Volume 41, Issue 2, Pages 251-258 (August 2004) Prostaglandin E2 inhibits transforming growth factor β1-mediated induction of collagen α1(I) in hepatic stellate cells  Alex Y. Hui, Andrew J. Dannenberg, Joseph J.Y. Sung, Kotha Subbaramaiah, Baoheng Du, Peter Olinga, Scott L. Friedman  Journal of Hepatology  Volume 41, Issue 2, Pages 251-258 (August 2004) DOI: 10.1016/j.jhep.2004.04.033

Fig. 1 TGF-β1 induces COX-2 mRNA expression. LX-1 cells were incubated in the presence of 2.5 ng/ml of TGF-β1 for 3–24 h. Total RNA was then extracted and 1 μg subjected to reverse transcription and real time quantitative PCR. The results are expressed as relative fold induction compared to untreated cells (control). Data are shown as mean±SE of three independent experiments. *P<0.05 compared with control. Journal of Hepatology 2004 41, 251-258DOI: (10.1016/j.jhep.2004.04.033)

Fig. 2 TGF-β1 induces COX-2 protein expression. LX-1 cells were incubated in the presence of 2.5 ng/ml of TGF-β1 or vehicle for 3, 6, or 12 h. Total protein was then extracted and 30 μg subjected to immunoblotting for COX-2 and β-actin. Journal of Hepatology 2004 41, 251-258DOI: (10.1016/j.jhep.2004.04.033)

Fig. 3 COX-2 contributes to both basal and TGF-β1-mediated production of PGE2. LX-1 cells were incubated in the presence of vehicle alone, different concentrations of TGF-β1 (1–10 ng/ml), 5 μM NS398 or the combination of TGF-β1 and NS398 for 18 h. PGE2 release was assayed by enzyme immunoassay. Data shown as mean±SE of three independent experiments. *P<0.05; **P<0.01 compared with control. Journal of Hepatology 2004 41, 251-258DOI: (10.1016/j.jhep.2004.04.033)

Fig. 4 TGF-β1 stimulates collagen α1(I) mRNA expression. LX-1 cells were incubated in the presence of vehicle alone or 2.5 ng/ml of TGF-β1 for 3–24 h. Total RNA was then extracted and 1 μg subjected to reverse transcription and real time quantitative PCR. The results are expressed as relative fold induction compared to control. Data shown as mean±SE of three independent experiments. *P<0.01 compared with control. Journal of Hepatology 2004 41, 251-258DOI: (10.1016/j.jhep.2004.04.033)

Fig. 5 Divergent effects of NS398 and PGE2 on basal expression of collagen α1(I) mRNA. LX-1 cells were incubated in the presence of 5 μM NS398 or 0.5 μM PGE2 for 18 h. Total RNA was then extracted and 1 μg subjected to reverse transcription and real time quantitative PCR. The results are expressed as relative fold induction compared to untreated cells (control). Data shown as mean±SE of three independent experiments. *P<0.01 compared with control. Journal of Hepatology 2004 41, 251-258DOI: (10.1016/j.jhep.2004.04.033)

Fig. 6 PGE2 inhibits basal expression of HSP47 mRNA. LX-1 cells were incubated with presence of 0.5 μM PGE2 for 18 h. Total RNA was then extracted and 1 μg subjected to reverse transcription and real time quantitative PCR. The results are expressed as relative fold induction compared to untreated cells (control). Data shown as mean±SE of three independent experiments. *P<0.01 compared with control. Journal of Hepatology 2004 41, 251-258DOI: (10.1016/j.jhep.2004.04.033)

Fig. 7 PGE2 causes dose-dependent suppression of TGF-β1-induced collagen α1(I) expression. LX-1 cells were incubated in the presence of vehicle alone, TGF-β1 (2.5 ng/ml) alone or TGF-β1 plus increasing concentrations of PGE2 for 18 h. Total RNA was then extracted and 1 μg subjected to reverse transcription and real time quantitative PCR. The results are expressed as relative fold induction compared to untreated cells (control). Data shown as mean±SE of three independent experiments. *P<0.01 compared with TGF-β1-treated cells. Journal of Hepatology 2004 41, 251-258DOI: (10.1016/j.jhep.2004.04.033)

