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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 19, 2002 as doi:10.1096/fj.02-0195fje. |
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2
* Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario La Fe, E-46009 Valencia, Spain; and
Departamento de Bioquímica, Facultad de Medicina, Universidad de Valencia, E-46010 Valencia, Spain
2Correspondence: Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario La Fe. Avda. Campanar 21, E-46009-Valencia, Spain. E-mail: Jose.Castell{at}uv.es
SPECIFIC AIM
Drug-metabolizing cytochromes P450 (CYPs) are down-regulated in the liver by cytokines during host inflammatory responses, which ultimately influences the fate and therapeutic efficacy of many drugs. We have studied the molecular mechanism behind down-regulation of the major human drug-metabolizing CYP (CYP3A4) by the proinflammatory cytokine interleukin-6 (IL-6), with special attention to the major transduction pathways and transcription factors involved.
PRINCIPAL FINDINGS
1. Activation of the glycoprotein gp130 receptor is at the origin of CYP3A4 down-regulation by IL-6
IL-6 shares with other proinflammatory cytokines the common receptor subunit gp130, which is a key element in signal transduction. We have shown that activation of the gp130 receptor by an IL-6-independent mechanism (agonistic-mAb) leads to CYP3A4 down-regulation, whereas blocking the receptor binding site with an antagonistic mAb prevents IL-6-mediated CYP repression.
2. CYP3A4 down-regulation by IL-6 is independent of the JAK/STAT pathway
The gp130 receptor subunit is capable of activating several signal transduction pathways, but for induction of acute-phase proteins (APPs), the JAK/STAT pathway is recognized as the most important one. We investigated whether STAT3 could be an essential player in the down-regulation of CYP3A4. We made use of a dominant-negative STAT3 adenoviral expression vector and showed how it is possible to block fibrinogen and haptoglobin induction by IL-6, leaving unaffected the down-regulation of CYP3A4 in differentiated human hepatoma cells.
3. IL-6-activated kinases (ERK1/2 and p38) through SHP-2/Ras/MAPK pathway do not play an important role in CYP3A4 down-regulation
The second best-characterized IL-6 transduction pathway downstream gp130 involves activation of SHP-2, Ras, and MAP kinases. Several targets for MAP kinases are known, but ERK1/2 and p38 are activated by IL-6 in human hepatoma cells. To assess the relevance of this pathway in down-regulation of CYP3A4, we made use of specific kinase inhibitors: PD98059 for ERKs and SB20358 for p38. Our experimental evidence did not support a relevant role of these kinases in the IL-6-mediated down-regulation of CYP3A4.
4. IL-6 down-regulates CYP3A4 through translational induction of repressive C/EBPß-LIP protein
Another target of proinflammatory cytokines is the transcription factor C/EBPß. Its expression can be induced through transcriptional and post-translational mechanisms. Moreover, cytokines can modify the relative abundance of the C/EBPß protein isoforms, which can be translated from the same C/EBPß mRNA. When we investigated the effect of IL-6 on C/EBPß in differentiated human hepatoma cells, we found a moderate up-regulation in mRNA and a dramatic increase in the low molecular weight isoform C/EBPß-LIP, which proved to be a repressor of CYP3A4 as demonstrated by adenovirus-mediated overexpression of this protein (Fig. 1
).
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5. C/EBPß-LIP can antagonize and reduce significantly the basal activating effect of C/EBP
and C/EBPß-LAP
Our next aim was to decipher the mechanism by which LIP inhibits CYP3A4 expression during inflammation. Experimental evidence supports a competition model among LIP and other C/EBP-activating forms such as C/EBP and C/EBPß-LAP. However, competition by LIP will be effective only if C/EBP and C/EBPß-LAP both act as constitutive transcriptional activators of CYP3A4. With the aid of adenoviral vectors, we demonstrated that overexpression of C/EBP or C/EBPß-LAP in human hepatoma cells causes up-regulation of CYP3A4 in its native chromatin context. The proposed competition mechanism was investigated by cotransfecting cells with activating C/EBP forms (C/EBP or C/EBPß-LAP) and the LIP factor. Experiments (summarized in Fig. 2
) clearly demonstrated that an increase in LIP expression can reduce the activating effect of C/EBP and C/EBPß-LAP on CYP 3A4 gene to 55%. This decrease is similar to the one observed after IL-6 treatment of cells.
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6. The mechanism envisaged is operative in human hepatocytes
To assess the relevance of our observations to human liver, we examined whether LIP could effectively impair CYP3A4 expression in cultured human hepatocytes, recognized as the model closest to human liver. Our experimental evidence demonstrated that a moderated increase in LIP expression reduced by 50% CYP3A4 mRNA levels. This finding confirms the repressive effect of LIP on CYP3A4 in a relevant model.
CONCLUSIONS
The mechanism by which the same inflammatory signal can simultaneously activate APPs and down-regulate other hepatic genes such as CYPs is intriguing, but little is known about the mechanism governing CYP down-regulation.
