Regulation of vasoactive intestinal polypeptide and galanin mRNA stabilities

MOLECULAR BRAIN RESEARCH ELSEVIER

Molecular Brain Research 39 (1996) 89-98

Research report

Regulation of vasoactive intestinal polypeptide and galanin mRNA stabilities Paul J. Tolentino, Lydia Villa-Komaroff * Division of Neuroscience, Department of Neurology, Program in Neuroscience, Children's Hospital and Harc'ard Medical School, Boston, MA 02I 15, USA Accepted 20 December 1995

Abstract

The stabilities of vasoactive intestinal polypeptide (VIP) and galanin mRNAs were examined in a human neuroblastoma cell line (NBFL) treated with agents that alter second-messenger pathways. VIP and galanin mRNA stabilities were estimated by the decay of steady-state levels of transcripts following transcriptional arrest with actinomycin D or 5,6-dichloro-1-/3-o-ribofuranosylbenzimidazole (DRB). In the presence of actinomycin D, phorbol ester treatment stabilized VIP mRNA while treatment with adenylate cyclase activators, calcium ionophore, or CNTF did not. In the presence of DRB, VIP mRNA was not stabilized in phorbol ester-treated cells but instead was stabilized in cells treated with adenylate cyclase activators. With either transcriptional inhibitor, stability of galanin mRNA was not significantly altered. The difference in the behavior of VIP mRNA in the presence of actinomycin D and DRB may result from their different mechanisms of action - - actinomycin D intercalates into nucleic acids while DRB is a kinase inhibitor. Using an assay for RNA stability that did not require transcriptional inhibitors, an in vitro transcribed VIP RNA fragment was relatively stable in extracts from phorbol ester-treated cells. Although treatment with phorbol ester alone resulted in stabilization of VIP mRNA, treatment with a combination of phorbol ester and adenylate cyclase activator, calcium ionophore, or CNTF did not - - implying a complex interaction of these second-messenger pathways in the regulation of RNA stability. Keywords: Neuropeptide; Phorbol ester; Post-transcriptional regulation; Vasoactive intestinal polypeptide; 3'-Untranslated region

1. Introduction

Vasoactive intestinal polypeptide (VIP) is a 28-amino acid peptide originally identified in porcine small intestine [38] and subsequently localized within neurons of the central and peripheral nervous systems [26], anterior pituitary cells [35], mast cells [14], eosinophils [1], and B and T lymphocytes [18]. Although the principal function of VIP is presumably as a neurotransmitter, VIP may also function as a regulator of energy metabolism in the columnar fields of the cerebral cortex, as a prolactin-releasing factor in the pituitary, and as a cytokine in leukocytes. VIP may also have growth factor-related functions; VIP enhances proliferation, differentiation, and survival of sympathetic neuroblasts in culture [34] and dramatically stimulates growth of cultured mouse embryos [20]. Galanin, a

* Corresponding author. Present address: Department of Neurology, Northwestern University, 633 Clark Street, Evanston, IL 60208-1111, USA. Fax: (l) (847) 491-4800. 0169-328X/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PI1 S01 6 9 - 3 2 8 X ( 9 6 ) 0 0 0 0 4 - 6

29-amino acid peptide also originally identified in porcine small intestine [47], has species-specific effects on smooth muscle contractility [15,17] and inhibitory effects on insulin secretion [29]. Like VIP, galanin has been found in both the central and peripheral nervous systems [7,28]. In some systems, both VIP and galanin expression are induced by axotomy. For example, axotomy induces both VIP and galanin expression in dorsal root ganglia [21,41] and in the superior cervical ganglion [30]. The mechanisms regulating VIP and galanin gene expression are of substantial interest given the diverse locations of VIP and galanin and the important functions that these peptides may serve. Transcriptional control elements in the 5'-flanking region of the VIP gene confer responsiveness to cAMP, phorbol ester, and CNTF [16,45,49]. Galanin gene expression in adrenal chromaffin cells is also stimulated by some of these agents, but the elements responsible for these effects have not been identified [36]. Along with transcriptional enhancer elements, post-transcriptional regulation of m R N A stability can be an important determinant of m R N A expression. Induction by phor-

90

P.J. Tolentino, L. Villa-Komaro~f/ Molecular Brain Research 39 (1996) 89 98

bol esters, calcium, and cAMP of a number of different mRNAs has been shown to involve post-transcriptional mechanisms [2,3,9,10,23,27,32,40,50,52,54]. Recently, the 3'-untranslated region (3'-UTR) of the VIP mRNA has been shown to destabilize VIP mRNA in explanted rat pituitaries [11]. To determine whether post-transcriptional regulation of VIP and galanin mRNA stability was occurring under conditions that alter steady-state mRNA levels, the stabilities of VIP and galanin mRNA were examined in the human neuroblastoma cell line NBFL after exposure to phorbol esters, calcium ionophore, adenylate cyclase activators, and CNTF.

2. Materials and methods 2.1. NBFL cell culture

NBFL human neuroblastoma cells were cultured in Dulbecco's modified Eagle's medium (4.5 g/1 glucose) supplemented with I0% fetal calf serum, 5% horse serum, and 50 U / m l penicillin/50 ~ g / m l streptomycin (GIBCO, BRL) at 37°C under 5% CO2/95% air in a humidified chamber. Cells were passaged every 5 - 7 days in 100-mm tissue culture dishes (Falcon) at a 1:6 split ratio. For stimulation experiments, the medium was withdrawn from the cultures (75-90% confluent) and replaced with fresh medium containing the calcium ionophore A23187 (Sigma, 5 p~M from a 5 mM stock in DMSO), forskolin (Sigma, 5 /.tM from a I0 mM stock in DMSO) combined with isobutylmethylxanthine (IBMX; Sigma, 250 ~ M from a 500 mM stock in DMSO), phorbol 12-myristate 13-acetate (PMA; Sigma, 100 nM from a 100 ~ M stock in DMSO), or rat recombinant ciliary neurotrophic factor (CNTF; Regeneron, 10 n g / m l from a 10 /~g/ml stock in PBS). For control experiments, the medium was withdrawn from the cultures and replaced with either fresh medium alone or fresh media containing 0.1% DMSO. Transcriptional arrest was achieved by addition of actinomycin D (Sigma, 5 ~ g / m l from a 10 m g / m l stock in DMSO) or DRB (Calbiochem, 50 /~g/ml from a 50 m g / m l stock in DMSO). These concentrations of transcriptional inhibitors were sufficient for blocking transcriptional activity of the VIP and galanin genes (unpublished observations).

