Fig 1.
SAA induces IKK and JNK activation in HSCs.
A-B. Activated human HSCs were treated with recombinant human SAA (5 μM) for the indicated times or with rhTNFα for 15 minutes. Extracts were subjected to western blot analysis using antibodies that recognize IκBα, phospho-p65 (S536), phospho-c-Jun or actin (A). Results were confirmed by in vitro kinase assay using GST-IκBα, GST-p65, GST-p65 (S536A) and GST-c-Jun substrates (B). C. Activated human HSCs were treated with recombinant human sFasR (10 μM) for the indicated times or with rhTNFα for 15 minutes. Extracts were analyzed by western blot using antibodies against IκBα or GAPDH. D. Activated human HSCs were infected with adenoviruses expressing NF-κB driven luciferase followed by treated with the indicated concentrations of SAA or with rhTNFα (10 ng/ml) for 6h. Lucerifase activity is expressed as fold induction in comparison to untreated control. E. Primary rat HSCs were activated for 2, 4 and 8 days and treated with SAA (5 μM) or rmTNFα (30 ng/ml) for 15 minutes. Extracts were subjected to western blot analysis using antibodies that recognize IκBα, phospho-p65 (S536), phospho-c-Jun or actin. F. Primary rat HSCs were activated for infected with adenoviruses expressing NF-κB driven luciferase at day 3 or day 7 after plating. 18 hours after infection, cells were treated with with SAA (5 μM) or rmTNFα (30 ng/ml) for 15 minutes. NF-κB driven luciferase was determined as described above.
Fig 2.
SAA induces chemokine and MMP 9 expression in HSCs in an NF-κB- and JNK-dependent manner in HSCs.
Activated human HSCs were treated with rhSAA in the prescence or absence of JNK inhibitor SP600125 (20 μM), or after infection with adenoviruses expressing either IκBsr or GFP. A. 24 hours after rhSAA treatment, supernatants were analyzed for MCP-1, IL-8 and RANTES secretion by ELISA. B-C. 6 hours after rhSAA treatment, RNA was extracted and analyzed for mRNA levels of MCP-1, IL-8 and RANTES (B) or MMP9 (C) by quantitiative real time PCR (*p<0.05 vs. SP600125 treatment or IκBsr infection, resp.). D. 24 hours after SAA treatment, proteins were extracted and MMP9 activity was analyzed by zymography.
Fig 3.
SAA induces HSC proliferation in an Erk-, JNK- and Akt-dependent manner.
A-B. Activated rat HSCs were treated with rhSAA (5 μM) for the indicated times. Phosphorylation of Erk at threonine 202 and tyrosine 204 (A), and Akt at serine 473 (B) was determined by western blot analysis in the absence or presence of MEK inhibitor PD98059 or PI-3 kinase inhibitor LY294002, respectively. C. Serum starved activated rat HSCs were pretreated with PD98059 (5 μM), LY294002 (10 μM) or SP600125 (20 μM) followed by stimulation with rhSAA for 24 hours. 18 hours after stimulation cells were pulsed with 1 uCi/ml 3H-thymidine followed by TCA precipitation and measurement in a scinitillation counter.
Fig 4.
SAA induces apoptosis in HSCs after NF-κB inhibition.
Activated rat HSCs were infected with adenoviruses expressing either GFP or IκBsr. 24 hours later, HSCs were treated with rhSAA (5 μM). A. After 18h or treatment, cell death was determined by LDH activity assay. B. Caspase 3 and PARP cleavage were determined by western blot analysis after 8 hours of rmSAA1 treatment. C. After 8 hours of rhSAA treatment, cells were stained with Annexin V (green) and propidium iodide (PI, red) to visualize apoptosis and necrosis. D. Primary mouse hepatocytes were serum-starved for 12h and treated with rmSAA (5 μM), ActD (0.2 μg/ml) or rmTNFα (30 ng/ml) for 18. Cell death was determined by LDH activity assay (*p<0.05 vs. untreated vehicle control). E. Primary mouse hepatocytes were treated with rmSAA (5 μM) for the indicated times and rmTNFα (30 ng/ml) for 120 min. Phosphorylation of Erk, Akt, p65 and c-Jun was determined by western blot analysis.
Fig 5.
SAA does not signal in HSCs through TLR4, IL-1R, TNF-R1 or SR-BI.
A. Activated human HSCs were pretreated with pertussis toxin followed by treatment with rhSAA (5 μM). Activation of JNK, IKK and Erk pathways was analyzed by immunoblot for p-c-Jun, p-p65 and pErk. B. Activated human HSCs were pretreated with SR-BI blocking antibody or control antibody followed by rhSAA (5 μM) stimulation. JNK activation was analyzed by western blot using phospho-specific antibodies for its substrate c-Jun. C. Skin fibroblasts from TLR4-sufficient C3H/HeOuJ and TRL4-deficient C3H/HeJ mice were stimulated with rhSAA (5 mM), rmTNFα (30 ng/ml) or LPS (100 nM) followed by immunoblot for p-p65. D. Skin fibroblasts from wild-type mice, TNFRI-knockout mice, IL-1 receptor knockout mice and from TNFRI and IL-1 receptor double knockout mice were treated with rhSAA (5 μM), rmIL-1β (5 ng/ml), rmTNFα (30 ng/ml) or irradiated with UV (100 J/m2) Activation of IKK and JNK was determined by western blot analysis for their phosphorylation of their targets p65 and c-Jun, respectively. E. Mouse embryonic fibroblasts from wild type mice, TRAF-2 deficient mice and RIP-1 deficient mice were treated rhSAA (5 μM), rmIL-1β (5 ng/ml), rmTNFa (30 ng/ml) or irradiated with UV (20 J/m2). Activation of IKK and JNK was determined by western blot analysis for their phosphorylation of their targets p65 and c-Jun, respectively.
Fig 6.
SAA1 and SAA3 levels are elevated during hepatic fibrogenesis.
A. Hepatic fibrosis was induced in 8 week old Balb/c mice by bile duct ligation (n = 3 mice per time point) or by treatment with CCl4 (8 injections of 0.5 μl CCl4/g body weight, dissolved in olive oil, every 3 days, n = 4 mice/group). Following RNA extraction and reverse transcription, mRNA levels of SAA1 and SAA3 were determined by quantitative real time PCR. Levels are expressed as fold-induction in comparison to sham or oil control mice after normalization to 18s. B. RNA was isolated from quiescent mouse HSCs (18 hours after plating, n = 3 independent isolations) or activated mouse HSCs (5 days after plating, n = 3 independent isolations). mRNA levels of SAA1 and SAA3 were determined by quantitative real time PCR. Levels are expressed as fold-induction in comparison to quiescent HSCs after normalization to 18s.