Biomedical subjects

R L Malek

Publications and source records attributed to R L Malek.

6 recordsLinked to original sources

PubMed1999

Adenosine A(2A) receptor mRNA regulation by nerve growth factor is TrkA-, Src-, and Ras-dependent via extracellular regulated kinase and stress-activated protein kinase/c-Jun NH(2)-terminal kinase.

We have shown previously that nerve growth factor (NGF) down-regulates adenosine A(2A) receptor (A(2A)AR) mRNA in PC12 cells. To define cellular mechanisms that modulate A(2A)AR expression, A(2A)AR mRNA and protein levels were examined in three PC12 sublines: i) PC12nnr5 cells, which lack the high affinity NGF receptor TrkA, ii) srcDN2 cells, which overexpress kinase-defective Src, and iii) 17.26 cells, which overexpress a dominant-inhibitory Ras. In the absence of functional TrkA, Src, or Ras, NGF-induced down-regulation of A(2A)AR mRNA and protein was significantly impaired. However, regulation of A(2A)AR expression was reconstituted in PC12nnr5 cells stably transfected with TrkA. Whereas NGF stimulated the mitogen-activated protein kinases p38, extracellular regulated kinase 1 and 2 (ERK1/ERK2), and stress-activated protein kinase/c-Jun NH(2)-terminal kinase (SAPK/JNK) in PC12 cells, these kinases were activated only partially or not at all in srcDN2 and 17.26 cells. Inhibiting ERK1/ERK2 with PD98059 or inhibiting SAPK/JNK by transfecting cells with a dominant-negative SAPKbeta/JNK3 mutant partially blocked NGF-induced down-regulation of A(2A)AR expression in PC12 cells. In contrast, inhibiting p38 with SB203580 had no effect on the regulation of A(2A)AR mRNA and protein levels. Treating SAPKbeta/JNK3 mutant-transfected PC12 cells with PD98059 completely abolished the NGF-induced decrease in A(2A)AR mRNA and protein levels. These results reveal a role for ERK1/ERK2 and SAPK/JNK in regulating A(2A)AR expression.

Animals↗

PubMed1999

Ciliary neurotrophic factor and phorbol ester each decrease selected STAT3 pools in neuroblastoma cells by proteasome-dependent mechanisms.

Many cytokines and growth factors activate common signal transduction pathways and yet are able to elicit distinct cell-specific responses. We are defining mechanisms regulating signalling molecules in order to understand how cytokines can produce unique responses. It was found that individual members of the signal transducer and activator of transcription (STAT) family are regulated by ciliary neurotrophic factor (CNTF) and by protein kinase C. Treatment of SH-SY5Y human neuroblastoma cells with the phorbol ester, 12- O -tetradecanoylphorbol 13-acetate (TPA), for 4-5 h caused a 60% decline in both STAT2 and STAT3 levels and no decline in levels of STATs 1, 5 or 6, or in Jaks 1 or 2. The decline in STAT3 was inhibited by treatment with MG132, an inhibitor of proteasome-dependent protein degradation. Treatment of cells with CNTF induced a rapid tyrosine phosphorylation of STAT3 followed by a time-dependent decay of this signal. Loss of tyrosine phosphorylated STAT3 was inhibited by MG132 but did not require protein kinase C activity. These results suggest that STAT3 availability can be controlled by proteasome-dependent pathways activated either by protein kinase C or by cytokines.

Ciliary Neurotrophic Factor↗

PubMed1999

Molecular cloning, tissue-specific expression, and chromosomal localization of a novel nerve growth factor-regulated G-protein- coupled receptor, nrg-1.

A novel and differentially expressed gene, named nrg-1, was identified by EST expression profiling and subsequently isolated as a 2.2-kb full-length clone from a rat PC12 cell cDNA library. Sequence analysis reveals that nrg-1 encodes a putative seven transmembrane spanning domain protein with structural features characteristic of receptors belonging to the G-protein-coupled receptor gene superfamily. The 400-amino-acid protein encoded by nrg-1 exhibits a high degree of sequence identity (40-44%) to the Edg receptor family; members include Edg-1, Edg-2, Edg-3, Edg-4, and H218. Both Northern analysis andEST expression profiling revealed that whole-tissue distribution of nrg-1 mRNA is restricted, found almost exclusively in brain. Transcripts of nrg-1 could be ubiquitously detected in different regions, with very prominent expression in lower brain regions such as the midbrain, pons,medulla, and spinal cord. In PC12 cells, nerve growth factor induces neuronal differentiation and repressed expression of nrg-1. Two other agents that differentiate PC12 cells, fibroblast growth factor and dibdutyryl cAMP, down-regulated nrg-1 mRNA levels. Epidermal growth factor, and agent that does not induce differentiation, did not repress nrg-1 mRNA levels. In a PC12 cell mutant that is deficient in protein kinase A activity (AB.11), all three differentiating agents were unable to down-regulate nrg-1 mRNA. Hence, protein kinase A appears to be an obligatory cellular component in nrg-1 mRNA regulation. Chromosomal mapping employing a rat somatic cell readiation hybrid panel demonstrated that nrg-1 is linked to marker D8Rat54 and tightly associated with H218 on chromosome 8.

Amino Acid Sequence↗

PubMed1999

A role of p75 in NGF-mediated down-regulation of the A(2A) adenosine receptors in PC12 cells.