2.2.1.1. cVIP. A VIP cDNA was synthesized using oligonucleotides that span positions 241-863 of the human VIP mRNA sequence [22] (5'-GGGAAGCTTCATGCTGATGGAGTTTTC-3' and 5 ' - G G G G G A T C C C T A C T C T T C CAAAAACGTCTC-Y (underlined are additional sequences for HindIII and BamHI cloning)). This VIP cDNA was cloned into pUC 18 and designated cVIP. HindIII/BamHI digestion of cVIP produces a 0.63 kilobasepairs (kbp) VIP cDNA fragment that was used for generation of radiolabeled probes for Northern blots. 2.2.1.2. 3VIP. A VIP cDNA encoding 20 adenosine residues at the 3'-end of the message sequence was synthesized using oligonucleotides that span positions 335 of the human VIP mRNA sequence [22] to 8965 of the human VIP gene [53] ( 5 ' - G G G G A G C T C G C A G T A A CATCTCAGAAGACC-Y and 5'-GGGAAGCTTTTTTTTTTTTTTTTTTTTTTGCAATC-Y (underlined are additional sequences for SacI and HindIII cloning)). The Y-primer above corresponds to the Y-end of the VIP mRNA described by Lamperti et al. [25] that extends beyond the 3'-end of the VIP mRNA originally described [22]. The 1.1 kbp RT-PCR product was digested with EcoRI/HindIII, producing a 0.88 kbp EcoRI/HindIII fragment that corresponds to the Y-untranslated region (3'-UTR) of the VIP mRNA, excluding the first 43 nucleotides that reside within the VIP 3'-UTR between the stop codon and the EcoRI site. This VIP cDNA was cloned into pGEM-3Z (Promega) and was designated 3VIP. Linearization of 3VIP by HindIII digestion, fbllowed by in vitro RNA synthesis [39] using T7 RNA polymerase (Promega) and [c~-32P]UTP (DuPont/NEN) generated a radiolabeled, polyadenylated, sense strand 0.88 kb VIP RNA fragment corresponding to most of the VIP 3'-UTR. 2.2.2. Galanin Plasmid hGal 45.2, containing a 0.7 kbp human galanin cDNA fragment cloned into the EcoRI site of Bluescript KS (Stratagene), was provided by Lee Kaplan (Massachusetts General Hospital, Boston, MA). P s t l / S ~ I digestion of hGal 45.2 produced a 0.49 kbp galanin cDNA fragment used for generation of radiolabeled probes for Northern blots. 2.3. RNA isolation and Northern blot analysis

2.2. Plasmid constructs 2.2.1. VIP plasmids Existing sequence information was used to generate two different VIP cDNA-containing plasmids: cVIP and 3VIP. VIP cDNAs were generated by RT-PCR using total RNA from NBFL cells and VIP mRNA-specific oligonucleotides (Biopolymer Facility, Children's Hospital, Boston, MA) described below. RT-PCR was performed as described [48].

Cells were washed twice in PBS and harvested with l ml of 0.05% trypsin/0.53 mM EDTA (GIBCO, BRL) per 100-ram tissue culture dish. The trypsin/EDTA was inactivated with 2 ml of fresh media and diluted with 7 ml of ice-cold PBS. The cells were then pelleted in a clinical centrifuge and washed twice in ice-cold PBS. Total cytoplasmic RNA was extracted following NP-40 lysis [19]. Polyadenylated RNA was purified from total cytoplasmic RNA samples with oligo-dT cellulose [25]. Total nuclear

P.J. Tolentino, L. Villa-Komaroff/ Molecular Brain Research 39 (1996) 89-98

RNA was prepared using the method of Chomczynski and Sacchi from the nuclear pellet obtained after NP-40 lysis of the cells [13]. Northern blot analysis was performed as described [37] with slight modifications. Total RNA (10 /xg) was fractionated on 1% agarose/6% formaldehyde gels, transferred to Hybond-N ÷ charged nylon membrane (Amersham), and fixed to the membrane with 0.05 M NaOH [4]. Filters were hybridized in a solution containing 50% formamide, 5 × NPC (1 × NPC = 0.18 M sodium chloride/0.010 M sodium phosphate/0.001 M CDTA, pH 7.7), 1% SDS, 5% dextran sulfate, 100 /xg/ml sheared denatured salmon sperm DNA, and the appropriate probe at 45-60°C for 12 h. DNA probes were labeled to a specific activity of 1 × 10 9 d.p.m.//xg using a random oligonucleotide priming kit (Boehringer Mannheim) with [ c~-32P]dCTP (3000 C i / m m o l , DuPont/NEN). Membranes were washed in 0.1 × N P C / 0 . 1 % SDS at 55-60°C and exposed to X-ray film (Kodak XAR-5) with an intensifying screen at - 8 0 ° C . Quantitative analysis of the autoradiograms was performed via a scanner system using Image-Pro Plus (Media Cybernetics) with film exposures within the linear range of detection. The VIP probe hybridized to a 1700 base transcript while the galanin probe hybridized to a 900 base transcript. The ethidium bromide signals of ribosomal RNAs (18s rRNA for cytoplasmic RNA blots, 45s rRNA for nuclear RNA blots) were quantified from the negative exposure of photographs (Type 55 P / N film, Polaroid) of the gels under UV illumination. The signal intensity of the ribosomal RNAs were used to correct for loading differences. 2.3.1. Cell extract preparation

NBFL cells were harvested from tissue culture plates using trypsin/EDTA, as described for RNA isolation. After two washes with ice-cold PBS, the cell pellet was homogenized in a 4-fold (vol./vol.) excess of homogenization buffer (20 mM Tris-HC1, pH 7.5, 50 mM KC1, 5 mM EGTA, 5 mM MgC12, 0.1 mM DTT, 1 /xg/ml leupeptin (Sigma), and 0.1 mM phenylmethylsulfonylfluoride (Sigma), 4°C). The cell pellet was homogenized in 1.5 ml polypropylene tubes with a polypropylene pestle. After centrifugation ( > 13 000 × g) for 2 min in a microfuge at 4°C, the supernatant was re-centrifuged for 30 min. The supernatant was collected, rapidly frozen in liquid nitrogen, and stored at - 7 0 ° C . Protein concentrations were determined using a BCA protein assay (Pierce). 2.3.2. RNA degradation in cell extracts

The degradation of RNA in cell extracts was based on an assay for c-myc RNA decay [31]. Five micrograms of NBFL cell extracts was combined with 10 pg of radiolabeled, in vitro synthesized VIP 3'-UTR RNA (calculated from specific activity of incorporated [c~-32p]UTP) in a final volume of 50 /xl of degradation reaction buffer (20 mM Tris-HC1, pH 8.0, 5 mM KC1, and 0.15 mM NaC1) and incubated at 37°C. At 0, 0.5, 1, and 2 h of incubation,

91

l0 /xl aliquots of the reaction mixture were collected and added to 100 ~1 of stop solution (2 mM EDTA, 20 mM Tris-HC1, pH 8.0, 0.5% sodium dodecyl sulfate, 50 /xl phenol, and 50 /zl of CHC13/isoamyl alcohol (25:1). After extraction, the samples were ethanol-precipitated and resolved on a 1% agarose/6% formaldehyde gel. Gels were dried onto Whatman 3MM paper and exposed to X-ray film.