Activation of the gp130 receptor subunit by IL-6-type cytokines is the key step for initiation of the cytoplasmic signal transduction cascades leading to induction of APPs. Our results support the notion that activation of the gp130 receptor is also at the root of CYP down-regulation by IL-6.
The gp130 receptor is able to activate two major signal transduction pathways (the JAK/STAT- and SHP-2/Ras/MAPK-mediated pathways) as well as other poorly characterized transduction routes. Consequently, we investigated whether these signaling pathways were essential in the down-regulation of CYP3A4. Experiments with a dominant-negative STAT3 adenoviral expression vector demonstrated that CYP3A4 down-regulation by IL-6 is independent of the JAK/STAT pathway in human hepatoma cells. Moreover, our results with specific inhibitors for ERK1/2 and p38 suggested the SHP-2/Ras/MAPK pathway is not decisive in this down-regulation.
Inflammatory stimuli and cytokines can also act via other targets such as the CCAAT enhancer binding proteins (C/EBPs). The expression levels of various C/EBPs are differentially modulated in response to inflammatory cytokines—among them, IL-6. In differentiated human hepatoma cells we found a moderate up-regulation in C/EBPß mRNA and a dramatic increase in the C/EBPß-LIP protein isoform. The C/EBPß mRNA directs production of two isoforms: a 35 kDa LAP (liver-enriched transcriptional activating protein) and a 20 kDa LIP (liver-enriched transcriptional inhibitory protein). LIP is proposed to function as a dominant-negative regulator of full-length C/EBP proteins, as it lacks most of the trans-activation domain but contains the DNA binding and dimerization domains. In consonance with this view, it has been shown that LIP is involved in the down-regulation of the alpha1(I) collagen gene by TNF- and in down-regulation of the closely related C/EBP gene during the acute-phase response. Our experiments with an adenoviral vector for LIP agree with this proposed negative role. By overexpressing LIP in hepatoma cells, it was possible to demonstrate a clear inhibitory effect on CYP3A4 expression.
To explain LIP-mediated repression, a competition model between LIP and other C/EBP forms (such as C/EBP and C/EBPß-LAP) has been suggested. However, this competition model will be effective only if C/EBP and C/EBPß-LAP both act as constitutive transcriptional activators of CYP3A4. Overexpression of these factors in human hepatoma cells with the aid of adenoviral vectors evidenced the positive role exerted by C/EBP and C/EBPß-LAP in regulating human CYP3A4 in its native chromatin context. Previous reports showing trans-activation of CYP3A4 promoter constructs by C/EBP would agree with our findings. A positive role of C/EBPß-LAP on CYP3A4 had not previously been reported.
The proof-of-concept of the competition mechanism was investigated by cotransfection experiments with adenoviral constructs. Results demonstrated that an increase in LIP expression could reduce significantly the activating effect of C/EBP and C/EBPß-LAP on CYP3A4. A similar competition mechanism (i.e., displacement of C/EBP from the D site in the albumin promoter and trans-activation interference) has been proposed for albumin down-regulation during liver regeneration.
An interesting point deserving further attention is the signaling pathway linking IL-6 with increased LIP translation. A leaky ribosome scanning mechanism proposes that a portion of ribosomes scan the C/EBPß mRNA until the third AUG (LIP) codon is reached. Initiation at this site may be controlled by specific cytoplasmic proteins that interact with the 5' region of C/EBPß mRNA, such as the CUG triplet repeat binding protein 1 (CUGBP1), or by PKR and mTOR signaling pathways, which control the eukaryotic translation initiation factors eIF-2 and eIF-4E, respectively. Nevertheless, a signaling mechanism linking gp130 activation and the regulation of C/EBPß mRNA translation remains to be demonstrated.
That many different cytokines down-regulate CYPs in the liver does not imply that a single common mechanism is responsible for all these repressive responses. Thus, TNF- is believed to represses CYP1A1 via redox regulation of nuclear factor 1; IL-2 down-regulates rat CYP2C11 and CYP3A2 expression, probably via induction of the proto-oncogene c-myc and IL-1 inhibits CYP2C11 transcription via binding of NF-
B to a low-affinity binding site in its promoter. Nuclear receptors have been claimed to be relevant factors in the mechanism of CYP repression, as a decreased expression of PXR and CAR was observed after stimulation with IL-6 and down-regulation of several CYPs was blocked in PPAR-/- mice after treatment with LPS. In the case of IL-6, the results of our research point to an increased expression in C/EBPß-LIP as the determining event in human hepatocytes. This truncated C/EBPß protein, by competing with other constitutive C/EBP-activating factors, particularly C/EBP, down-regulates expression of human CYP3A4 (Fig. 3
).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0195fje; to cite this article, use FASEB J. (September 5, 2002) 10.1096/fj.02-0195fje 
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