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First published online March 29, 2004
doi: 10.1242/10.1242/jcs.01002

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Induction of nitric oxide synthase-2 proceeds with the concomitant downregulation of the endogenous caveolin levels

¹ Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, 28040 Madrid, Spain
² Fundación Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain
³ Instituto de Bioquímica (Centro Mixto Consejo Superior de Investigaciones Cientificas-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain

View larger version (29K): [in a new window]   Fig. 1. Induction of NOS2 proceeds with the concomitant downregulation of the cav-1, cav-2 and cav-3 levels in mouse C2C12 myotubes treated with LPS/IFN-. Myotubes were treated with LPS/IFN- and the changes in NOS2, cav-1, cav-2 and cav-3 were analysed by immunoblot and quantified using the UVI-band software. Induction of NOS2 together with the accumulation of nitrites is depicted in (A) The intensity of the NOS2 bands at five different times as integrated using the UVI-band software is shown at the top. Accumulated nitrites in a 54 hour period is shown at the bottom. The disappearance of cav-1 (continuous line), cav-2 (dotted line) and cav-3 (dashed line) in myotubes is represented in (B), both as the immunodetected bands (top) and as a plot (bottom). Protein levels are expressed as percentage of the maximum; the initial amount of caveolin is therefore 100%. ß-Tubulin is also shown as a control of total protein in each sample. The results shown are representative of four experiments (means±s.d.).

View larger version (77K): [in a new window]   Fig. 2. Subcellular localization of NOS2 and the various caveolin isoforms in mouse C2C12 myotubes in control experiments (A) and in cells treated with the proinflammatory mixture of LPS/IFN-ß (B). (Left) Myotubes were fixed with paraformaldehyde and methanol, and incubated with antibodies against cav-1, cav-2, cav-3 or NOS2 and either left untreated (A) or treated with LPS/IFN- for 36 hours (B). (Middle) Subcellular distribution of NOS2 in myotubes untreated (A) or treated with LPS/IFN- for 36 hours (B). The merge signal is depicted on the right. The caveolin fluorescence was visualized by confocal microscopy at an excitation wavelength of 488 nm and is shown in green, whereas the NOS2 was obtained after excitation at 543 nm and is shown in red. Overlap of green and red labeling is depicted in yellow; overlap of green and blue labeling is depicted in light blue; overlap of red and blue is depicted in violet. Scale bar, 50 µM. In all cases, the position of the cell nuclei (blue) was obtained after staining with Hoechst and excitation at 405 nm.

View larger version (56K): [in a new window]   Fig. 3. Localization of NOS2-GFP to the Golgi apparatus (A) and particulate immunofluorescence of NOS2-GFP both in myoblasts and myotubes (B). Live C2C12 myoblasts were grown on glass coverslips, transfected with a NOS2-GFP construct and incubated with the Golgi marker BODIPY-Texas red ceramide (A). In addition, C2C12 myoblasts and myotubes were grown on glass coverslips and transfected with GFP alone (left) or with a construct of NOS2 fused to the GFP reporter (right) (B). (B, far right) Magnification of myotubes transfected with a NOS2-GFP construct. The subcellular localization was analysed by laser confocal microscopy at an excitation wavelength of 488 nm. Scale bar, 10 µM (bottom), 50 µM (all others). In all cases, the position of the cell nuclei (blue) was obtained after staining with Hoechst and excitation at 405 nm.

View larger version (34K): [in a new window]   Fig. 4. Treatment of myotubes with ·NO donors induce changes in the levels of cav-3, but not of cav-1 or cav-2. C2C12 myoblasts were differentiated into myotubes and incubated with LPS (2 µg ml-1) plus IFN- (100 U ml-1), the ·NO donor DETA-NONOate (100 µM), the ·NO inhibitors 1400W (100 µM) and nitro-Arg (100 µM) for 36 h (cav-1 and cav-2) or 48 h (cav-3) in different combinations. The cells were scraped and the changes in cav-1 (white bars), cav-2 (single-slashed bars) and cav-3 (double-slashed bars) protein levels were determined by immunodetection (A). In addition, activated C2C12 myotubes were incubated with the ·NO donor DETA NONOate, the lipophilic cGMP analog 8-bromo-cGMP (20 µM), with the soluble guanylate cyclase inhibitor ODQ (10 µM) or with cycloheximide (CHX; 10 µM) in various combinations for 48 hours, and the levels of cav-3 were determined by immunodetection (B). In every case, the total amount of protein loaded was similar, as judged by ß-tubulin quantification. *P<0.001 vs the corresponding condition in the absence of ·NO donor. Averaged results are shown, being representative of at least three independent experiments (±s.d.).