3. Results 3.1. Regulated expression o f VIP and galanin mRNAs in N B F L cells

The effects on VIP and galanin mRNA steady-state levels in response to different agents known to activate second-messenger pathways were examined. In these experiments, the existing medium on NBFL cells was replaced with fresh media containing the different agents. Replacing the existing media with fresh media (or fresh media containing 0.1% DMSO) resulted in a low level of induction of both VIP and galanin mRNAs (Fig. 1A). DMSO (0.1%), the vehicle for several of the agents described below, had no effect on VIP or galanin mRNA expression (unpublished observations). For the experiments described below, the "control" condition was defined by the level of mRNA expression after replacement with fresh media containing 0.1% DMSO. Although the addition of fresh media alone resulted in a moderate induction of both VIP and galanin mRNA expression, fresh media supplemented with a variety of agents stimulated RNA expression even more strongly. In these studies, the following agents were utilized: (1) PMA, a phorbol ester that activates protein kinase C; (2) forskolin/IBMX, an adenylate cyclase activator and a phosphodiesterase inhibitor, respectively, which increase cAMP levels within cells; (3) A23187, a calcium ionophore; (4) CNTF, a neurotrophic factor known to induce VIP mRNA in NBFL cells [45]. All agents increased VIP mRNA levels (Fig. 1A). The range of VIP mRNA induction varied from 235 _+ 22% with PMA treatment to 1148 _+ 118% with forskolin/IBMX treatment. In contrast to the > 10-fold range of VIP mRNA expression, the effects of second-messenger pathway manipulation on galanin mRNA expression were more modest (Fig. 1A) - the most potent induction of galanin mRNA occurred with PMA treatment (189 _+ 32% of control expression), while A23187 treatment actually reduced the amount of galanin transcript (68 _+ 4% of control expression). Since manipulation of second-messenger pathways is known to affect VIP gene transcription, the effect of these manipulations on the VIP precursor hnRNA in total nuclear RNA was examined. VIP precursor hnRNA was identified by Northern blot analysis of total nuclear RNA using the same radiolabeled VIP cDNA probe used in the

P.J. Tolentino, L. Villa-Komaroff / Molecular Brain Research 39 (1996) 89-98

92

cytoplasmic Northern blots described above. The VIP cDNA hybridized to a transcript of about 9000 bases corresponding to the full-length VIP mRNA precursor transcript (Fig. 1B). The VIP precursor hnRNA was undetectable in total nuclear RNA from untreated NBFL cells. However, VIP precursor hnRNA was detected in total nuclear RNA from NBFL cells given fresh media alone or with the different agents. As observed in cytoplasmic RNA, the weakest induction of VIP precursor hnRNA (136 + 12% of control expression) was associated with PMA treatment, while the strongest induction of VIP precursor hnRNA was associated with forskolin/IBMX treatment (979 __ 101% of control expression). Fig. 1C compares the degree of VIP mRNA induction in total cytoplasmic RNA and the degree of VIP precursor hnRNA induction in total nuclear RNA. After treatment with forskolin/IBMX, A23187, or CNTF, induction of VIP mRNA reflected the induction of its nuclear precursor. However, after PMA treatment, the degree of VIP mRNA induction exceeded the degree of VIP precursor hnRNA induction, implying that transcriptional activation and other

A

0

Ct

nuclear mechanisms could not completely account for the full extent of VIP mRNA induction. One possible explanation for the discrepancy between the levels of induction of the nuclear precursor and that of the mature cytoplasmic message is PMA-induced stabilization of the mature message.

3.2. Stability of VIP and galanin transcripts in NBFL cells To assess whether the stability of VIP a n d / o r galanin mRNA was altered in cells treated with PMA, forskolin/IBMX, A23187, or CNTF, the decay of steadystate levels of VIP and galanin mRNA was measured after transcriptional arrest with actinomycin D or DRB. Actinomycin D induces transcriptional arrest by intercalation into DNA [43], while DRB blocks transcription by inhibiting RNA polymerase II [12]. To observe the decay of the steady-state levels of VIP and galanin mRNA after transcriptional arrest, NBFL cells were treated with PMA, forskolin/IBMX, A23187, or CNTF for 6 b as described in the Methods. Actinomycin D was then added and the

B

P F/I A2 CN

0

Ct

P F/I A2 CN

pre-VlP

VIP

Galanin

fll C 1400 1200< Z 1000rr

-~ aoo~ 8 6002 0 '~ 400 0"4 200

Ctrl

PMA

F/I

A2

CNTF

Fig. l. Effects of second-messenger pathway manipulation on VIP and galanin mRNA expression in NBFL cells. Media on subconfluent NBFL cells was replaced with fresh media alone or supplemented with 0.1% DMSO (Control or Ct), 100 nM PMA (P), 5 /~M forskolin/250 /~M IBMX (F/I), 5 /~M A23187 (A2), or 5 n g / m l CNTF (CN). Subconfluent NBFL cells receiving no media change were designated as (0). After incubation for 6 h, i0 /~g of total cytoplasmic RNA or total nuclear RNA were subjected to Northern blot analysis. A: Hybridization of radiolabeled VIP and galanin cDNAs to total cytoplasmic RNA. Loading differences on the blot are reflected by the intensity of the ethidium signal of the 18s rRNA. B: Hybridization of radiolabeled VIP cDNA to total nuclear RNA. Loading differences on the blot are reflected by the intensity of the ethidium signal of the nuclear 45s rRNA. C: Quantification of VIP mRNA induction in total cytoplasmic RNA (gray boxes) and VIP precursor hnRNA in total nuclear RNA (hatched boxes), calculated as percent of control (Ct) expression, n = 4. A 2-tailed Student's t-test was used to compare differences in cytoplasmic VIP mRNA induction versus nuclear VIP precursor hnRNA induction for each experimental condition. * P < 0.01.

