PubMed Health⌕ Search

Biomedical subjects

P D Gardner

Publications and source records attributed to P D Gardner.

At least 19 recordsLinked to original sources
PubMed2001

The signal transduction pathway underlying ion channel gene regulation by SP1-C-Jun interactions.

During neuronal differentiation, an exquisitely controlled program of signal transduction events takes place, leading to the temporally and spatially regulated expression of genes associated with the differentiated phenotype. A critical class of genes involved in this phenomenon is that made up of genes encoding neurotransmitter-gated ion channels that play a central role in signal generation and propagation within the nervous system. We used the well established PC12 cell line to investigate the molecular details underlying the expression of the neuronal nicotinic acetylcholine receptor class of ion channels. Neuronal differentiation of PC12 cells can be induced by nerve growth factor, leading to an increase in neuronal nicotinic acetylcholine receptor gene expression. Nerve growth factor initiates several signal transduction cascades. Here, we show that the Ras-dependent mitogen-activated protein kinase and phosphoinositide 3-kinase pathways are critical for the nerve growth factor-mediated increase in the transcriptional activity of a neuronal nicotinic acetylcholine receptor gene promoter. In addition, we show that a component of the Ras-dependent mitogen-activated protein kinase pathway, nerve growth factor-inducible c-Jun, exerts its effects on receptor gene promoter activity most likely through protein-protein interactions with Sp1. Finally, we demonstrate that the target for nerve growth factor signaling is an Sp1-binding site within the neuronal nicotinic acetylcholine receptor gene promoter.

Animals↗
PubMed2000

Interactions between regulatory proteins that bind to the nicotinic receptor beta4 subunit gene promoter.

The genes encoding the alpha3, alpha5 and beta4 subunits of nicotinic acetylcholine receptors are tightly clustered within the genome. As these three subunits constitute the predominant acetylcholine receptor subtype expressed in the peripheral nervous system, their genomic proximity suggests a regulatory mechanism ensuring their coordinate expression. We previously identified two transcriptional regulatory elements within the beta4 promoter. One of these elements, a CT box, interacts with the regulatory factors heterogeneous nuclear ribonucleoprotein K and Puralpha. Another element, a CA box, interacts with Sp1 and Sp3. The binding site for a fifth factor, Sox10, overlaps the CT and CA boxes. As the CT and CA boxes are adjacent, we postulated that the proteins that bind to the elements interact. Here we report that the CT box-binding factors interact with each other as do the CA box-binding factors. However, there are no direct associations between the two pairs of proteins. Interestingly though, Sox10 directly interacts with all four proteins, suggesting a central role in beta4 gene expression for this member of the Sox family of regulatory factors.

Cyclic AMP Response Element-Binding Protein↗
PubMed2000

Synergistic transcriptional activation by Sox10 and Sp1 family members.

Neuronal nicotinic acetylcholine receptors (nAChR) are expressed at specific times during development and in discrete neuronal populations. Transcriptional regulation of the receptor genes clearly plays a key role in the molecular pathway underlying the expression of these critical synaptic components. In an effort to understand this regulation, we focus upon the genes encoding three receptor subunits: alpha3, alpha5 and beta4. These subunits are genomically clustered and constitute the predominant nAChR subtype expressed in the peripheral nervous system. We and others demonstrated that the general transcription factors, Sp1 and Sp3, can transactivate the promoter of each subunit gene. Further, we showed that the regulatory factor Sox10 transactivates the alpha3 and beta4 promoters and does so in a cell-type-specific manner. Interestingly, the Sp- and Sox10-binding sites on the beta4 promoter are located immediately adjacent to each other, raising the possibility that the two sets of factors functionally interact to regulate receptor gene expression. Consistent with this hypothesis, we demonstrated that the proteins can directly interact. Here, we extend these observations and show that Sox10 and the Sp factors functionally interact, leading to synergistic transcriptional activation in a cholinergic cell line. Finally, evidence for the existence of cell-type-specific co-regulators for Sp1 and Sox10 is presented.

Animals↗
PubMed1999

Cell type-specific activation of neuronal nicotinic acetylcholine receptor subunit genes by Sox10.