View larger version (35K): [in a new window]   Fig. 5. Association of the three caveolin isoforms to NOS2 purified and immunoprecipitated from mouse C2C12 myotubes. Separation of the total lysate of LPS/IFN--treated C2C12 myotubes into a soluble [Supernatant (Sup.)] or particulate [Pellet (Pel.)] fraction and analysis of the NOS2, cav-1, cav-2 and cav-3 distribution as judged by immunodetection (A). Additionally, NOS2 was immunoprecipitated from C2C12 myotubes treated with LPS/IFN- and the association with CaM, cav-1, cav-2 and cav-3 was determined (B). Finally, NOS2 was affinity purified using an ADP/Sepharose column (C). In this experiment, a lane was loaded with the amount of total cell lysate (Lys.) to give a similar NOS2 immunoreactivity to that of the purified sample (Pur.). Under this circumstance, the amount of cav-1, cav-2 and cav-3 that was retained bound to the purified NOS2 was compared with that present in the total lysate as determined by immunoblot (C).

View larger version (26K): [in a new window]   Fig. 6. Synthesis of ·NO in mouse C2C12 myotubes treated with LPS/IFN- when incubated with antisense phosphorothioates (ASP) of cav-1, cav-2 and cav-3 (A), and abrogation of caveolin-1 downregulation by protein kinase inhibitors (B). C2C12 myotubes were treated for 8 hours with antisense phosphorothioate oligonucleotides complementary to the first 21 bases of the mRNA encoding cav-1, cav-2 and cav-3. After the treatment, the muscle cells were challenged with LPS/IFN- for 36 hours and the amount of ·NO that accumulates was determined with the Griess assay. The antisense oligonucleotides (ASP) were added individually (cav-1, cav-2 and cav-3) or in combination (cav-123). A scrambled oligo corresponding to the cav-1 base sequence was also used as a control. The absence of changes in NOS2 and ß-tubulin in each case is confirmed by immunodetection (A, bottom). The results shown are representative of ten individual experiments. In order to determine the pathway of downregulation followed by cav-1 and cav-2 in the presence of the LPS/IFN- mixture, the cells were incubated with various protein kinase inhibitors for 48 hours (B). Subsequently, the levels of cav-1 were determined by immunodetection. The concentration of nitrites in the supernatant in the presence of 10 µM Erk inhibitor PD was also determined (right). *P<0.001 vs the LPS/IFN- condition. Error bars represent deviation from the average.

View larger version (32K): [in a new window]   Fig. 7. Changes induced in NOS2, cav-1 and cav-3 levels in muscle (quadriceps) of wild-type (NOS2+/+) and knock-out (NOS2-/-) mice upon treatment with LPS/IFN-. The LPS/IFN- proinflammatory stimulus was maintained in animals for 36 hours and the muscular tissue was processed. The intensity of each immunodetected protein band was quantified using the UVI-band software and represented as a vertical bar chart. The results shown are representative of two individual experiments, with four mice used in each condition tested.

View larger version (22K): [in a new window]   Fig. 8. Changes in the cav-1 levels observed with the LPS/IFN- proinflammatory stimulus in mouse hepatocytes, HepG2 cells and Raw 264.7 macrophages. Mice were injected with LPS (1.5 µg LPS g-1) and IFN- (8 U g-1), and the changes in the cav-1 levels were determined by immunodetection. HepG2 and Raw 264.7 were grown and challenged with the proinflammatory stimuli.

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