P.J. Tolentino, L. Villa-Komaroff / Molecular Brain Research 39 (1996) 89-98

A 1O0

8O "~

A

B

B

100

100-1~ 8012

60-

93

60-

8O

80

6o-

60-

40-

40-

20-

20~

g

< 40z rr E 20-

40-

20-

Z

rr E

0 Time (hrs) Fig. 2. Stability of VIP and galanin mRNA after transcriptional arrest with actinomycin D. Media on subconfluent NBFL cells was replaced with fresh media supplemented with 0.1% DMSO (11), 100 nM PMA (O), 5 /xM forskolin/250 /zM IBMX (D), 5 p~M A23187 (©), or 5 n g / m L CNTF ( • ) . After incubation for 6 h, the media was further supplemented with 5 /xg/ml actinomycin D. At the time of actinomycin D addition (t = 0) and l, 3, and 6 h thereafter, total cytoplasmic RNA was prepared and subjected to Northern blot analysis. Quantitative differences in loading of the denaturing gels were corrected by the intensity of the ethidium signal of the 18s rRNA. Northern blots were hybridized with a radiolabeled VIP cDNA or with a radiolabe'led galanin cDNA. A: VIP mRNA steady-state levels, expressed as mean percent_+ S.E.M. of mRNA expression at t = 0 . B: Galanin mRNA steady-state levels _+S.E.M., expressed as mean percent_+ S.E.M. of mRNA expression at t = 0 (n = 4).

decay of steady-state levels of VIP and galanin mRNAs was measured (Fig. 2). The pattern of VIP mRNA decay reveals two findings (Fig. 2A). First, VIP mRNA decayed relatively rapidly during the first 3 h after actinomycin D addition, but little to no further degradation of VIP mRNA was observed beyond 3 h. Second, VIP mRNA was relatively unstable under control conditions or in cells treated with forskolin/IBMX, A23187, or CNTF. However, VIP mRNA was very stable in cells treated with PMA - - the same condition where the degree of cytoplasmic VIP mRNA induction exceeded the degree of VIP precursor hnRNA induction (Fig. 1C). Galanin mRNA was generally more stable than VIP mRNA, and its pattern of decay was not as strongly affected by the different agents as that observed for VIP mRNA (Fig. 2B). With transcription arrested by DRB (Fig. 3), most of the VIP mRNA degradation also occurred in the first 3 h after DRB addition, with little or no degradation observed beyond 3 h. Surprisingly, when DRB was used to inhibit transcription, VIP mRNA was stabilized in forskolin/IBMX-treated cells but not in PMA-treated cells (Fig. 3A). However, forskolin/IBMXinduced stabilization of VIP mRNA is inconsistent with the previous observation that forskolin/IBMX treatment resulted in equivalent inductions of VIP precursor hnRNA and VIP mRNA (Fig. 1C). Galanin mRNA did not degrade significantly over the duration of the experiment (Fig. 3B). Stabilization of mRNA after 2 - 3 h of actinomycin Dor DRB-induced transcriptional arrest has been observed in other systems and has been attributed to the loss of expres-

~ 4 5 6

0

Fig. 3. Stability of VIP and galanin mRNA after transcriptional arrest with DRB. Media on subcontinent NBFL cells was replaced with fresh media supplemented with 0.1% DMSO (11), 100 nM PMA (O), 5 /xM forskolin/250 /xM IBMX (D), 5 /zM A23187 (~), or 5 n g / m l CNTF ( • ) . After incubation for 6 h, the media was further supplemented with 50 /xg/ml DRB. At the time of DRB addition (t = 0) and 1, 3, and 6 h thereafter, total cytoplasmic RNA was prepared and subjected to Northern blot analysis. Quantitative differences in loading of the denaturing gels were corrected by the intensity of the ethidium signal of the 18s rRNA. Northern blots were hybridized with a radiolabeled VIP cDNA or with a radiolabeled galanin cDNA. A: VIP mRNA steady-state levels, expressed as mean percent_+S.E.M, of mRNA expression at t = 0 . B: Galanin mRNA steady-state levels, expressed as mean percent + S.E.M. of mRNA expression at t = 0 (n = 4).

sion of a labile protein required for mRNA degradation [42]. Assuming first-order kinetics of decay for the first 3 h after transcriptional arrest, estimates of VIP mRNA halflives in the presence of actinomycin D or DRB are presented in Table 1A. The divergent results, especially in PMA- and forskolin/IBMX-treated cells, observed with actinomycin D and DRB suggest that the pathways for VIP mRNA degradation are significantly altered by one or both of the transcriptional inhibitors. Resolution of the discordant findings observed with actinomycin D and DRB

Table 1 Half-lives of VIP mRNA in NBFL cells Treatment

Single treatment a Control PMA Forskolin/IBMX A23187 CNTF PMA co-treatment b Forskolin/IBMX A23187 CNTF

VIP mRNA half-life(h) Actinomycin D

DRB

4 ± 1 > 6 1.5 _+0.1 1.9_+0.2 3.0 _+0.4

4.4_+ 1 3.4_+0.7 >6 2.2_+0.3 3.3_+0.9

3.7 _+0.3 1.7_+0.2 1.5 _+0.1

The half-life of VIP mRNA under different conditions was determined, approximating first-order kinetics for the first 3 h after addition of transcriptional inhibitor, a Half-lives of VIP mRNA in NBFL cells under conditions of single-stimulation, measured in the presence of actinomycin D or DRB. b Half-lives of VIP mRNA in NBFL cells under conditions of dual-stimulation, measured in the presence of actinomycin D.

P.J. Tolentino, L. Villa-Komaroff / Molecular Brain Research 39 (1996) 89-98

94

required an assay of VIP mRNA stability independent of these transcriptional inhibitors.

3.4. Effect of co-actic, ating second-messenger systems in NBFL cells

3.3. Stabili O, of a VIP RNA fragment in extracts" from NBFL cells

To determine whether pathways activated by PMA stabilized VIP mRNA under all conditions or whether other signaling pathways in the cells modified PMA-induced stabilization, NBFL cells were treated with PMA in combination with forskolin/IBMX, A23187, or CNTF (Fig. 5). Co-stimulation with PMA and A23187 or CNTF induced both VIP precursor hnRNA and VIP mRNA to a greater extent than was observed with either A23187 or CNTF alone (Fig. 5A), and the level of VIP mRNA induction was matched by an equivalent induction of VIP precursor hnRNA (Fig. 5B). In contrast, co-stimulation with PMA and forskolin/IBMX induced VIP precursor hnRNA to the same extent as lorskolin/IBMX alone; however, VIP mRNA was induced to a greater extent than was observed with forskolin/IBMX alone (Fig. 5A). After co-stimulation with PMA and forskolin/IBMX, the degree of VIP mRNA induction exceeded the degree of VIP precursor hnRNA (Fig. 5B), implying that nuclear precursor induction was insufficient to account for the induction of the mature transcript, and suggesting that selective stabilization of cytoplasmic VIP mRNA was occurring. To determine whether VIP mRNA was stabilized in cells treated with both PMA and forskolin/IBMX, NBFL cells were co-stimulated with PMA and forskolin/IBMX, A23187, or CNTF for 6 h. Actinomycin D was then added and the decay of steady-state levels of VIP mRNA was measured. The combination of PMA and forskolin/IBMX stabilized VIP mRNA relative to combinations of PMA with A23187 or CNTF (Fig. 6). As had been observed under conditions of single stimulation and actinomycin

