Abstract
Injection of rats with bacterial lipopolysaccharide down-regulates P450 (P450) 2C11 (2C11) mRNA to about 20% of its control levels after only 6 hr, and this level is maintained for at least 48 hr. Although we and others have demonstrated that this effect may be at least partially mediated by the cytokines interleukin-1, interleukin-6, and tumor necrosis factor-α, as well as by glucocorticoids, the time courses and potencies of 2C11 repression by each single mediator suggested that no cytokine alone is responsible for the entire time course of 2C11 suppression during inflammation. Here, we show that transforming growth factor-β, hepatocyte growth factor, and interleukin-11 are potent inhibitors of 2C11 expression. In all three cases, 0.1 ng/ml was enough to down-regulate 2C11 mRNA levels to 50% of control. Interleukin-8, a cytokine that is secreted during the acute phase response but does not influence the liver acute phase response, did not affect 2C11 expression. The various mediators have different time courses of 2C11 down-regulation, indicating that the roles of each may be different at different phases of the response.
Infections and tissue inflammation stimulate a complex series of reactions. The liver is one of the principal targets of inflammatory mediators, responding to a variety of different factors with the secretion of a battery of acute phase proteins (APPs1) (Baumann and Gauldie, 1994). The inflammatory mediators that induce APP expression in liver cells have been grouped in four different categories: 1) interleukin (IL)-6-type cytokines (IL-6, IL-11, leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor; 2) IL-1-type cytokines (IL-1α, IL-1β, and tumor necrosis factors-α and -β); 3) glucocorticoids; and 4) growth factors such as transforming growth factor-β (TGF-β), hepatocyte growth factor (HGF), and fibroblast growth factor (for a review, see Baumann and Gauldie, 1994).
Various laboratories have shown that several members of the cytochrome P450 (P450) family are suppressed during the acute phase response (APR) (Morgan, 1997). The suppression of P450 2C11 (2C11), the major component of microsomal P450s in male rat liver, seems to be mediated at least in part by cytokines like IL-1, tumor necrosis factor-α, IL-6, interferon-α, and glucocorticoids (Morgan, 1997; Iber et al., 1997). However, none of these effectors by itself could down-regulate the 2C11 to less than 10% of its control level over the whole time period from 6 to 36 hr after treatment, as is seen with LPS treatment (Morgan, 1989), suggesting that 2C11 down-regulation is likely mediated by several different effectors. Furthermore, the above mediators account only for three of the four different main groups of mediators affecting the liver during APR (Baumann and Gauldie, 1994). In fact, secretion of the growth factor TGF-β, a member of the fourth group mediating the APR, is one of the earliest events in an inflammatory response (Wahl et al., 1989a). Because growth factors like TGF-β are involved in the inflammatory response, and in the APR liver, we investigated the influence of TGF-β and HGF on 2C11 expression.
Materials and Methods
Materials.
Male Sprague-Dawley rats ( 200–300 g weight) from Harlan Sprague Dawley Inc. (Indianapolis, IN) were used for hepatocyte isolation. Cell culture medium (Waymouth’s MB 752/1) and additives were purchased from Life Technologies, Inc. (Bethesda, MD). ECL was purchased from Amersham Life Science (Arlington Heights, IL). TGF-β, HGF, IL-11, and IL-8 were purchased from R&D Systems Inc. (Minneapolis, MN). Matrigel was prepared as described previously (Chen et al., 1995).
Hepatocyte Isolation and Culture.
Isolation of rat hepatocytes was performed by in situcollagenase perfusion (Liddle et al., 1992). Hepatocytes were plated in Waymouth’s medium containing 0.15 μM insulin (Liddleet al., 1992) on 60-mm culture dishes (Falcon; Becton-Dickinson, Lincoln Park, NJ) coated with 0.3 ml of Matrigel (7 mg/ml) at a density of 3–4 × 106 live cells per dish. Medium was changed to remove the dead cells 4 h after plating, and every 48 h thereafter. Cells were cultured for 5 days before the addition of the various inflammatory mediators, so that 2C11 expression could be recovered.
Isolation of Total RNA and Slot-Blot Assays.
Total hepatocyte RNA was prepared by the method of Chomczynski and Sacchi (1987). The relative abundance of 2C11 mRNA in the samples was measured by a slot-blot hybridization assay as described previously (Morgan, 1989). All results were normalized to the contents of poly(A+) RNA in the samples, measured by probing slot-blots with an oligo(dT)30 probe (Wright and Morgan, 1991). Bound probes were detected by autoradiography and quantified by analysis on a Lynx video densitometer (Applied Imaging, Santa Clara, CA). The loads of total RNA used had been previously determined to be in the linear range for the assay.
Microsomal Protein Isolation and Western Blotting.
Microsomes were isolated by differential ultracentrifugation (Iberet al., 1997). The amount of 2C11 protein in the microsomes was assayed by Western blotting under conditions of linearity using a polyclonal antibody to 2C11, which was made specific to 2C11 by immunoabsorption with female microsomal P450 as described previously (Morgan, 1985). The bound second antibody-horseradish peroxidase complex was visualized by a chemiluminescence method (ECL) according to the manufacturer’s recommendations and quantitated by video densitometry.
Presentation of Results and Statistical Analyses.
Values for each experiment were calculated as a percentage of the mean value (arbitrary units) for an appropriate control group. One-way analysis of variance and Newman-Keuls test were used to test for significant differences between the means of different groups. All results are expressed as the mean ± SE for each group.
