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First published online 29 July 2008
doi: 10.1242/jcs.027151

Journal of Cell Science 121, 2751-2758 (2008)
Published by The Company of Biologists 2008
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P311 functions in an alternative pathway of lipid accumulation that is induced by retinoic acid

James K. Leung1, Sylvaine Cases2 and Thiennu H. Vu1,*

1 Lung Biology Center and Department of Medicine, University of California, San Francisco, CA 94143, USA
2 Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA


Figure 1
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  		Fig. 1. P311 expression induces morphological changes in cells seeded at high density. (A) Semiquantitative RT-PCR showing low levels of expression of the Myc-tagged P311 transcripts in C3H10 cells that contain the Tet-On transactivator and a Tet-responsive mP311-myc transgene (mP311-myc) without (–) doxycycline (Dox) treatment, and increased levels of mP311-myc transcripts after induction with Dox (+). Control cells (C3H10 cells containing the Tet-On transactivator and a Tet-responsive empty vector) showed no expression of the mP311-myc transcripts. β-actin transcripts were used as a loading standard. (B,C) Control cells seeded at high density (106 cells per 100-mm plate) have the typical spindle-shaped morphology of C3H10T1/2 cells in culture. (D,E) cells stably expressing P311 seeded at the same high density show a different morphology exhibiting cell enlargement and a dark perinuclear ring. Scale bars, 40 µm (B,D), 10 µm (C,E).

 

Figure 2
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  		Fig. 2. P311 increases lipid accumulation. (A,B) C3H10 control and cells stably expressing P311 were pulsed with fatty-acid complexes. Lipids were then extracted and measured for relative levels of triglycerides (A) and cholesterol (B). There is a significant (approximately threefold; P<0.05) increase in cholesterol and triglyceride levels in cells stably expressing P311. Mean ± s.d. from triplicate samples are shown. (C,D) Staining with the lipophilic dye Oil-Red-O of C3H10 control (C) and P311 stable (D) cells pulsed with fatty-acid complexes for 24 hours demonstrates the presence of lipid droplets in cells stably expressing P311 (D). (E-L) Phase-contrast (E,I,G,K) and epifluorescence (F,J,H,L) images of C3H10 cells stably expressing P311 (E-H) and NIH3T3-L1 adipocytes (I-L) immunostained with either adipophilin (E,F,I,J) or TIP47 (G,K,H,L) antibody demonstrating staining of lipid droplets in NIH3T3-L1 adipocytes with both adipophilin and TIP47 antibody, but no staining of lipid droplets in C3H10 P311 with either antibody. Scale bars, 5 µm (C,D).

 

Figure 3
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  		Fig. 3. P311 gene silencing does not affect lipid accumulation in NIH3T3-L1 cells. (A) P311 mRNA expression levels during adipogenic differentiation of NIH3T3-L1 cells as determined by quantitative real-time RT-PCR. There is almost a complete lack in P311 transcripts at the beginning of induction (Day 2) followed by a gradual increase to base-line levels at the completion of differentiation (Day 8). Mean ±s.d. from triplicate samples are shown. Asterisks indicate statistically significant (P<0.01) pair wise comparison between Day 0 and Day 2 samples (*) and between Day 0 and Day 4 samples (**). There is no significant difference between Day 0 and Day 8 samples. (B) NIH3T3-L1 preadipocytes infected with control lentivirus or lentivirus expressing a short hairpin RNA (shRNA) targeting P311 were examined for relative P311 RNA expression levels by quantitative real-time RT-PCR. There is an ~90% decrease in P311 transcripts in P31-shRNA-lentivirus-infected cells relative to control-lentivirus-infected cells. Mean ± s.d. from triplicate samples are shown. The differences between P311 shRNA and control samples are statistically significant (P<0.05). (C-F) Phase contrast (C and E) and epifluorescence (D and F) images of control (C,D) and P311-shRNA-lentivirus-infected NIH3T3-L1 (E,F) cells following induction of adipogenic differentiation demonstrate that both control and P311-shRNA-lentivirus-infected cells (marked by GFP) accumulate lipid droplets (arrows) to similar extent. Scale bar, 10 µm. (G) The relative amount of lipids in control versus P311-shRNA-lentivirus-infected NIH3T3-L1 cells following induction of adipogenic differentiation, assessed by Nile-Red-stained cells followed by flow cytometry, indicates no significant change in lipid accumulation in the P311-shRNA-lentivirus-infected population. Control and P311-shRNA-lentivirus-infected NIH3T3-L1 cells were sorted for GFP and Nile Red fluorescence. The mean fluorescence intensities of the Nile Red channel in the GFP+ populations were determined. Values are normalized to the average of the mean fluorescence intensities of the control GFP+ samples. Mean ± s.d. from triplicate samples are shown.