RNA decay in the absence of transcriptional inhibitors was examined by measuring the degradation of a radiolabeled VIP RNA fragment in extracts from NBFL cells, A VIP mRNA 3'-UTR RNA fragment was chosen for these studies because experiments in explanted anterior pituitaries suggested that the VIP 3'-UTR contains sequences that destabilize VIP mRNA [11]. The VIP 3'-UTR RNA fragment was relatively stable in cell extracts from PMAtreated NBFL cells, compared to cell extracts from NBFL cells receiving all other treatments; there was a 30 min lag in the decay of the VIP 3'-UTR RNA fragment in extracts from PMA-treated cells that was not observed in other cell extracts, and so there was a higher level of intact VIP 3'-UTR RNA fragment in extracts from PMA-treated cells at all time points (Fig. 4). A non-adenylated galanin RNA fragment was equally unstable in all cell extracts (unpublished observations). Thus, the stabilization of the VIP 3'-UTR RNA fragment in cell extracts from PMA-treated NBFL cells was not due to the nonspecific absence of RNAse activity. The stabilization of the VIP 3'-UTR RNA fragment in cell extracts from PMA-treated cells was consistent with the stabilization of the endogenous VIP mRNA in PMA-treated NBFL cells observed after transcriptional arrest with actinomycin D. Furthermore, these experiments suggested that the sequences responsible for PMA-induced stabilization of VIP mRNA are present within the VIP 3'-UTR. Time 0

•

•

0.5

100.

1

- Ctrl

80-

- PMA

60.

g 13- F/I

<~ - A 2

A - CNTF

n

40.

20.

0

. . . .

0

I

0.5

~ '

'

'

I

1

. . . .

I

1.5

. . . .

J

2

Time (Hours) Fig. 4. Stability of an in vitro transcribed VIP 3'-UTR RNA fragment in NBFL cell extracts. A radiolabeled RNA fragment corresponding to the majority of the VIP 3'-UTR was generated from plasmid 3VIP. For preparation of cell extracts, media on subconfluent NBFL cells was replaced with fresh media containing 0.1% DMSO (Ctrl, m), 100 nM PMA (PMA, 0 ) , 5 /xM forskolin/250 /xM IBMX ( F / I , []), 5 #M A23187 (A2, ~), or 5 n g / m L CNTF (CNTF, • ) . After 6 h, the cell extracts were prepared. The stability of the in vitro synthesized VIP 3'-UTR RNA fragment was determined in these cell extracts. On the left are sample autoradiographs showing the degradation pattern of the radiolabeled VIP 3'-UTR RNA in different cell extracts. On the right is a quantification of radiolabeled VIP 3'-UTR RNA levels, expressed as a mean percent ± S.E.M. of signal at t 0, over 2 h of incubation in the different cell extracts (n = 5).

P.J. Tolentino, L. Villa-Komaroff / Molecular Brain Research 39 (1996) 89-98

95

lOO I

D-induced transcriptional arrest, VIP mRNA decayed relatively rapidly during the first 3 h after actinomycin D addition, with little to no degradation observed beyond 3 h.

I~1 60-

g A

Ct

P

F/I

< z 40-

F/I A2 A2 CN CN PMA PMA PMA

E 20-

VIP Time (hrs)

18s

pre-VlP

:

Fig. 6. Stability of VIP mRNA after co-stimulation with PMA and other agents and transcriptional arrest with actinomycin D. Media on subconfluent NBFL cells was replaced with fresh media supplemented with 100 nM P M A / 5 IxM forskolin/250 txM IBMX (O), 100 nM P M A / 5 ~M A23187 ( ~ ), or 100 nM PMA/5 n g / m l CNTF ( [] ). After incubation for 6 h, the media was further supplemented with 5 ~ g / m l actinomycin D. At the time of actinomycin D addition (t = 0) and 1, 3, and 6 h thereafter, total cytoplasmic RNA was prepared and subjected to Northern blot analysis. Quantitative differences in loading of the denaturing gels were corrected by the intensity of the ethidium signal of the 18s rRNA. Northern blots were hybridized with a radiolabeled VIP cDNA, and VIP mRNA steady-state levels are expressed as percent_+ S.E.M. of mRNA expression at t = 0 (n = 3).

B Assuming first-order kinetics of decay for the first 3 h after actinomycin D addition, estimates of VIP mRNA half-lives under the co-stimulation conditions in the presence of actinomycin D are presented in Table lB.

< Z

rr O

4. Discussion

c O

O O

F/I + PMA

A23187 + PMA

CNTF + PMA

Fig. 5. Effect of co-stimulation of PMA with other agents on cytoplasmic VIP mRNA and nuclear VIP precursor hnRNA expression. Media on subconfluent NBFL cells was replaced with fresh media supplemented with 0.2% DMSO (Control or Ct), 100 nM PMA (P), 5 /xM forskolin/250 /xM IBMX (F/l), 100 nM P M A / 5 /xM forskolin/250 /xM IBMX ( F / I / P M A ) , 5 /xM A23187 (A2), 100 nM P M A / 5 /xM A23187 (A2/PMA), 5 n g / m l CNTF (CN), or 100 nM P M A / 5 n g / m l CNTF (CN/PMA). After incubation for 6 h, 10 /xg of total cytoplasmic RNA or total nuclear RNA were subjected to Northern blot analysis. A: Hybridization of radiolabeled VIP eDNA to total cytoplasmic RNA (VIP) or to total nuclear RNA (pre-V1P) Northern blots. Loading differences for cytoplasmic and nuclear RNAs were reflected by the ethidium signal of the 18s rRNA and 45s rRNA, respectively. B: Quantification of VIP mRNA induction in total cytoplasmic RNA (gray boxes) and VIP precursor hnRNA in total nuclear RNA (hatched boxes), calculated as percent of control (Ct) expression, n = 4. A 2-tailed Student's t-test was used to compare differences in cytoplasmic VIP mRNA induction versus nuclear VIP precursor hnRNA induction for each experimental condition. * * P < 0.001.

