Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Apr;14(4):1334-45.
doi: 10.1091/mbc.e02-07-0378.

Differential induction of nuclear factor-kappaB and activator protein-1 activity after CD40 ligation is associated with primary human hepatocyte apoptosis or intrahepatic endothelial cell proliferation

Jalal Ahmed-Choudhury  1 , Clare L Russell, Satinder Randhawa, Lawrence S Young, David H Adams, Simon C Afford
Affiliations

Differential induction of nuclear factor-kappaB and activator protein-1 activity after CD40 ligation is associated with primary human hepatocyte apoptosis or intrahepatic endothelial cell proliferation

Jalal Ahmed-Choudhury et al. Mol Biol Cell. 2003 Apr.

Erratum in

  • Mol Biol Cell.2003 May;14(5):following table of contents. Choudhury JA [corrected to Ahmed-Choudhury J]

Abstract

CD40, a tumor necrosis factor receptor superfamily member, is up-regulated on intraheptatic endothelial cells (IHEC) and epithelial cells during inflammatory liver disease, and there is evidence that the functional outcome of CD40 ligation differs between cell types. Ligation of CD40 on cholangiocytes or hepatocytes results in induction of Fas-mediated apoptosis, whereas ligation of IHEC CD40 leads to enhanced chemokine secretion and adhesion molecule expression. We now report that differential activation of two transcription factors, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), in primary human hepatocytes or IHEC, is associated with and may explain, in part, the different responses of these cell types to CD40 ligation. CD40 ligation induced a rise in NF-kappaB activity in hepatocytes,which peaked at 2 h and returned to baseline by 24 h; however, IHEC CD40 ligation resulted in a sustained up-regulation of NF-kappaB (>24 h). In hepatocytes, CD40 ligation led to sustained up-regulation of AP-1 activity >24 h associated with increased protein levels of RelA (p65), c-Jun, and c-Fos, whereas no induction of AP-1 activity was observed in IHECs. Analysis of mitogen-activated protein kinase phosphorylation (phospho-extracellular signal-regulated kinase 1/2 and phospho-c-Jun NH(2)-terminal kinase 1/2) and expression of inhibitor kappaBalpha were entirely consistent, and thus confirmed the profiles of NF-kappaB and AP-1 signaling and the effects of the selective inhibitors assessed using electrophoretic mobility shift assay or Western immunoblotting. CD40 ligation resulted in induction of apoptosis in hepatocytes after 24 h, but on IHECs, CD40 ligation resulted in proliferation. Inhibition of (CD40-mediated) NF-kappaB activation prevented IHEC proliferation and led to induction of apoptosis. Selective extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase inhibitors reduced levels of apoptosis in (CD40-stimulated) hepatocytes by approximately 50%. We conclude that differential activation of these two transcription factors in response to CD40 ligation is associated with differences in cell fate. Transient activation of NF-kappaB and sustained AP-1 activation is associated with apoptosis in hepatocytes, whereas prolonged NF-kappaB activation and a lack of AP-1 activation in IHECs result in proliferation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
