acetyltransferase and of vesicular acetylcholine transporter in NG108-15 cells

Treatment of the cholinergic cell line NG108-15 with retinoic acid or cAMP results in an increase of choline acetyltransferase activity (ChAT) whereas none of these agents influences the amount of the vesicular acetylcholine transporter (VAChT) as judged from vesamicol binding and immunoblot studies. We suggest that immaturity of posttranslational events controlling the expression of VAChT protein is responsible for the apparent absence of coregulation of ChAT and VAChT protein expression.     

Exploring the Regulation of the Expression of ChAT and VAChT Genes in NG108-15 Cells: Implication of PKA and PI3K Signaling Pathways

Involvement of different protein kinases regulated by cAMP and implication of muscarinic receptors in the regulation of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) mRNA levels and ChAT activity has been studied in NG108-15 cells. Dibutyryl cAMP enhanced both ChAT and VAChT mRNA levels and stimulated ChAT activity. Muscarinic stimulation or inhibition did not change ChAT activity or the receptor subtype mRNA pattern. MEK1/2 did not affect the regulation of ChAT and VAChT mRNA levels. However, PKA plays a major role in regulating ChAT and VAChT mRNA levels, because H89 decreased both. Strikingly, inhibition of PI3K by LY294002 had two opposite effects: ChAT mRNA level was decreased and VAChT mRNA level was increased. Such a result consolidates the observation that ChAT and VAChT genes, despite their unusual organization in a single cholinergic locus, can be differentially or synergistically regulated, depending on the activated signaling pathways.     

Retinoic acid-mediated enhancement of the cholinergic/neuronal nitric oxide synthase phenotype of the medial septal SN56 clone: establishment of a nitric oxide-sensitive proapoptotic state

It is unclear what mechanisms lead to the degeneration of basal forebrain cholinergic neurons in Alzheimer's or other human brain diseases. Some brain cholinergic neurons express neuronal nitric oxide (NO) synthase (nNOS), which produces a free radical that has been implicated in some forms of neurodegeneration. We investigated nNOS expression and NO toxicity in SN56 cells, a clonal cholinergic model derived from the medial septum of the mouse basal forebrain. We show here that, in addition to expressing choline acetyltransferase (ChAT), SN56 cells express nNOS. Treatment of SN56 cells with retinoic acid (RA; 1 microM) for 48 h increased ChAT mRNA (+126%), protein (+88%), and activity (+215%) and increased nNOS mRNA (+98%), protein (+400%), and activity (+15%). After RA treatment, SN56 cells became vulnerable to NO excess generated with S-nitro-N-acetyl-DL-penicillamine (SNAP) and exhibited increased nuclear DNA fragmentation that was blocked with a caspase-3 inhibitor. Treatment with dexamethasone, which largely blocked the RA-mediated increase in nNOS expression, or inhibition of nNOS activity with methylthiocitrulline strongly potentiated the apoptotic response to SNAP in RA-treated SN56 cells. Caspase-3 activity was reduced when SNAP was incubated with cells or cell lysates, suggesting that NO can directly inhibit the protease. Thus, whereas RA treatment converts SN56 cells to a proapoptotic state sensitive to NO excess, endogenously produced NO appears to be anti-apoptotic, possibly by tonically inhibiting caspase-3.     

1,25-Dihydroxyvitamin D3 interaction with dexamethasone and retinoic acid: effects on procollagen messenger ribonucleic acid levels in rat osteoblast-like cells

We previously have reported that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], dexamethasone, and retinoic acid inhibit collagen synthesis in rat osteoblast-like cell primary cultures. We also have found that dexamethasone increases 1,25-(OH)2D3 receptor levels in these cells. Furthermore, this increase in 1,25-(OH)2D3 receptor level is paralleled by an enhanced inhibition of collagen synthesis when dexamethasone and 1,25-(OH)2D3 are used in combination. In contrast, retinoic acid at high doses decreases 1,25-(OH)2D3 receptor level in rat osteoblast-like cells and attenuates 1,25-(OH)2D3 inhibition of collagen synthesis. In the present study, we have used a [32P]cDNA probe for rat pro alpha 1 (I) to determine if these osteotropic agents act by modulating steady state procollagen mRNA levels. Hybridization with a [32P]cDNA probe for human actin was used as a control. We find that the steady state levels of procollagen mRNA are decreased in all cases, while there are negligible changes in actin mRNA levels. Dexamethasone, at the low dose of 13 nM, acts synergistically with 1,25-(OH)2D3 in decreasing procollagen mRNA levels. The effects of retinoic acid and 1,25-(OH)2D3 are additive at low doses (13 and 130 nM); however, at a high dose of retinoic acid (1.3 microM), combined treatment with 1,25-(OH)2D3 does not reduce procollagen mRNA levels beyond the decrease due to retinoic acid alone. The reduction in procollagen mRNA level after each of these treatments falls in the same range as inhibition of collagen synthesis measured at the protein level. These data suggest that the synthesis of collagen under these treatments is controlled primarily through modulation of steady state procollagen mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.     

Mapping of a retinoic acid-responsive element in the promoter region of the complement factor H gene.