Nerve growth factor (NGF) induces differentiation of the rat pheochromocytoma clone (PC12) by activating the high affinity receptor, p140(trkA), linked to mitogen-activated protein kinase. While the physiological role of the low affinity NGF receptor (p75) has not been clearly defined, this receptor promotes activation of nuclear factor (NF) kappaB in Schwann cells. PC12 cells express the A(2A) adenosine receptor (AR), whose expression is significantly decreased by NGF treatment. In this study, we determined whether TrkA or p75 is involved in NGF-mediated regulation of A(2A)AR expression. NGF treatment decreased A(2A)AR in a time-dependent manner, with maximal effects observed by 1 day, and continued down-regulation of the receptor for up to 3 days in the presence of NGF. The decrease in A(2A)AR was associated with a more delayed decrease in the steady-state levels of the A(2A)AR mRNA. Down-regulation of the A(2A)AR at 1 day was mimicked by activators of NFkappaB, such as H(2)O(2), and ceramide, and was attenuated by the inhibitor pyrrolidine dithiocarbamate or following transient transfection of PC12 cells with a dominant negative IkappaBalpha mutant. Moreover, NGF stimulated nuclear accumulation of p65 subunits of NFkappaB (but not p50 subunits) in PC12 cells, as determined by electrophoretic mobility shift assays and by Western blotting. In contrast, inhibition of TrkA by AG879 or of TrkA-dependent mitogen-activated protein kinase mitogen-activated protein kinase kinase with PD98059 blocked PC12 cell differentiation without affecting A(2A)AR down-regulation, suggesting dissociation between these two phenomena. Taken together, these data provide strong support for the involvement of the p75/NFkappaB pathway in NGF-mediated down-regulation of A(2A)AR in PC12 cells.

Animals↗

PubMed1998

Nerve growth factor regulation of m4 muscarinic receptor mRNA stability but not gene transcription requires mitogen-activated protein kinase activity.

Nerve growth factor (NGF) up-regulated steady-state levels of m4 muscarinic acetylcholine receptor (mAChR) mRNA in PC12 cells. Up-regulation of mRNA levels was associated with a corresponding increase in mAChR binding sites. Two other growth factors, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), up-regulated m4 mRNA and mAChR binding sites. Treatment of PC12 cells with NGF and bFGF, but not EGF, has previously been demonstrated to result in sustained activation of mitogen-activated protein kinase (MAPK). Analogously, NGF and bFGF, but not EGF, increased the stability of m4 mRNA in PC12 cells. In HER-PC12 cells, a clonal PC12 cell transfectant overexpressing EGF receptors and displaying sustained MAPK activation upon receptor stimulation, EGF treatment stabilized the m4 transcript. A synthetic inhibitor of MAPK kinase, PD98059, inhibited growth factor-induced stabilization of the m4 transcript in both PC12 and HER-PC12 cells. These findings demonstrate that the MAPK pathway is involved in transcript stabilization. Cycloheximide pretreatment abolished the post-transcriptional effect of NGF, indicating that de novo protein synthesis was required for the observed increase in m4 mRNA stability. By contrast, cycloheximide had no discernible post-transcriptional effect if added after NGF treatment, suggesting that an inducible yet stable protein factor was involved in m4 mRNA decay. An unusually well conserved 137 nucleotides of m4 3'-untranslated region has been identified by sequence comparison with other mRNAs that are post-transcriptionally regulated by NGF. In PC12 cells that heterologously overexpress this region, we demonstrate that NGF no longer stabilizes endogenous m4 mRNA. This conserved region probably represents an NGF-responsive element involved in mRNA stability regulation. Finally, transcription of the m4 gene can be induced by all three growth factors but is not dependent on MAPK activity, unlike growth factor-induced m4 mRNA stabilization.

Animals↗

PubMed1997

Opposing regulation of ciliary neurotrophic factor receptors on neuroblastoma cells by distinct differentiating agents.

We have used SH-SY5Y neuroblastoma cells as a model for differentiating neurons to examine the mechanisms that regulate responses to the neuropoietic cytokine ciliary neurotrophic factor (CNTF). Retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) each induced differentiation of SH-SY5Y cells. Cells treated for 24 h with retinoic acid (10 microM) showed a threefold increase in 125I-CNTF binding sites and were up to five times more sensitive to CNTF than untreated cells in stimulating the tyrosine phosphorylation of the transcription factor STAT3. TPA (10 nM) induced a transient 42% decrease in 125I-CNTF binding sites after 4 h of treatment that recovered to near control levels after 7 h of continuous exposure. TPA-treated cells showed a decreased sensitivity to CNTF and a sevenfold decrease in levels of STAT3. The retinoic acid-induced increase in 125I-CNTF binding could be prevented by administration of either cycloheximide or actinomycin D, whereas neither agent altered the TPA-induced decrease in 125I-CNTF binding. In addition, levels of mRNA for both the CNTF receptor alpha and gp130 subunits increased twofold as measured by RNase protection after treatment with retinoic acid for 30 h. The increase in CNTF receptor alpha subunit mRNA was not due to a decrease in its turnover rate, and therefore, was likely due to an increase in gene expression. Thus, retinoic acid and TPA regulate CNTF receptors on neuroblastoma cells differently, and the results demonstrate the importance of transcriptional control of CNTF receptors and also implicate translational and post-translational mechanisms in the regulation of cytokine receptors and responses on neurons.

Antineoplastic Agents↗