EMSA analysis showing changes in DNA binding activity of NF-κB in the nuclear extracts of hepatocytes (A) and IHECs (B) stimulated via CD40 or TNF-α (positive control). Aliquots of nuclear extracts (20 μg of protein) from cultured and stimulated primary human hepatocytes and IHECs were subjected to EMSA analysis with 32P-labeled oligonucleotide probe consensus sequence to NF-κB. In hepatocytes, NF-κB activation was detected at 2 h but was largely undetectable at 24 h (A), whereas NF-κB activation was sustained >24 h in the IHECs (B). (A) Hepatocyte NF-κB: lane 1, negative control (labeled probe alone); lane 2, 2-h TNF-α stimulation; lane 3, unstimulated control; lane 4, 2-h CD40 stimulation; lane 5, supershift by using anti-RelA mAb; lane 6, cold competition with 100× excess of unlabeled probe; lane 7, 24-h CD40 stimulation. (B) IHEC NF-κB: lane 1, negative control (labeled probe alone); lane 2, 2-h unstimulated control; lane 3, 2 h after TNF-α stimulation; lane 4, 2-h CD40 stimulation; lane 5, 24-h CD40 stimulation; lane 6, cold competition with 100× excess of unlabeled probe.
Figure 2
Figure 2
Western immunoblots showing changes in RelA (p65) levels in CD40 stimulated or 2-h TNF-α (positive control) stimulated hepatocyte (A) and IHEC (B) nuclear extracts. Aliquots of nuclear extracts (40 μg of protein) from cultured and stimulated primary human hepatocytes and IHECs were subjected to Western blot analysis. Relative changes in p65 levels among the two cell types, as determined by densitometric analysis, and various experimental conditions are shown in the histograms (n = 4). Consistent with the EMSA data in Figure 1, increased levels of hepatocyte Rel A (p65) detected at 2 h were back to baseline at 24 h (A), whereas increased levels of RelA (p65) were sustained >24 h in the IHECs (B).
Figure 3
Figure 3
Western Immunoblots showing changes in levels of cytoplasmic IκBα in CD40-stimulated or 2-h TNF-α (positive control)–stimulated hepatocytes (A) and IHECs (B). Aliquots of cytoplasmic extracts (40 μg of protein) from cultured and stimulated primary human hepatocytes and IHECs were subjected to Western blot analysis. Relative changes in IκBα levels among the two cell types, as determined by densitometric analysis, and various experimental conditions are shown in the histograms (n = 3). Consistent with the EMSA data in Figure 1 and the Western blot data in Figure 2, increased levels of hepatocyte IκBα detected after 2 h of CD40 stimulation were back to baseline at 24 h (A), whereas increased levels of IκBα were sustained >24 h in the IHECs (B). (A) Hepatocyte IκBα: lane 1, unstimulated control; lane 2, 2-h TNF-α stimulation; lane 3, 2-h CD40 stimulation; lane 4, 24-h CD40 stimulation. (B) IHEC IκBα: lane 1, unstimulated control; lane 2, 2-h TNF-α stimulation; lane 3, 2-h CD40 stimulation; and lane 4, 24-h CD40 stimulation.
Figure 4
Figure 4
EMSA analysis showing changes in DNA binding activity of AP-1 in the nuclear extracts of hepatocytes (A) and IHECs (B) stimulated via CD40 or TNF-α (positive control). Aliquots of nuclear extracts (20 μg of protein) from cultured and stimulated primary human hepatocytes and IHECs were subjected to EMSA analysis with 32P-labeled oligonucleotide probe consensus sequence to AP-1. In hepatocytes, increased AP-1 binding was detected in response to CD40 at 2 and 24 h (A), whereas in IHECs little AP-1 activation was detected in response to CD40 at either 2 or 24 h (B). (A) Hepatocyte AP-1: lane 1, negative control (labeled probe alone); lane 2, unstimulated control; lane 3, 2-h TNF-α stimulation; lane 4, 2-h CD40 stimulation; lane 5, 24-h CD40 stimulation; lane 6, cold competition with 100× excess of unlabeled probe. (B) IHEC AP-1: lane 1, negative control (labeled probe alone); lane 2, 2-h TNF-α stimulation; lane 3, unstimulated control; lane 4, 2-h CD40 stimulation; lane 5, 24-h CD40 stimulation; and lane 6, cold competition with 100× excess of unlabeled probe. (C) Supershift EMSA analysis showing the presence of AP-1 components c-Jun and c-Fos in nuclear extracts of hepatocytes stimulated for 24 h with CD40. Aliquots of nuclear extracts (20 μg of protein) from cultured and stimulated primary human hepatocytes were subjected to supershift EMSA analysis containing 2 μg of antibodies against c-Jun or c-Fos components of AP-1 and 32P-labeled oligonucleotide probe consensus sequence of AP-1.