The regulatory factor Sox10 is expressed in neural crest derivatives during development as well as in the adult CNS and peripheral nervous system. Mutations of the human Sox10 gene have been identified in patients with Waardenburg-Hirschsprung syndrome that is characterized by defects in neural crest development. Previous studies suggested that Sox10 might function as an important transcriptional regulator of neural crest development. No natural target genes of Sox10 have yet been identified. Although human Sox10 activates a synthetic promoter consisting of a TATA box and multiple Sox consensus sequences, no transcriptional activity of the rat Sox10 homolog has been detected. Here we report that the neuronal nicotinic acetylcholine receptor beta4 and alpha3 subunit gene promoters are transactivated by rat Sox10 in a cell type-specific manner. The alpha3 and beta4 subunits, in combination with the alpha5 subunit, make up the predominant nicotinic receptor subtype expressed in the peripheral nervous system. Transfections using Sox10 mutants indicate that the C-terminal region is dispensable for its ability to activate the beta4 and alpha3 promoters. Rat Sox10 was originally identified as an accessory protein of the POU domain protein Tst-1/Oct6/SCIP in glial cells. Tst-1/Oct6/SCIP was shown previously to activate the alpha3 promoter. We now demonstrate that it can transactivate the beta4 promoter as well. However, we were unable to detect any synergistic effects of Sox10 and Tst-1/Oct6/SCIP on beta4 or alpha3 promoter activity. Finally, we present data suggesting that recombinant Sox10 protein can directly interact with a previously characterized regulatory region of the beta4 gene.

3T3 Cells↗
PubMed1998

Differential effects of heterogeneous nuclear ribonucleoprotein K on Sp1- and Sp3-mediated transcriptional activation of a neuronal nicotinic acetylcholine receptor promoter.

The neuronal nicotinic acetylcholine receptor gene family consists of 11 members, alpha2-alpha9 and beta2-beta4. Three of the genes, those encoding the alpha3, alpha5, and beta4 subunits, are clustered tightly within the genome. These three subunits constitute the predominant acetylcholine receptor subtype expressed in the peripheral nervous system. The genomic proximity of the three genes suggests a regulatory mechanism ensuring their coordinate expression. However, it is likely that gene-specific regulatory mechanisms are also functioning because the expression patterns of the three genes, although similar, are not identical. Previously we identified regulatory elements within the beta4 promoter region and demonstrated that these elements interact specifically with nuclear proteins. One of these elements, E1, interacts with the regulatory factor Puralpha as well as three other unidentified DNA-binding proteins with molecular masses of 31, 65, and 114 kDa. Another element, E2, interacts with Sp1 and Sp3. Because E1 and E2 are immediately adjacent to one another, we postulated that the proteins that bind to the elements interact to regulate beta4 gene expression. Here we report the identification of the 65-kDa E1-binding protein as heterogeneous nuclear ribonucleoprotein K and demonstrate that it affects the transactivation of beta4 promoter activity by Sp1 and Sp3 differentially.

Animals↗
PubMed1997

Sp1 and Sp3 regulate expression of the neuronal nicotinic acetylcholine receptor beta4 subunit gene.

Neuronal nicotinic acetylcholine receptors play important roles in signal transduction within the nervous system. The receptors exist in a variety of functionally distinct subtypes that are determined by their subunit structures. The subunits are encoded by 11 genes, alpha2-alpha9 and beta2-beta4. Three of the genes, alpha3, alpha5, and beta4, are tightly clustered, and their encoded proteins make up the predominant receptor subtype in the peripheral nervous system. The tight linkage of the genes suggests there may be a common regulatory mechanism underlying their expression. However, although their expression patterns significantly overlap, they are not identical, indicating that independent regulatory mechanisms must also exist. Our studies have focused upon the gene encoding the beta4 subunit for which we have identified several transcriptional regulatory elements. One of these elements, E2, specifically interacts with the general transcription factor Sp1. Here we show that another member of the Sp family of factors, Sp3, can specifically interact with E2 whereas two other members, Sp2 and Sp4, cannot. Co-transfection experiments indicate that Sp3 can transactivate a beta4 promoter/reporter gene construct and, furthermore, that Sp1 and Sp3 can transactivate the beta4 reporter construct synergistically. The transactivation is dependent upon an intact E2 and may involve direct interactions between Sp1 and Sp3.

Animals↗
PubMed1997

Transcriptional regulation of neuronal nicotinic acetylcholine receptor genes. A possible role for the DNA-binding protein Puralpha.