 

Figure 4
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  		Fig. 4. P311 is a retinoic-acid-responsive gene in lung fibroblasts. (A) Quantitative RT-PCR showing induction of P311 mRNA in lung fibroblasts isolated from early postnatal mice following treatment with retinoic acid (RA; 1 µM), but not with fatty acids (FA; 0.1 µM) for 1 week. Mean ± s.d. from triplicate samples are shown. Asterisks indicate statistically significant (P<0.01) pair wise comparisons between 1 µM RA-treated and untreated samples (*) and between 1 µM RA+FA-treated and untreated samples (**). (B) Quantitative RT-PCR showing that treatment with a higher concentration of FA (1 µM) for 48 hours did also not induce P311 mRNA in lung fibroblasts isolated from early postnatal mice, and that the induction of P311 mRNA by RA is dose-dependent. Note that treatment with 1 µM RA at 48 hours induced P311 mRNA to a similar extent as treatment for 1 week as shown in panel A. Mean ± s.d. from triplicate samples are shown. Asterisks indicate statistically significant (P<0.01) pair-wise comparisons between 200 nM RA+FA-treated and 12.5nM RA+FA-treated samples (*) and between 1 µM RA+FA-treated and 12.5 nM RA+FA-treated samples (**).

 

Figure 5
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  		Fig. 5. Retinoic acid increases lipid accumulation in lung fibroblasts. (A) Lung fibroblasts cultured for 1 week to deplete endogenous lipid droplets were exposed to fatty-acid complexes (0.1 µM) and retinoic acid (RA; 1 µM) for 1 week, stained with Nile Red and analyzed by flow cytometry. The shift in mean fluorescence intensity following fatty acid (FA) and RA treatment indicates increased Nile-Red staining and, hence, increased cellular lipids. (B) Graph of mean fluorescence intensities normalized to the average mean fluorescence intensity of untreated samples. The mean ± s.d. from triplicate samples are shown. Asterisks indicate statistically significant (P<0.01) pair wise comparisons between FA treated and untreated samples (*) and between RA treated and untreated samples (**).

 

Figure 6
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  		Fig. 6. P311 gene silencing affects retinoic-acid-mediated lipid accumulation in lung fibroblasts. (A) Lung fibroblasts cultured for 1 week and infected with control lentivirus or P311-shRNA lentivirus were examined for relative P311 mRNA expression levels by quantitative real-time RT-PCR. There is an ~80% decrease in P311 transcripts in P311-shRNA-lentivirus-infected cells relative to control-lentivirus-infected cells. Mean ± s.d. from triplicate samples are shown. The differences between P311-shRNA and control samples are statistically significant (P<0.01). (B) Epifluorescence images of control and P311-shRNA-lentivirus-infected cells exposed to fatty-acid complexes (1 µM) and RA (1 µM) for 48 hours, showing that P311-shRNA-lentivirus-infected cells (identifiable by co-expression of GFP) accumulated less lipid droplets (stained with Nile Red) relative to control-lentivirus-infected cells (arrows). (C) The relative amount of lipids in control- versus P311-shRNA-lentivirus-infected cells, assessed by Nile-Red-stained cells followed by flow cytometry, indicates an ~85% decrease in lipid accumulation in the P311-shRNA-lentivirus-infected population. Fatty-acid- (1 µM) and retinoic-acid- (1 µM) treated control- and P311-shRNA-lentivirus-infected cells were sorted for GFP and Nile-Red fluorescence after 48 hours. The mean fluorescence intensities of the Nile-Red channel in the GFP-positive populations were determined. Values were normalized to the average of the mean fluorescence intensities of the control GFP+ samples. Mean ± s.d. from triplicate samples are shown. The differences between P311 shRNA and control samples are statistically significant (P<0.01).

 

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