Figure 5
Figure 5
Western immunoblots showing changes in c-Jun levels in CD40-stimulated or 2-h TNF-α (positive control)–stimulated hepatocyte (A) and IHEC (B) nuclear extracts. Aliquots of nuclear extracts (40 μg of protein) from cultured and stimulated primary human hepatocytes, and IHECs were subjected to Western blot analysis. Relative changes in c-Jun levels among both cell types, as determined by densitometric analysis and various experimental conditions, are shown in the histograms (n = 3). In hepatocytes, increased c-Jun was detected at 2 and 24 h after CD40 activation (A), whereas in IHEC no activation in response to CD40 was detected at either time point (B).
Figure 6
Figure 6
Western immunoblots showing changes in c-Fos levels in CD40-stimulated or 2 h TNF-α (positive control)–stimulated hepatocyte (A) and IHEC (B) nuclear extracts. Aliquots of nuclear extracts (40 μg of protein) from cultured and stimulated primary human hepatocytes and IHECs were subjected to Western blot analysis. Relative changes in c-Fos levels among both cell types, as determined by densitometric analysis, are shown in the histograms (n = 3). In hepatocytes, increased c-Fos was detected at 2 and 24 h after CD40 activation (A), whereas in IHEC no activation in response to CD40 was detected at either time point (B) consistent with the observations for c-Jun (Figure 5).
Figure 7
Figure 7
Western immunoblots showing changes in levels of cytoplasmic phospho-ERK1/2 and phospho-JNK1/2 in CD40-stimulated or 2-h TNF-α (positive control)–stimulated hepatocytes (A) and IHECs (B) and/or treatment with specific inhibitors to ERK (50 μM PD98059) and JNK (100 μM DMAP). Aliquots of cytoplasmic extracts (40 μg of protein) from cultured and stimulated primary human hepatocytes, and IHECs were subjected to Western blot analysis. In hepatocytes, increased levels of phospho-ERK1/2 and phospho-JNK1/2 were detected at 2 and 24 h after CD40 activation (A) (top and bottom, lanes 3 and 4), whereas in IHECs no activation of these phospho-MAPKs in response to CD40 was detected at either time point (B) (top and bottom). Use of the selective inhibitors to ERK or JNK in CD40-stimulated hepatocytes prevented activation of phospho-ERK1/2 and phospho-JNK1/2, respectively (A) (top and bottom, lanes 7 and 8). (A) Top, hepatocyte phospho-ERK1/2: lane 1, unstimulated control; lane 2, 2-h TNF-α stimulation; lane 3, 2-h CD40 stimulation; lane 4, 24-h CD40 stimulation; lane 5, unstimulated control + ERK inhibitor; lane 6, 2-h TNF-α stimulation + ERK inhibitor; lane 7, 2-h CD40 stimulation + ERK inhibitor; and lane 8, 24-h CD40 stimulation + ERK inhibitor. Bottom, hepatocyte phospho-JNK1/2: lane 1, unstimulated control; lane 2, 2-h TNF-α stimulation; lane 3, 2-h CD40 stimulation; lane 4; 24-h CD40 stimulation; lane 5, unstimulated control + JNK inhibitor; lane 6, 2-h TNF-α stimulation + JNK inhibitor; lane 7, 2-h CD40 stimulation + JNK inhibitor; and lane 8, 24-h CD40 stimulation + JNK Inhibitor. (B) Top, IHEC phospho-ERK1/2: lane 1, unstimulated control; lane 2, 2-h CD40 stimulation; lane 3, 24-h CD40 stimulation; and lane 4, 2-h TNF-α stimulation. Bottom, IHEC phospho-JNK1/2: lane 1, unstimulated control; lane 2, 2-h CD40 stimulation; lane 3, 24-h CD40 stimulation; and lane 4, 2-h TNF-α stimulation.
Figure 8
Figure 8
Levels of apoptosis in primary cultures of hepatocytes and IHECs stimulated via CD40 or TNF-α. Histogram shows the percentage of ISEL-positive apoptotic hepatocytes or IHECs after CD40 or TNF-α stimulation. Black bars show results for hepatocytes and the white bars show results for IHECs (n = 5 for each cell type). Although TNF-α triggered similar levels of increased apoptosis in both cell types, CD40 activation only caused an increase in apoptosis in hepatocytes.