Nicotinic acetylcholine receptors constitute a multigene family (alpha2-alpha9, beta2-beta4) expressed in discrete temporal and spatial patterns within the nervous system. The receptors are critical for proper signal transmission between neurons and their targets. The molecular mechanisms underlying receptor gene expression have not been completely elucidated but clearly involve regulation at the level of transcription. We previously identified a novel 19-base pair (bp) transcriptional regulatory element in the promoter region of the rat beta4 subunit gene. This 19-bp element interacts specifically with DNA-binding proteins enriched in nuclear extracts prepared from adult rat brain. Using a combination of cellulose-phosphate, DNA-cellulose, and DNA sequence-specific affinity chromatographies, we purified the 19-bp element binding activity approximately 19,000-fold. Analysis by denaturing gel electrophoresis revealed the presence of four polypeptides in the most purified fraction, ranging in molecular masses between 31 and 114 kDa. Peptide sequence analysis revealed that one of the polypeptides is the bovine homologue of the transcriptional regulatory factor, Puralpha. Electrophoretic mobility shift assays indicated that Puralpha interacts directly and specifically with the 19-bp element. In addition, mobility shift assays using an anti-Puralpha monoclonal antibody revealed the presence of Puralpha, or an immunologically related protein, in nuclear extracts prepared from brain tissue. We hypothesize that the interaction between Puralpha and the 19-bp element is critical for proper expression of the beta4 subunit gene.

Amino Acid Sequence↗
PubMed1996

Transcriptional regulation of neuronal nicotinic acetylcholine receptor genes. Functional interactions between Sp1 and the rat beta4 subunit gene promoter.

To date, 11 members (alpha2-alpha9 and beta2-beta4) of the neuronal nicotinic acetylcholine receptor gene family have been identified. These genes encode subunits that form distinct receptors with different pharmacological and physiological profiles in temporally and spatially restricted patterns within the nervous system. Distinct molecular mechanisms probably orchestrate the expression of various receptor subtypes, yet little is known of specific transcriptional regulatory elements and their associated factors that are responsible for this segregated pattern of expression. Here we report the identification of an element, in the 5'-flanking region of the rat beta4 subunit gene, containing a CA box that is necessary for beta4 promoter activity in a transiently transfected cholinergic cell line, SN17. This element was shown to interact with a protein(s) in SN17 nuclear extracts that is antigenically related to the transcriptional activator Sp1. Furthermore, co-transfection experiments confirmed that Sp1 can transactivate a beta4 promoter-reporter gene construct, indicating that Sp1 is necessary, at least in part, for transcriptional activation of the beta4 subunit gene.

Animals↗
PubMed1995

A novel regulatory element of a nicotinic acetylcholine receptor gene interacts with a DNA binding activity enriched in rat brain.

Nicotinic acetylcholine receptors are ligand-gated ion channels that play a critical role in signal transmission in the nervous system. The genes encoding the various subunits that comprise functional acetylcholine receptors are expressed in distinct temporal and spatial patterns. Studies to understand the molecular mechanisms underlying the differential expression of the receptor subunit genes have led to the identification, in this report, of a 19-base pair cis-acting element that is required for transcriptional activation of the rat beta 4 subunit gene. Screening of computer data bases with the 19-base pair element revealed the sequence to be unique among known transcriptional regulatory elements. Loss of this element resulted in drastically reduced beta 4 promoter activity in transfected cholinergic SN17 cells. Furthermore, this element specifically interacts with nuclear proteins prepared from both SN17 cells and adult rat brain. UV cross-linking experiments indicated the presence, in SN17 nuclear extracts, of a prominent protein species (approximately 50 kDa) that interacts specifically with the 19-base pair element. These results lead us to hypothesize that interactions between the 50-kDa protein and the novel 19-base pair element are necessary for transcriptional activation of the beta 4 subunit gene.

Animals↗
PubMed1995

Electrical activity suppresses nicotinic acetylcholine receptor gamma subunit promoter activity.