Retinoic acid receptors are members of the steroid/thyroid hormone receptor superfamily. Pursuant to the discovery that dexamethasone increases complement factor H expression, we examined the effects of retinoic acid on this gene. Both H mRNA and protein levels are increased by retinoic acid in L cells. Using the luciferase reporter gene system we have identified a region of the H promoter required for the retinoic acid response. This region contains an imperfect palindrome of the TGACC motif, present in thyroid hormone and estrogen-responsive elements. We demonstrate specific binding of the retinoic acid receptor beta to this sequence of the H gene by DNA-protein gel retardation assay. Therefore, these studies extend the sphere of influence of the retinoids to complement, an intrinsic component of the humoral immune system.     

Autonomic microganglion cells: a source of acetylcholine in the rat carotid body.

Hypoxic chemosensitivity of peripheral arterial chemoreceptors and the ventilatory response to O2 deprivation increases with postnatal development. Multiple putative neurotransmitters, which are synthesized in the carotid body (CB), are thought to mediate signals generated by hypoxia. Acetylcholine (ACh) is believed to be a major excitatory neurotransmitter participating in hypoxic chemosensitivity. However, it is not known whether ACh originates from type I cells in the CB. In these studies, we tested the hypothesis that choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) mRNAs are expressed in the CB and that mRNA levels would increase with postnatal maturation or exposure to hypoxia. Semiquantitative in situ hybridization histochemistry and immunohistochemistry were used to localize cholinergic markers within neurons and cells of the rat CB, the nodose-petrosal-jugular ganglion complex, and the superior cervical ganglion up to postnatal day 28. We show that the pattern of distribution, in tissue sections, is similar for both ACh markers; however, the level of VAChT mRNA is uniformly greater than that of ChAT. VAChT mRNA and immunoreactivity are detected abundantly in the nodose-petrosal-jugular ganglion complex in a number of microganglion cells embedded in nerve fibers innervating the CB for all postnatal groups, whereas ChAT mRNA is detected in only a few of these cells. Contrary to our hypothesis, postnatal maturation caused a reduction in ACh trait expression, whereas hypoxic exposure did not induce the upregulation of VAChT and ChAT mRNA levels in the CB, microganglion, or within the ganglion complex. The present findings indicate that the source of ACh in the CB is likely within autonomic microganglion cells and cholinergic nerve terminals.     

Inhibitors of nitric oxide synthase attenuate nerve growth factor-mediated increases in choline acetyltransferase expression in PC12 cells

NGF can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF-mediated neurotrophic responses. To investigate the role of NO in NGF-activated expression of cholinergic phenotype, PC12 cells were treated with either the nonselective NOS inhibitor L-NAME (N (omega)-nitro-L-arginine methylester) or the inducible NOS selective inhibitor MIU (s-methylisothiourea), and the effect on NGF-stimulated ChAT mRNA levels and ChAT specific activity was determined. NGF increased steady-state levels of mRNA and protein for both inducible and constitutive isozymes of NOS in PC12 cells, and led to enhanced NOS activity and NO production. MIU and, to a lesser extent, L-NAME blocked neurite outgrowth in nerve growth factor (NGF)-treated PC12 cells. Both L-NAME and MIU attenuated NGF-mediated increases in choline transferase (ChAT)-specific activity and prevented the increase in expression of ChAT mRNA normally produced by NGF treatment of PC12 cells. The present study indicates that NO may be involved in the modulation of signal transduction pathways by which NGF leads to increased ChAT gene expression in PC12 cells.     

Recoverin in Cultured Human Retinoblastoma Cells: Enhanced Expression During Morphological Differentiation

Abstract: Recoverin is a calcium-binding protein expressed in retinal photoreceptors. It appears to delay the termination of the phototransduction cascade by blocking the phosphorylation of photoexcited rhodopsin. The goal of this study was to determine if recoverin mRNA and protein are expressed in cultured human Y79 retinoblastoma cells, so that this cell line could be used as a model to study the mechanism of recoverin gene expression in the retina. A cDNA encoding human recoverin was PCR cloned and used for prokaryotic expression of recoverin protein. Polyclonal antibodies raised against pure recombinant recoverin were used for western blotting and immunocytochemistry of Y79 cells grown as attachment cultures in the presence of the differentiating agents dibutyryl cyclic AMP (dbcAMP) or butyrate. Northern blot analysis was performed on mRNA extracted from Y79 cells that were also treated with the differentiating agents. In Y79 cell monolayer cultures, recoverin was immunolocalized to the cell cytoplasm, and immunoreactivity was increased dramatically by the addition of 2 m M butyrate to the culture medium. Butyrate treatment also caused an increase in the development of neurite-like cellular processes. Addition of 4 m M dbcAMP resulted in a moderate increase in both recoverin immunoreactivity and number of cellular processes. Western and northern blots of butyrate and dbcAMP-treated Y79 cell cultures demonstrated an increase in recoverin protein and RNA expression, respectively, comparable with that observed with immunocytochemistry. These data suggest that, under the influence of the differentiating agent butyrate, Y79 cells exhibit an increase in expression of the photoreceptor protein recoverin and a concomitant morphological differentiation toward a neuronal phenotype.