Cyclic AMP-guanine exchange factor activation inhibits JNK-dependent lipopolysaccharide-induced apoptosis in rat hepatocytes

Abstract

Lipopolysaccharide (LPS) is known to damage hepatocytes by cytokines released from activated Kupffer cells, but the ancillary role of LPS as a direct hepatotoxin is less well characterized. The aim of this study was to determine the direct effect of LPS on hepatocyte viability and the underlying signaling mechanism. Rat hepatocyte cultures treated overnight with LPS (500 ng/mL) induced apoptosis as monitored morphologically (Hoechst 33258) and biochemically (cleavage of caspase 3 and 9 and the appearance of cytochrome C in the cytoplasm). LPS-induced apoptosis was additive to that induced by glycochenodeoxycholate or Fas ligand, was associated with activation of c-Jun N-terminal kinase B (JNK) and p38 mitogen-activated protein kinases (MAPK), and inhibition of protein kinase (AKT). Inhibition of JNK by SP600125, but not of p38 MAPK by SB203580 attenuated LPS-induced apoptosis, indicating JNK dependency. CPT-2-Me-cAMP, an activator of cAMP-GEF, decreased apoptosis due to LPS alone or in combination with glycochenodeoxycholate or Fas ligand. CPT-2-Me-cAMP also prevented LPS-induced activation of JNK and inhibition of AKT. Taken together, these results suggest that LPS can induce hepatocyte apoptosis directly in vitro in a JNK-dependent manner and activation of cAMP-GEF protects against the LPS-induced apoptosis most likely by reversing the effect of LPS on JNK and AKT.

Figure 1

LPS induces apoptosis in hepatocytes. Hepatocytes were treated overnight with 500 ng/mL LPs and cells were evaluated morphologically by Hoechst staining for the presence of apoptosis. Representative photomicrographs (100×) are shown; the arrows depict apoptotic hepatocytes (A). The amount of apoptosis was quantified by counting the number of hepatocytes with apoptotic morphology and is expressed as a percentage of the amount of apoptosis seen in the control and represents mean ± sD [n = 3–5] (B). Biochemical evidence of apoptosis was also obtained by immunoblotting for the 17 kD or 37 kD cleavage product of caspase 3 or caspase 9, respectively (C and D). Cytosolic preparations were immunoblotted for the appearance of mitochondrial cytochrome C (E). *Significantly different from control, P < 0.05. Abbreviations: LPS, lipopolysaccharide; SD, standard deviation.

Figure 2

LPS-mediated apoptosis is additive to that induced by bile acids or Fas ligand. Hepatocytes were treated with 500 ng/mL LPs overnight followed by treatment with 100 μM GCDC for 2 hours (A) or 50 ng/mL Fas L for 4 hours (B). Cells were evaluated morphologically by Hoechst staining for the presence of apoptosis. Results are expressed as a percentage of the amount of apoptosis seen in the control and represent mean ± SD (n = 3–5). *Significantly different from control, ^#Significantly different from LPs alone, P < 0.05. Abbreviations: LPS, lipopolysaccharide; GCDC, glycochenodoxycholate; SD, standard deviation; FAS, Fas ligand.

Figure 3

LPS activates stress activated kinases in hepatocytes. Cultured rat hepatocytes were treated with 500 ng/mL LPS for the time points indicated and cell lysates were prepared. The lysates were immunoblotted for phospho JNK (A and D), phospho p38 MAPK (B and D), and phospho AKT (C and D). The blots were developed by chemiluminescence, digitized and quantified. Results of quantification of at least 3 separate experiments are shown in A B and C and representative gels in D. Hepatocytes were pretreated for 30 min with either a JNK (SP 600125, 15 μM) or p38 MAPK inhibitor (SB 203580, 5 μM) and then overnight with 500 ng/mL of LPS (E). Cells were evaluated morphologically by Hoechst staining for the presence of apoptosis. Results are expressed as the percentage increase in apoptosis compared to control and represent mean ± SD (n = 3). *Significantly different from control, ^#Significantly different from LPS, P < 0.05. Abbreviations: LPS, lipopolysaccharide; JNK, c-Jun N-terminal kinase; pJNK, phospho JNK; MAPK, p38 mitogen-activated protein kinases; AKT, protein kinase; SD, standard deviation.

Figure 4

Activation of cAMP-GEFs attenuates LPs induced hepatocyte apoptosis. Hepatocytes were pretreated with 20 μM cPT2-Me-cAMP or vehicle for 30 minutes followed by treatment with vehicle or 500 ng/mL LPS overnight. Cells were evaluated morphologically by Hoechst staining for the presence of apoptosis (A). Results are expressed as a percentage of the amount of apoptosis seen in the control and represent mean ± SD (n = 4–6). Whole cell lysates were immunoblotted for the 17 kD cleaved fragment of caspase 3 and equal protein loading was verified by immunoblotting for actin (B). Hepatocytes were pretreated with 20 uM cPT-2-Me-cAMP for 30 minutes and then treated overnight with 500 ng/mL LPS ± pretreatment before exposing to 100 μM GCDC for 2 hours (C) or 50 ng/mL of Fas L for 4 hours (D). *Significantly different from control. ^#Significantly different from LPS, P < 0.05. Abbreviations: LPS, lipopolysaccharide; cAMP, cyclic adenosine monophosphate; CPT-2-Me-cAMP, 4-(4-chlorophenylthio)-2’-O-methyladenosine-3’5’cyclic monophosphate; GCDC, glycochenodoxycholate; SD, standard deviation.

Figure 5

Activation of cAMP-GEF inhibits LPs induced phosphorylation of JNK. Hepatocytes were treated with 500 ng/mL of LPs +/− pretreatment with 20 μM cPT-2-Me-cAMP for 4 or 24 hrs. Whole cell lysates were prepared. The lysates were immunoblotted for phospho JNK (A and C) and phospho AKT (B and C) and developed by chemiluminescence, digitized and quantified. Representative gels are shown in C and the results of quantification of at least 3 separate experiments are shown in A and B. *Significantly different from control, P < 0.05. ^#Significantly different from LPS, P < 0.05. Abbreviations: LPS, lipopolysaccharide; cAMP cyclic adenosine monophosphate; CPT-2-Me-cAMP,4-(4-chlorophenylthio)-2’-O-methyladenosine-3’5’cyclic monophosphate; JNK, c-Jun N-terminal kinase; AKT, protein kinase; SD, standard deviation.