Nicotinic acetylcholine receptors are pentameric, transmembrane, ligand-gated ion channels critical for neuromuscular signal transmission. Prior to innervation, the genes encoding these receptors are expressed in nuclei throughout the muscle fiber. Muscle innervation leads to a dramatic decrease in expression of these genes in extrasynaptic nuclei. This reduction in gene expression can be reversed by muscle denervation. The effects of denervation on receptor gene expression can be blocked by electrical stimulation of muscle using extracellular electrodes. The molecular mechanisms by which muscle electrical activity leads to altered patterns of nicotinic acetylcholine receptor gene expression are not well understood. Using an in vitro electrical stimulation paradigm to induce muscle activity, we have been able to mimic the effect of innervation on extrasynaptic acetylcholine receptor gene expression. We have found that a 93-bp region of 5'-flanking DNA, spanning nucleotides -150 to -57 relative to the transcription start site of the gamma subunit gene, is required for the suppression of gene expression in response to muscle activity. Sequences downstream of this region are transcriptionally active but are not responsive to muscle activity. However, these downstream sequences become responsive to muscle activity when placed under the control of the gamma subunit muscle-specific enhancer.

Animals↗
PubMed1995

Differential expression of sodium channels and nicotinic acetylcholine receptor channels in nnr variants of the PC12 pheochromocytoma cell line.

An important component of neuronal differentiation is the tightly controlled expression of a spectrum of ion channel proteins. Ion channels play a critical role in the generation and propagation of action potentials as well as in the cellular response to neurotransmitters, and thus are central in the transfer and integration of information in the nervous system. A model system amenable to analysis of ion channel expression and neuronal differentiation is the rat pheochromocytoma (PC12) cell line. Here, we have used electrophysiological and molecular biological approaches to analyze the expression of voltage-dependent sodium (Na) channels and nicotinic acetylcholine receptors (nAChR) in mutagenized variants (nnr cells) of the PC12 cell line. Our data reveal striking differences in the expression of these channels when compared to wild-type PC12 cells. Even in the absence of nerve growth factor (NGF), nnr cells express functional Na channels and Na channel mRNA at levels exceeding those in wild-type PC12 cells differentiated with NGF. In contrast, acetylcholine-induced currents were evident in only a small proportion of cells, presumably due to the altered pattern of expression of mRNAs encoding individual nAChR subunits. The altered ion channel expression in these variants provides an avenue for analyzing Na channel and nAChR channel function, as well as for identifying mechanisms governing their expression.

Animals↗
PubMed1994

Nerve growth factor increases the transcriptional activity of the rat neuronal nicotinic acetylcholine receptor beta 4 subunit promoter in transfected PC12 cells.

Neuronal nicotine acetylcholine receptors play a key role in synaptic transmission in the nervous system. Although complementary DNA clones encoding a family of acetylcholine receptor subunits have been isolated and subsequent anatomical studies indicate differences in the temporal and spatially restricted patterns of expression of each gene, the cellular and molecular mechanisms controlling the expression of these genes are unknown. As part of a long-term goal to elucidate these mechanisms, we have been identifying and characterizing regions of the receptor subunit genes involved in transcriptional regulation. Here, we report the localization of the transcription initiation site of the rat beta 4 subunit gene, demonstrate using transient transfection analysis of PC12 cells that sequences upstream of this site are capable of activating transcription of a heterologous gene, and show that this transcriptional activity is enhanced in PC12 cells by treatment with nerve growth factor.

Amino Acid Isomerases↗
PubMed1994

Nerve growth factor increases nicotinic ACh receptor gene expression and current density in wild-type and protein kinase A-deficient PC12 cells.

Although neuronal nicotinic ACh receptors (nAChR) play a key role in synaptic transmission and information transfer in the nervous system, little is known about the molecular mechanisms that govern the expression of the multiple subunits that form the receptors and determine their functional properties. Using electrophysiological and molecular biological approaches, we have investigated the NGF-mediated regulation of nAChR expression in rat pheochromocytoma (PC12) cells and protein kinase A (PKA)-deficient PC12 cells. We report that NGF treatment increases steady state levels of mRNA encoding the alpha 3, alpha 5, alpha 7, beta 2, and beta 4 subunits, increases the occurrence of ACh-induced single-channel activity in excised patches, and increases ACh-induced macroscopic current density, all by mechanisms independent of PKA activity.

Animals↗
PubMed1993

Localization of preproenkephalin mRNA-expressing cells in rat auditory brainstem with in situ hybridization.