The results presented in this report suggest that V1P mRNA is stabilized in NBFL cells treated with phorbol esters. Because galanin mRNA was much more stable than VIP mRNA, it was more difficult to determine whether galanin mRNA stability was significantly altered. Since the levels of VIP mRNA changed significantly under the conditions and time course of these experiments, the majority of the experiments focused on alterations of VIP mRNA stability. First, the difference in the induction of VIP precursor hnRNA and VIP mRNA by PMA treatment suggested that nuclear mechanisms could not completely account for the full extent of VIP mRNA induction. Second, the pattern of VIP mRNA degradation after treatment with actinomycin D revealed that PMA treatment resulted in stabilization of VIP mRNA. Third, an in vitro transcribed VIP RNA fragment was relatively stable in cell extracts from PMA-treated cells and was unstable in all other cell extracts. These studies indicate that VIP mRNA expression is influenced by post-transcriptional mechanisms as well as by transcriptional ones. The agents used in these studies activate VIP gene transcription via previously identified enhancer elements [16,45,49,51]. However, treatment with the phorbol ester PMA not only increased the level of VIP

96

P.,L Tolentino, L. Villa-Komarc~ff/ Molecular Brain Research 39 (1996) 89-98

precursor hnRNA, but also stabilized VIP mRNA in the cytoplasm. PMA-induced mRNA stabilization has been observed for a number of different transcripts. Stabilization of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA was one of the earliest models for PMA-induced mRNA stabilization [40]. Other mRNAs that have been shown to be stabilized by PMA treatment include transforming growth factor /31 [50], GAP-43 [33], ribonucleotide reductase RI [9] and R2 [2], and amyloid precursor protein mRNA [54]. On the other hand, PMA treatment has also been shown to destabilize other mRNAs, including mRNAs for the surface antigens CD4 and CD8 and the mRNAs for recombination activating genes 1 and 2 in early thymocytes [46], and the M1 muscarinic receptor message [27]. Though the stabilities of many mRNAs are regulated by PMA-sensitive pathways, PMA-treatment does not alter mRNA stability in a nonspecific manner; for example, the stability of c-myc mRNA is unaffected by PMA treatment [6]. The same sequence within the VIP mRNA 3'-UTR may regulate both instability and inducible stabilization, or different sequences may perform these functions independently, or interaction between different sequences may be required. The adenosine + uridine-rich element (ARE) within the GM-CSF mRNA 3'-UTR was the first sequence element shown to destabilize a normally stable mRNA [40]. Subsequent experiments have shown that the cisacting sequences of GM-CSF mRNA responsible for PMA-induced stabilization of the message are present in the GM-CSF mRNA Y-UTR; however the sequences that confer stability are outside of the ARE [24]. The 0.86 kb VIP mRNA 3'-UTR that was used in the cell extract experiment is approximately 71% AU-rich and contains 2 AUUUA elements and 13 AUUU(n > 3) regions. In other systems, the AU-richness of the ARE and the presence of the AUUUA pentamer have been identified as principal elements mediating mRNA stability [6,8,40]. Given the apparent diversity of the AREs that confer instability, it is possible that some of the pathways identified for the regulated degradation of other messages [5] may also act upon VIP mRNA. Actinomycin D has been used in many studies of PMA-induced mRNA stabilization [2,9,27,40,50,54]. In the present study, PMA-induced VIP mRNA stabilization was observed after actinomycin D-induced transcriptional arrest. However, stabilization of VIP mRNA was not observed after transcriptional arrest by DRB. Because the mechanism of action of actinomycin D involves intercalation into DNA at transcriptionally active sites [43], it is unlikely that actinomycin D would have direct effects on second-messenger signaling pathways within the cell. On the other hand, DRB is an inhibitor of casein kinase II [55] and at least one other kinase [44]. It has been suggested that the mechanism of action of DRB-induced transcrip-

tional arrest involves its activity as a kinase inhibitor [44,56] DRB may inhibit other kinase-sensitive pathways, including those that regulate RNA stability. This may explain why the observed stability of VIP mRNA in PMAor forskolin/IBMX-treated cells was dependent on whether actinomycin D or DRB was used to arrest transcription. The degradative pathways for a specific mRNA, e.g., VIP mRNA, may be qualitatively altered by the kinase inhibitor DRB, and the interaction between DRB and mRNA degradative pathways may be more pronounced when certain kinase (PKC, PKA) pathways are strongly activated at the same time. PMA-induced stabilization of VIP mRNA can be replicated using a non-translated VIP Y-UTR RNA fragment and cell extracts from NBFL cells treated with PMA. The paradigm of studying the degradation of an in vitro transcribed RNA fragment in cell extracts was originally used to study c-myc mRNA degradation [31] and has been used to examine the degradation of VIP RNA in anterior pituitary extracts [11]. PMA-induced stabilization of transforming growth factor /31 mRNA has also been demonstrated in vitro [50]. However, these experiments must be interpreted with caution. There are important differences between endogenous VIP mRNA and the VIP 3'-UTR RNA fragment; the VIP 3'-UTR RNA fragment is not full length, not capped, not associated with translating ribosomes, and may not be associated with the same proteins as endogenous VIP mRNA. There are also differences between the cytoplasm of the intact cells and the cell extracts, such as ionic conditions and the presence of membrane-associated intracellular proteins. Thus, the mechanisms of RNA degradation and PMA-induced stabilization in the cell extracts may not reflect the corresponding mechanisms within intact cells. Our results also indicate that PMA-stimulation is insufficient to ensure stabilization of VIP mRNA; rather, additional second-messenger signals apparently modulate the pathways of mRNA degradation. The combination of PMA and forskolin/IBMX resulted in VIP mRNA stability (tl/z = 3.7 + 0.3 h) that was intermediate between the stability of VIP mRNA with either agent alone (tl/2 > 6 h with PMA; t~/2 = 1.5 ___0.1 h with forskolin/IBMX). On the other hand, PMA treatment had no effect on the stability of VIP mRNA in the presence of A23187 (tl/2 = 1.9 _+ 0.2 h with A23187, t~/2 = 1.7 _+ 0.2 h with PMA and A23187). Furthermore, the combination of PMA and CNTF actually appeared to destabilize VIP mRNA (t~/2 = 1.5 + 0.1 h) compared to the stability of VIP mRNA observed when stimulated by CNTF alone (tl/2 = 3.0 + 0.4 h). The manipulations described in this report may reveal not only how VIP mRNA stability is regulated by second-messenger pathways, but also provide a framework for studying how other transcripts may be post-transcriptionally regulated through second-messenger pathways.

P.J. Tolentino, L. Villa-Komaroff / Molecular Brain Research 39 (1996) 89-98

Acknowledgements W e t h a n k J o n a t h a n Flax, N i n a Irwin, E d L a m p e r t i , a n d R o b i n M o z e l l for t e c h n i c a l a d v i c e and c o m m e n t s o n the m a n u s c r i p t . W e also t h a n k J. S t e p h e n F i n k for p r o v i d i n g the N B F L cell line. T h i s w o r k w a s s u p p o r t e d b y grants f r o m the T o b a c c o R e s e a r c h C o u n c i l ,

[15]

[16]

the Spinal C o r d

R e s e a r c h F o u n d a t i o n , N I H R01 N S 2 7 8 3 2 ,

and an N I H

c e n t e r G r a n t P30 H D 1 8 6 5 5 . P.J.T. w a s s u p p o r t e d by N R S A T32 M H 1 8 0 1 2 .

