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First published online 28 March 2006
doi: 10.1242/jcs.02867

Journal of Cell Science 119, 1622-1631 (2006)
Published by The Company of Biologists 2006
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NMDA induces post-transcriptional regulation of {alpha}2-guanylyl-cyclase-subunit expression in cerebellar granule cells

Sandra Jurado1, Fernando Rodríguez-Pascual2, José Sánchez-Prieto1, Francisco M. Reimunde2, Santiago Lamas2 and Magdalena Torres1,*

1 Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, Madrid, E-28040 Spain
2 Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Ramiro de Maeztu 9, Madrid, E-28040 Spain


Figure 1
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  		Fig. 1. Time-course of the upregulation of guanylyl cyclase {alpha}2 mRNA induced by NMDA in cerebellar granule cells. NMDA 100 µM was added to the culture medium and the total RNA extracted at the time points indicated. The RNA was reverse transcribed using random hexamers and the amount of {alpha}2 mRNA and 18S rRNA was determined by quantitative PCR using specific primers and probes for both. All the results were normalised against the 18S rRNA values and represent the mean ± s.e.m. of three experiments performed in triplicate. *P<0.001, significant difference from control values.

 

Figure 2
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  		Fig. 2. (A,B) Effect of (A) cycloheximide (CHx) and (B) actinomycin D (ActD) on the activity of NMDA. Cells were incubated with the concentrations of actinomycin D or CHx indicated for 30 minutes before the addition of NMDA. The mRNA was then extracted 24 hours later and subjected to RT-PCR (using random hexamers) and quantitative PCR. The results are the mean ± s.e.m. of five experiments performed in triplicate. *P<0.001 and #P<0.001, significant difference from control values. (C) The half-life of {alpha}2 mRNA was assessed in control and NMDA-treated cells by using actinomycin D (10 µg/ml). RNA was harvested at selected time points and the relative abundance of {alpha}2 mRNA was determined by quantitative RT-PCR. The results are the mean ± s.e.m. of four experiments performed in triplicate.

 

Figure 3
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  		Fig. 3. (A) 3' UTR sequence of {alpha}2 mRNA. This sequence was obtained by direct sequencing and by cloning and sequencing of two overlapping PCR fragments. The two oligonucleotide pairs for PCR are indicated in bold and underlined, and are labelled as F1, R1 and F2, R2. ARE sequences are indicted in bold capital letters. (B) An agarose gel showing the PCR products obtained with the different oligonucleotide pairs. Lanes 1, 3 and 5 correspond to the PCR products obtained after RT. Lanes 2, 4 and 6 show that no amplification was observed in the absence of RT; dT, oligodT.

 

Figure 4
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  		Fig. 4. Interaction of the RNA probes from the 3' UTR of the rat {alpha}2 subunit of guanylyl cyclase with nuclear or cytosolic proteins extracted from granule cells. (A) A radiolabelled, in vitro transcribed RNA fragment corresponding to the 843 bp fragment obtained with F1 and R1 primers (see Fig. 3) was incubated with of cytosolic (Cyt) or nuclear (Nucl) protein extracts from control or NMDA-treated cells. After binding, RNA-protein complexes were treated with T1 RNase and resolved by non-denaturing gel electrophoresis as described in the Materials and Methods. Controls were performed without the protein extract (NP). (B) Radiolabelled, in vitro transcribed RNA fragments corresponding to that of described in panel A was incubated with nuclear (Nucl) protein extracts from NMDA-treated cells or control cells, in the presence or absence of the non-labelled probe (NLP). (C) Radiolabelled, in vitro transcribed RNA fragment corresponding to the 1176 bp fragment obtained with the F2 and R2 primers (see Fig. 3) was used in a similar experiment to that described in A. (D) The presence of endogenous {alpha}2 mRNA was assayed in the IP material from cytosolic or nuclear extracts obtained with anti-HuR or anti-AUF1 by real-time RT-PCR. The results are the mean ± s.e.m. of two experiments. *P<0.01 and +P<0.01, significant difference from control values (IgG).

 

Figure 5
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  		Fig. 5. NMDA-treatment downregulates AUF1 levels in granule cells. (A) Whole-cell lysate (20 µg) of (1) control or (2) NMDA-treated cells, lamin B1 was immunodetected as loading control. (B) Nuclear or cytosolic extracts (N or C, respectively) from control or NMDA-treated cells. Lamin B1 (a nuclear protein) and GAPDH were immunodetected as loading control.

 

Figure 6
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  		Fig. 6. NMDA stimulates the NO-cGMP pathway in granule cells. (A) NMDA increases intracellular Ca2+ levels, as measured with the fluorimetric probe FLUO-4AM. (B) NMDA increases intracellular NO as measured with the fluorimetric probe DAF-FM. (C-E) NMDA also stimulates (C) cGMP, (D) VASP phosphorylation and (E) CREB phosphorylation. For immunocytochemistry, cells were incubated in the presence of 0.5 mM IBMX for 30 minutes and then fixed or stimulated with 100 µM NMDA for10 minutes before processing as described in Materials and Methods.. For phosphorylation studies, cells were incubated with 100 µM NMDA for 15 minutes in the presence or absence of 20 µM Rp-8-pCPT-cGMPS added 30 minutes before the stimulation. VASP and CREB phosphorylation was analysed with specific antibodies as described in the Materials and Methods. *P<0.001, significant difference from control values.

 

Figure 7
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  		Fig. 7. (A-C) Specific inhibitors of the NO-cGMP pathway (B) affect nuclear AUF1 levels and (A) abolish the effects of NMDA on nuclear AUF1 levels and on (C) the accumulation of {alpha}2 mRNA. Cells were incubated with the indicated compounds alone or in combination with 100 µM NMDA for 24 hours (ODQ and KT5823 were added 30-60 minutes before NMDA). Subsequently, proteins or RNA were extracted and processed as described in the Materials and Methods. (KT5823, 1 µM; MK-801, 5 µM; ODQ, 10 µM; and Sp-8-Br-PET-cGMPS, 50 µM). The results are the mean ± s.e.m. of four experiments. *P<0.001, significant difference from control values.

 

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