Fig. 8 PGE2 inhibits collagen α1(I) mRNA expression in TGF-β1-pretreated LX-1 cells. LX-1 cells were incubated in the presence of vehicle alone or 2.5 ng/ml of TGF-β1 for 18 h, or pre-treated with 2.5 ng/ml of TGF-β1 for 6 h before addition of 0.5 μM of PGE2 for further 12 h. Total RNA was then extracted and 1 μg subjected to reverse transcription and real time quantitative PCR. The results are expressed as relative fold induction compared to untreated cells (control). Data shown as mean±SE of three independent experiments. *P<0.01 compared with control and **P<0.01 compared with TGF-β1-treated cells. Journal of Hepatology 2004 41, 251-258DOI: (10.1016/j.jhep.2004.04.033)

Fig. 9 COX-2 inhibition stimulates TGF-β1-induced collagen α1(I) expression. LX-1 cells were incubated in the presence of vehicle, 2.5 ng/ml of TGF-β1, 5 μM of NS398 for 6 h or 5 μM of NS398 for 3 h followed by 2.5 ng/ml of TGF-β1 for 3 h. Total RNA was then extracted and 1 μg subjected to reverse transcription and real time quantitative PCR. The results are expressed as relative fold induction compared to control. *P<0.05 compared to control and **P<0.05 compared to control or NS398-treated cells. Journal of Hepatology 2004 41, 251-258DOI: (10.1016/j.jhep.2004.04.033)

Fig. 10 PGE2 has no effect on TGF-β1-induced expression Smad mRNAs. LX-1 cells were treated with vehicle, 2.5 ng/ml TGF-β1 or TGF-β1 plus 0.5 μM PGE2 for 18 h. Total RNA was then extracted and 1 μg subjected to reverse transcription and real time quantitative PCR. The results are expressed as relative fold induction compared to untreated cells (control). Data shown as mean±SE of three independent experiments. *P<0.05 and **P<0.01 compared with control. There was no significant difference between TGF-β1-treated and TGF-β1 and PGE2-treated cells. Journal of Hepatology 2004 41, 251-258DOI: (10.1016/j.jhep.2004.04.033)

Fig. 11 Expression of PGE2 receptor mRNAs by LX-1 cells. Total RNA of untreated LX-1 cells was extracted and subjected to RT-PCR for receptors EP1-4. RNA substrate that had not undergone reverse transcription was used as a negative control (N) of the PCR. The positive controls (std) were cDNAs of the respective receptors. Journal of Hepatology 2004 41, 251-258DOI: (10.1016/j.jhep.2004.04.033)

Fig. 12 Effect of TGF-β1 on COX-2 mRNA expression in primary rat HSC. Primary rat HSC were incubated in the presence of 7.5 ng/ml of TGF-β1 for 18 h. Total RNA was then extracted and 1 μg subjected to reverse transcription and real time quantitative PCR. The results are expressed as relative fold induction compared to untreated cells (control). Data are shown as mean±SE of three independent experiments. *P<0.01 compared with control. Journal of Hepatology 2004 41, 251-258DOI: (10.1016/j.jhep.2004.04.033)

Fig. 13 PGE2 inhibits collagen α1(I) mRNA expression in TGF-β1-pre-treated rat HSC. Rat HSC were incubated in the presence of vehicle alone or 7.5 ng/ml of TGF-β1 for 18 h, or pre-treated with 7.5 ng/ml of TGF-β1 for 6 h before addition of 1 μM of PGE2 for further 12 h. Total RNA was then extracted and 1 μg subjected to reverse transcription and real time quantitative PCR. The results are expressed as relative fold induction compared to untreated cells (control). Data shown as mean±SE of three independent experiments. *P<0.01 compared with control and **P<0.05 compared with TGF-β1-treated cells. Journal of Hepatology 2004 41, 251-258DOI: (10.1016/j.jhep.2004.04.033)