Figure 9
Figure 9
Effect of CD40 ligation and NF-κB inhibition on IHEC proliferation. (A) Histogram shows the proliferative index expressed as the percentage of Ki67-positive cells up to 72 h for either unstimulated IHECs or after CD40 stimulation (n = 8). CD40 stimulation led to a marked increase in cell proliferation in the endothelial cells. (B) Histogram shows the proliferative index expressed as the percentage of Ki67-positive cells for unstimulated IHECs or cells stimulated via CD40 in the presence or absence of the specific and potent NF-κB inhibitor, CAPE (25 μg/ml). The addition of CAPE completely inhibited the endothelial cell proliferation in response to CD40 activation. The results represent data from at least three separate experiments.
Figure 10
Figure 10
Effects of specific NF-κB, ERK, and/or JNK inhibitors on levels of apoptosis in hepatocytes or IHECs after CD40 stimulation. (A) Histogram shows the percentage of ISEL-positive apoptotic cells in either unstimulated or CD40-stimulated primary human hepatocytes or IHECs in the presence or absence of the NF-κB inhibitor, CAPE (25 μg/ml). The addition of CAPE to IHECs allowed CD40 to trigger similar levels of apoptosis to those induced in the hepatocytes. The results summarize data from at least four separate experiments. (B) Western immunoblot showing inhibition of nuclear RelA levels by a specific NF-κB inhibitor in CD40-stimulated IHECs. Aliquots of nuclear extracts (40 μg of protein) from cultured and stimulated primary human IHECs were subjected to Western blot analysis. Relative changes in RelA levels as determined by densitometric analysis, and various experimental conditions are shown in the histograms (n = 3). Use of the selective inhibitor to NF-κB, CAPE (25 μg/ml), in CD40-stimulated IHECs prevented increase in nuclear RelA (p65) levels. (C) Histogram shows the percentage of CD40-stimulated and unstimulated primary human hepatocytes positive by ISEL staining in the presence or absence of the selective ERK and/or JNK inhibitors PD98059 (50 μM) and DMAP (100 μM), respectively. The inhibitors reduced hepatocyte apoptosis in response to CD40 activation, however, levels of apoptosis were still above baseline. The results summarize data from at least four separate experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abmayr SM, Worman JL. Preparation of nuclear and cytoplasmic extracts from mammalian cells. In: Asubel, Brent FM, Kingston R, Moore RE, Seidman DD, Struhl JG, editors. Current Protocols in Molecular Biology. 1991. K. John Wiley & Sons, New York, 12.1.1–12.1.9. - PubMed
    1. Afford SC, Ahmed-Choudhury J, Randhawa S, Russell C, Youster J, Crosby HA, Eliopoulos A, Hubscher SG, Young LS, Adams DH. CD40 activation-induced, Fas-dependent apoptosis and NF-κB/AP-1 signaling in human intrahepatic biliary epithelial cells. FASEB J. 2001;15:2345–2354. - PubMed
    1. Afford SC, Hubscher S, Strain AJ, Adams DH, Neuberger JM. Apoptosis in the human liver during allograft rejection and end-stage liver disease. J Pathol. 1995;176:373–380. - PubMed
    1. Afford SC, Rhandawa S, Eliopoulos AG, Hubscher SG, Young LS, Adams DH. CD40 activation induces apoptosis in cultured human hepatocytes via induction of cell surface FasL expression and amplifies Fas mediated hepatocyte death during allograft rejection. J Exp Med. 1999;189:441–446. - PMC - PubMed
    1. Akira S. Roles of STAT3 defined by tissue-specific gene targeting. Oncogene. 2000;19:2607–2611. - PubMed

Publication types

MeSH terms