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First published online April 24, 2006
doi: 10.1242/10.1242/jcs.02895

Journal of Cell Science 119, 1896-1902 (2006)
Published by The Company of Biologists 2006
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Cbfa-1 mediates nitric oxide regulation of MMP-13 in osteoblasts

Carlos Zaragoza1,2,*, Esther López-Rivera1,2, Concepción García-Rama1, Marta Saura3, Antonio Martínez-Ruíz1,2, Tania R. Lizarbe1,2, Fernando Martín-de-Lara2 and Santiago Lamas1,2

1 Fundación Centro Nacional de Investigaciones Cardiovasculares (CNIC), Melchor Fernández Almagro 3, 28029 Madrid, Spain
2 Centro de Investigaciones Biológicas (CSIC), Instituto `Reina Sofía' de Investigaciones Nefrológicas, Ramiro de Maeztu 9, 28040 Madrid, Spain
3 Departamento de Fisiología, Facultad de Medicina, Universidad de Alcalá, Ctra de Barcelona, Km 33.5, 28871 Madrid, Spain


Figure 1
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  		Fig. 1. MMP13 and iNOS protein levels during MC3T3-E1 differentiation. MC3T3-E1 cells were analyzed at the time points indicated in the presence of 50 µg/ml ascorbic acid and 10 mM ß-glycerol phosphate. MMP-13 (upper panel) and iNOS (lower panel) were detected by immunoblot (n=3), whereas NO production was detected by Griess reaction (lower graph) (n=3).

 

Figure 2
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  		Fig. 2. NO reduces MMP-13 protein and MMP-13 activity during MC3T3-E1 differentiation. MC3T3-E1 cells were induced to differentiate with ascorbic acid (AA) and ß-glycerol phosphate (ßGP), and analyzed at the time points indicated in the presence or absence of 200 µM 1400W. (A) Immunoblot of MMP-13 and MT1-MMP (n=3). (B) Gelatin zimography (n=3). (C) Detection of MMP-13 activity by using a fluorogenic substrate from culture supernatants (n=3, mean ± s.d.).

 

Figure 3
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  		Fig. 3. The NO-cGMP-PKG pathway transcriptionally regulates MMP-13 expression during MC3T3-E1 differentiation. MC3T3-E1 cells were induced to differentiate and analyzed at the time points indicated. (A) MMP-13 promoter activity in MC3T3-E1 transiently transfected with pMMP-13 WT, and incubated with 100 µM DEA-NO, or 200 µM of the iNOS inhibitor 1400W (n=3, mean ± s.d., *P<0.05 versus control). (B) Measurement of MMP-13 promoter activity in MC3T3-E1 cells transiently transfected with pMMP-13 WT, and incubated with 100 µM DEA-NO, 10 µM 8-Br-cGMP, 20 µM ODQ, and 100 µM DEA-NO plus 20 µM ODQ (n=3, mean ± s.d., *P<0.05 versus control, #P<0.05 versus DEA-NO). (C) Measurement of MMP-13 promoter activity in MC3T3-E1 cells transiently transfected with pMMP-13 WT and co-transfected with a dominant-positive construct of PKG1-{alpha} (black bars) (n=3, mean ± s.d., *P<0.05 versus control). MMP-13 mRNA was evaluated in these cells by RT-PCR analysis using GAPDH as a control (inset shows a representative experiment and a densitometric analysis of three independent experiments, mean ± s.d., *P<0.05 versus absence of PKG).

 

Figure 4
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  		Fig. 4. The NO-cGMP-PKG pathway regulates MMP-13 expression through the Cbfa-1 responsive element OSE-2. Measurement of MMP-13 promoter activity in MC3T3-E1 cells transiently transfected with pMMP-13 WT or pMMP-13 OSE-2. (A) In the absence or presence of 8-Br-cGMP (8BrcGMP). (B) In the absence or presence or DEA-NO (n=3, mean ± s.d., *P<0.05 versus non-treated with 8-Br-cGMP or DEA-NO, #P<0.05 pMMP-13 OSE-2 versus pMMP-13 WT non-stimulated with 8-Br-cGMP or DEA-NO, @P<0.05 pMMP-13 OSE-2 versus pMMP-13 WT stimulated with 8-Br-cGMP or DEA-NO).

 

Figure 5
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  		Fig. 5. Effect of the NO-cGMP-PKG pathway on Cbfa-1 regulation in MC3T3-E1 cells. (A) Immunoblot analysis of Cbfa-1 over a time course of MC3T3-E1 differentiation (n=3). (B) Immunoblot analysis of Cbfa-1 over a time course of MC3T3-E1 differentiation in the presence of DEA-NO or 8Br-cGMP (n=3). (C) Immunocytochemistry analysis of Cbfa-1 (red) at day 7 of MC3T3-E1 differentiation. Cells were induced to differentiate (ii), or not (i), or induced to differentiate in the presence of 200 µM 1400W (iii). Nuclei were visualized with Hoechst (blue) (n=3). Bars, 25 µm. (D) Cbfa-1 expression from nuclear extracts and cytosolic extracts of MC3T3-E1 after 7 and 14 days of differentiation. PARP was detected as a control for nuclear integrity (n=3). (E) Recombinant Cbfa-1 (2 µg) was incubated with 100 units of PKG or 100 units of denatured PKG (bPKG) in phosphorylation buffer (see Materials and Methods), containing 1 µCi of [32P]{gamma}-ATP, in the presence or absence of 100 µM cGMP. Samples were electrophoresed and phosphorylation was visualized by autoradiography (n=2).

 

Figure 6
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  		Fig. 6. Cbfa-1 and MMP-13 expression are reduced in iNOS-deficient embryos. Immunohistological detection of Cbfa-1 (A,C,E,G,I,K,M,O) and MMP-13 (B,D,F,H,J,L,N,P) on sections from iNOS wild-type (WT) (left side) and iNOS-deficient (right side) 17-day-old embryos. Experiments were performed in triplicate on sections from three different embryos. Skulls (E,F,GH), ribs (I,J,K,L) and vertebrae (M,N,O,P) are magnified. m, Meckel's cartilage; mb, maxillary bone; nb, nasal bone; hbc, cartilage of the hyoid bone; cc, cricoid cartilage. (Q) Immunoblot detecting the levels of Cbfa-1, MMP-13 and GAPDH from WT (+/+) and iNOS-deficient (NOS2, -/-) mouse embryo lysates (n=3).

 

Figure 7
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  		Fig. 7. Cbfa-1 is required for NO-induced MMP-13 expression in differentiating MC3T3-E1 cells. MC3T3-E1 cells were differentiated and, on day 7, Cbfa-1 and GAPDH expression were silenced by RNA interference (siRNA). Cbfa-1, MMP-13 and GAPDH expression were evaluated by immunoblot. The capacity of Cbfa-1-silenced cells to produce NO was evaluated by measuring nitrite accumulation from culture supernatants (lower panel: n=3, mean ± s.d., *P<0.05 versus day 0).

 

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© The Company of Biologists Ltd 2006