[17]

[18]

References [19] [1] Aliakbari, J., Sreedharan, S.P., Turck, C.W. and Goetzl, E.J., Selective localization of vasoactive intestinal peptide and substance P in human eosinophils, Biochem. Biophys. Res. Commun., 148 (1987) 1440-1445. [2] Amara, F.M., Chen, F.Y. and Wright, J.A., Phorbol ester modulation of a novel cytoplasmic protein binding activity at the 3'-untranslated region of mammalian ribonucleotide reductase R2 mRNA and role in message stability, J. Biol. Chem., 269 (1994) 6709-6715. [3] Bartlett, J.D., Luethy, J.D., Carlson, S.G., Sollott, S.J. and Holbrook, N.J., Calcium ionophore A23187 induces expression of the growth arrest and DNA damage inducible CCAAT/enhancer-binding protein (C/EBP)-related gene, gadd153, J. Biol. Chem., 267 (1992) 20465-20470. [4] Beckers, T., Schmidt, P. and Hilgard, P., Highly sensitive northern hybridization of rare mRNA using a positively charged nylon membrane., BioTechniques, 16 (1994) 1074-1078. [5] Beelman, C.A. and Parker, R., Degradation of mRNA in eukaryotes, Cell, 81 (1995) 179-183. [6] Bohjanen, P.R., Petryniak, B., June, C.H., Thompson, C.B. and Lindsten, T., An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3' untranslated region of lymphokine mRNA, Mol. Cell. Biol., 11 (1991) 3288-3295. [7] Ch'ng, J.L.C., Christofides, N.D., Anand, P., Gibson, S.J., Allen, Y.S., Su, H.C., Tatemoto, K., Morrison, J.F.B., Polak, J.M. and Bloom, S.R., Distribution of galanin immunoreactivity in the central nervous system and the responses of galanin-containing neuronal pathways to injury, Neuroscience, 16 (1985) 343-354. [8] Chen, C.-Y.A. and Shyu, A.-B., Selective degradation of early-response-gene mRNAs: functional analyses of sequence features of the AU-rich elements, Mol. Cell. Biol., 14 (1994) 8471-8482. [9] Chen, F.Y., Amara, F.M. and Wright, J.A., Regulation of mammalian ribonucleotide reductase R1 mRNA stability is mediated by a ribonucleotide reductase R 1 mRNA T-untranslated region cis-trans interaction through a protein kinase C-controlled pathway, Biochem. J., 302 (1994) 125-132. [10] Chen, M., Schnermann, J., Smart, A.M., Brosius, F.C., Killen, P.D. and Briggs, J.P., Cyclic AMP selectively increases renin mRNA stability in cultured juxtaglomerular granular cells, J. Biol. Chem., 268 (1993) 24138-24144. [11] Chew, L.-J., Murphy, D. and Carter, D.A., Alternatively polyadenylated vasoactive intestinal peptide mRNAs are differentially regulated at the level of stability, Mol. Endocrinol., 8 (1994) 603-613. [12] Chodosh, L.A., Fire, A., Samuels, M. and Sharp, P.A., 5,6-dichlorol-/3-D-ribofuranosylbenzimidazole inhibits transcription elongation by RNA polymerase II in vitro, J. Biol. Chem., 264 (1989) 22502257. [13] Chomczynski, P. and Sacchi, N., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156-159. [14] Cutz, E., Chan, W., Track, N.S., Goth, A. and Said, S.I., Release of

[20]

[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31 ] [32]

[33]

97

vasoactive intestinal polypeptide in mast cells by histamine liberators, Nature, 275 (1978) 661-662. Ekblad, E., H~kanson, R., Sundler, F. and Wahlestedt, C., Galanin: neuromodulatory and direct contractile effects on smooth muscle preparations, Br. J. Pharmacol., 86 (1985) 241-246. Fink, J.S., Verhave, M., Walton, K., Mandel, G. and Goodman, R.H., Cyclic AMP- and phorbol ester-induced transcriptional activation are mediated by the same enhancer element in the human vasoactive intestinal peptide gene, J. Biol. Chem., 266 (1991) 3882-3887. Fox, J.E.T., McDonald, T.J., Kostolanska, F. and Tatemoto, K., Galanin: An inhibitory neural peptide of the canine small intestine, Life Sci., 39 (1986) 103-110. Gomariz, R.P., Leceta, J., Garrido, E., Garrido, T. and Delgado, M., Vasoactive intestinal peptide (VIP) mRNA expression in rat T and B lymphocytes, Regul. Peptides, 50 (1994) 177-184. Greenberg, M.E. and Ziff, E.B., Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene, Nature, 311 (1984) 433438. Gressens, P., Hill, J.M., Gozes, I., Fridkin, M. and Brenneman, D.E., Growth factor function of vasoactive intestinal peptide in whole cultured mouse embryos, Nature, 362 (1993) 155-158. HiSkfelt, T., Wiesenfeld-Hallin, Z., Villar, M. and Melander, T., Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy, Neurosci. Lett., 83 (1987) 217-220. Itoh, N., Obata, K., Yanaihara, N. and Okamato, H., Human preprovasoactive intestinal polypeptide contains a novel PHI-27-1ike peptide, PHM-27, Nature, 304 (1983) 547-549. Iwai, Y., Akahane, K., Pluznik, D.H. and Cohen, R.B., Ca 2+ ionophore A23187-dependent stabilization of granulocyte-macrophage colony-stimulating factor messenger RNA in murine thymoma EL-4 cells is mediated through two distinct regions in the 3'-untranslated region, J. lmmunol., 150 (1993) 4386-4394. Iwai, Y., Bickel, M., Pluznik, D.H. and Cohen, R.B., Identification of sequences within the murine granulocyte-macrophage colonystimulating factor mRNA 3'-untranslated region that mediate mRNA stabilization induced by mitogen treatment of EL-4 thymoma cells, J. Biol. Chem., 266 (1991) 17959-17965. Lamperti, E.D., Rosen, K.M. and Villa-Komaroff, L., Characterization of the gene and messages for vasoactive intestinal polypeptide (VIP) in rat and mouse, Mol. Brain Res., 9 (1991) 217-231. Larsson, L.-I., Localization of vasoactive intestinal polypeptide: a critical appraisal. In: S.I. Said (Ed.), Vasoactiue Intestinal Peptide, 1982, pp. 51-63. Lee, N.H., Earle-Hughes, J. and Fraser, C.M., Agonist-mediated destabilization of ml muscarinic acetylcholine receptor mRNA, J. Biol. Chem., 269 (1994) 4291-4298. Lindh, B., Lundberg, J.M. and H~Skfelt, T., NPY-, galanin-, VIP/PHI-, CGRP- and substance P-immunoreactive neuronal subpopulations in cat autonomic and sensory ganglia and their projections, Cell Tissue Res., 256 (1989) 259-273. McDonald, T.J., Dupre, J., Tatemoto, K., Greenberg, G.R., Radziuk, J. and Mutt, V., Galanin inhibits insulin secretion and induces hyperglycemia in dogs, Diabetes, 34 (1985) 192-196. Mohney, R.P., Siegel, R.E. and Zigmond, R.E., Galanin and vasoactire intestinal peptide messenger RNAs increase following axotomy of adult sympathetic neurons, J. Neurobiol., 25 (1994) 108-118. Pei, R. and Calame, K., Differential stability of c-myc mRNAs in a cell free system, Mol. Cell. Biol., 8 (1988) 2860-2868. Perrone-Bizzozero, N.I., Cansino, V.V. and Kohn, D.T., Posttranscriptional regulation of GAP-43 gene expression in PC12 cells through protein kinase C-dependent stabilization of the mRNA, J. Cell. Biol., 120 (1993) 1263-1270. Perrone-Bizzozero, N.I., Neve, R.L., Irwin, N., Lewis, S., Fishcer, I. and Benowitz, L.I., Post-transcriptional regulation of GAP-43 mRNA levels during neuronal differentiation and nerve regeneration, Mol. Cell Neurosci., 2 (1991) 402-409.