Hair cells and auditory nerve dendrites in the inner ear are innervated by pontine neurons that have been demonstrated by immunochemical techniques to contain several neurotransmitters, including acetylcholine and the opioid peptide enkephalins and dynorphins. The functions of these nerve fibers are not known, but may involve modifying auditory sensitivity to low intensity stimuli. In the guinea pig the opioid pathways originate in the lateral superior olivary region. A recent study in the gerbil has reported cells expressing preproenkephalin mRNA present only in the ventral nucleus of the trapezoid body, and not in the superior olivary region. In the present study, a non-radioisotopically labeled in situ hybridization method was used to identify cells expressing mRNA coding for preproenkephalin in rat pontine neurons, specifically in the ventral nucleus of the trapezoid body. These cells may represent an enkephalin-containing medial olivocochlear system in the rat, the origin of the lateral system in the rat that differs markedly from the better-studied guinea pig and cat, or a non-olivocochlear enkephalin-containing system.

Animals↗
PubMed1993

Potential role of helix-loop-helix proteins in cardiac gene expression.

Because helix-loop-helix (HLH) transcription factors appear to play an important role in mesodermal development, we have investigated the potential role of these factors in cardiac gene expression. HLH proteins interact with DNA at consensus "E-box" sites and may be tissue specific or more widely expressed. We have examined cardiac cells for expression and regulation of widely expressed factors Pan1/Pan2 and the inhibitor of differentiation (Id) by RNase protection analysis. The effect of MyoD, Id, and Pan1/Pan2 expression on skeletal and cardiac promoters in cardiac cells was examined by transient cotransfection studies. Our results indicate that neonatal ventricular cells are a functional HLH environment, because MyoD can activate a skeletal muscle-specific promoter in these cells. MyoD, however, has no effect on the expression of several genes that are expressed in cardiac cells. In addition, Id may be an early response gene for signal transduction in cardiac cells, because increases in Id mRNA occurred within 30 minutes of stimulation with serum or phenylephrine. Activities of three cardiac promoter elements in primary ventricular myocytes were not downregulated by Id. Surprisingly, expression of Pan1 and Pan2 exhibited a strong negative effect on cardiac expression of the myosin light chain-2 promoter.

Animals↗
PubMed1991

Multiple binding sites for myogenic regulatory factors are required for expression of the acetylcholine receptor gamma-subunit gene.

The nicotinic acetylcholine receptor (AChR) is a critical component of the mechanism responsible for signal transduction between nerve and muscle. Induction of AChR gene expression occurs during myogenesis when myoblasts exit the cell cycle and fuse to form myotubes. Induction of muscle-specific genes during differentiation is believed to be mediated through the activities of members of the MyoD family of myogenic regulatory factors. At least two of the factors, MyoD and myogenin, function as transcriptional activators. Here we present evidence that MyoD is capable of activating transcription of the mouse AChR gamma subunit gene. We have identified seven potential MyoD-binding sites (E-boxes) within the 5'-flanking DNA of the gamma subunit gene. We demonstrate that two small regions of the 5'-flanking DNA, one containing a pair of E-boxes and the other containing a single E-box, are required for gamma subunit expression in mouse muscle cells. DNase I footprinting indicates that the E-box proximal to the transcriptional initiation site is capable of interacting directly with MyoD. Furthermore, site-directed mutational analysis indicates that the single E-box is necessary for transactivation by MyoD and expression of the gamma subunit promoter region in muscle cells.

Animals↗
PubMed1991

ACh receptor-rich membrane domains organized in fibroblasts by recombinant 43-kildalton protein.

Neurotransmitter receptors are generally clustered in the postsynaptic membrane. The mechanism of clustering was analyzed with fibroblast cell lines that were stably transfected with the four subunits for fetal (alpha, beta, gamma, delta) or adult (alpha, beta, epsilon, delta) type mouse muscle nicotinic acetylcholine receptors (AChRs). Immunofluorescent staining indicated that AChRs were dispersed on the surface of these cells. When transiently transfected with an expression construct encoding a 43-kilodalton protein that is normally concentrated under the postsynaptic membrane, AChRs expressed in these cells became aggregated in large cell-surface clusters, colocalized with the 43-kilodalton protein. This suggests that 43-kilodalton protein can induce AChR clustering and that cluster induction involves direct contact between AChR and 43-kilodalton protein.

Acetylcholine↗