98

P.J. Tolentino, L. Villa-Komaroff / Molecular Brain Research 39 (1996) 89 98

[34] Pincus, D.W., DiCicco-Bloom, E.M. and Black, I.B., Vasoactive intestinal peptide regulates mitosis, differentiation and survival of cultured sympathetic neuroblasts, Nature, 343 (1990) 564-567. [35] Reichlin, S., Neuroendocrine significance of vasoactive intestinal polypeptide, Ann. NY Acad. Sci., 527 (t988)431-449. [36] RiSkaeus, A., Pruss, R.M. and Eiden, L.E., Galanin gene expression in chromaffin cells is controlled by calcium and protein kinase signaling pathways, Endocrinology, 127 (1990) 3096-3102. [37] Rosen, K.M., Lamperti, E.D. and Villa-Komaroff, L., Optimizing the Northern blot procedure, BioTechniques, 8 (1990) 398-403. [38] Said, S.I. and Mutt, V.M., Potent peripheral and splanchnic vasodilator peptide from normal gut, Nature, 225 (1970) 863-864. [39] Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: a Laboratoo, Manual, 2nd edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [40] Shaw, G. and Kamen, R., A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation, Cell, 46 (1986) 659-667. [41] Shehab, S.A.S. and Atkinson, M.E., Vasoactive intestinal polypeptide (VIP) increases in the spinal cord after peripheral axotomy of the sciatic nerve originate from primary afferent neurons, Brain Res., 372 (1986) 37-44. [42] Sbyu, A.-B., Greenberg, M.E. and Belasco, J.G., The c-~)s transcript is targeted for rapid decay by two distinct mRNA degradation pathways, Genes Det,., 3 (1989) 62-70. [43] Sobell, H.M., Actinomycin and DNA transcription, Proc. Natl. Acad. Sci. USA, 82 (1985) 5328-5331. [44] Stevens, A. and Maupin, M.K., 5,6-dichloro-l-/3-I>ribofuranosylbenzimidazole inhibits a Hela protein kinase that phosphorylates an RNA polymerase II-derived peptide, Biochem. Biophys. Res. Commun., 159 (1989) 508-515. [45] Symes, A.J., Rao, M.S., Lewis, S.E., Landis, S.C., Hyman, S.E. and Fink, J.S., Ciliary neurotrophic factor coordinately activates transcription of neuropeptide genes in a neuroblastoma cell line, Proc. Natl. Acad. Sci. USA, 90 (1993) 572-576. [46] Takahama, Y. and Singer, A., Post-transcriptional regulation of early T cell development by T cell receptor signals, Science, 258 (1992) 1456-1462.

[47] Tatemoto, K. and Mutt, V., Isolation and characterization of the intestinal peptide porcine PHI (PHI-27), a new member of the glucagon-secretin family, Proc. Natl. Acad. Sci. USA, 78 (1981) 6603-6607. [48] Tolentino, P.J., Dikkes, P., Tsuruda, L., Ebert, K., Fink, J.S., Villa-Komaroff, L. and Lamperti, E.D., Quantitative analysis of the expression of a VIP transgene, Mol. Brain Res., 1995, in press. [49] Tsukada, T., Fink, J.S., Mandel, G. and Goodman, R.H., Identification of a region in the human vasoactive intestinal polypeptide gene responsible for regulation by cyclic AMP, J. Biol. Chem., 262 (1987) 8743-8747. [50] Wager, R.E. and Assoian, R.K., A phorbol ester-regulated ribonuclease system controlling transforming growth factor 131 gene expression in hematopoietic cells, Mol. Cell. Biol., 10 (1990) 59835990. [51] Waschek, J.A., Hsu, C.-M. and Eiden, L.E., Lineage-specific regulation of the vasoactive intestinal peptide gene in neuroblastoma cells is conferred by 5.2 kilobases of 5'-flanking sequence, Proc. Natl. Acad. Sci. USA, 85 (1988) 9547-9551. [52] Wodnar-Filipowicz, A. and Moroni, C., Regulation of interleukin 3 mRNA expression in mast cells occurs at the posttranscriptional level and is mediated by calcium ions, Proc. Natl. Acad. Sci. USA, 87 (1990) 777 781. [53] Yamagami, T., Ohsawa, K., Nishizawa, M., lnoue, C., Gotoh, E., Yanaihara, N., Yamamoto, H. and Okamoto, H., Complete nucleotide sequence of human vasoactive intestinal peptide/PHM-27 gene and its inducible promoter, Ann. NY Acad. Sci., 527 (1988) 87-102. [54] Zaidi, S.H.E. and Malter, J.S., Amyloid precursor protein mRNA stability is controlled by a 29-base element in the T-untranslated region, J. Biol. Chem., 269 (1994) 24007-24013. [55] Zandomeni, R. and Weinmann, R., Inhibitory effect of 5,6-dichlorol-/3-D-ribofuranosylbenzimidazole on a protein kinase, J. Biol. Chem., 259 (1984) 14804-14811. [56] Zandomeni, R., Zandomeni, M.C., Shugar, D. and Weinmann, R., Casein kinase type II is involved in the inhibition by 5,6-dichloro-l/3-D-ribofuranosylbenzimidazole of specific RNA polymerase II transcription, J. Biol. Chem., 261 (1986) 3414 3419.