Results and Discussion
Influence of TGF-β, HGF, IL-11, and IL-8 on the Expression of 2C11 mRNA.
Fig. 1A shows that 24 hr of treatment with TGF-β, HGF, or IL-11 caused a concentration-dependent reduction of 2C11 mRNA expression in the cultured hepatocytes. Remarkably, a concentration of 0.1 ng/ml for any mediator was enough to reduce 2C11 mRNA levels to 50% of control. Thus, all four major groups of effectors mediating the APR during inflammation in liver (Baumann and Gauldie, 1994) impair 2C11 mRNA and protein expression. Recent studies have shown that TGF-β regulates the basal and polycyclic aromatic hydrocarbon-induced P450 1A1 and 1B1 expression in primary hepatocytes (Doehr et al., 1997; Abdel-Razzak et al., 1994). Here, we observe that P450 (2C11) gene expression is down-regulated by TGF-β independently of any inducer mechanism, at concentrations that were 2–3 times lower then those used for inhibition of P450 1A1, 1A2, and 1B1 (Abdel-Razzak et al., 1994; Doehr et al. 1997).
For HGF and IL-11, this is the first time that these mediators have been shown to be involved in P450 regulation. IL-11 repressed 2C11 expression to a maximum extent of 50% (fig. 1A), whereas TGF-β and HGF repressed 2C11 expression by 80% at the highest concentrations tested. However, IL-11 with an EC50 of about 1 pg/ml appeared to be 10–100-fold more potent than the two growth factors. Although initially identified as a hematopoietic cytokine with thrombopoietic activity, IL-11 is now shown to be expressed in a variety of tissues and to be regulated by inflammatory cytokines in a complex and cell/tissue-specific manner (Du and Williams, 1997). IL-11 and IL-6 have overlapping biological functions and signaling pathways (Taga, 1997), and IL-11 regulates APP expression in rat liver similarly to IL-6 (Baumann and Schendel, 1991). We show here that IL-11 was about 10-fold more potent than IL-6 in down-regulation of 2C11 (fig. 1; Chen et al., 1995). Consistent with the finding that IL-6 produces a smaller maximum response of 2C11 than does IL-1 (Chen et al., 1995), IL-11 only repressed 2C11 expression by a maximum of 50% in the present study.
Although HGF was first identified for its mitogenic activities (Michalopoulos and DeFrances, 1997), recent studies have shown that serum levels of HGF are increased during injury (Okajima et al., 1997), inflammation (Matsuno et al., 1997), and sepsis (Sakon et al., 1996). The increase in HGF serum concentration found in humans during sepsis (Sakon et al., 1996) or in mice during inflammation (Matsuno et al., 1997) lies in the concentration range that we found to inhibit 2C11 expression. IL-8, an important cytokine during APR that does not effect liver APP, did not repress 2C11 expression at concentrations of up to 10 ng/ml (data not shown), showing that 2C11 down-regulation by the above mediators is specific.
Influence of IL-11, HGF, and TGF-β on 2C11 Protein Expression.
Fig. 1B shows that the above changes in 2C11 mRNA are reflected by similar changes in 2C11 protein. HGF and TGF down-regulate 2C11 protein to 35% and 46% of its control level after 24 hr, dropping to 21% and 17% after 48 hr, respectively. IL-11 inhibits 2C11 protein expression 46% after 24 and 48 hr (fig. 1B).
Time Dependence of TGF-β, HGF, and IL-11 Effects on 2C11 Expression.
To investigate the possible contributions of different cytokines to P450 down-regulation at different times during an APR, primary hepatocytes were treated with maximally effective concentrations of TGF-β, HGF, or IL-11 or with 7.5 ng/ml IL-1 for varying times (fig.2). All four agents caused a rapid suppression of 2C11 mRNA within 4 hr of treatment (fig. 2), with IL-1 and TGF-β producing the largest effect (about 30% of control at this time point). The maximal effect of IL-11 was reached by 8 hr of incubation, whereas the effects of TGF-β, HGF, and IL-1 reached their nadirs at 16, 24, and 24 hr, respectively.
Secretion of TGF-β from macrophages is one of the first events of the inflammatory response, resulting in the activation of a cascade of events including activation of IL-1 transcription (Wahl et al., 1989b). This, combined with the fact 2C11 is already inhibited after 4 hr by TGF-β, suggests that this APR mediator could play a crucial role in 2C11 inhibition by LPS, especially during the early phase. It is notable that the various APR mediators that suppress 2C11 expression do so with different time dependence values (fig. 2), suggesting that while many factors may contribute to P450 suppression, their relative contributions may be different at different times in the response. The curve accompanying the filled symbols in the figure represents the highest inhibition by a single mediator at the respective time (fig. 2). This curve, which does not take into account the possibility that different APR mediators could regulate 2C11 expression in an additive way, strongly resembles 2C11 down-regulation during LPS treatment in vivo (Morgan, 1989).
In summary, it appears that many different mediators released during inflammation, and belonging to all four classes defined by Baumann and Gauldie (1994), are capable of down-regulating P450 2C11 expression, suggesting that they may all participate in 2C11 regulation. It is unlikely that any one of them is solely responsible for the 2C11 down-regulation during inflammation, as the time dependence and magnitude of 2C11 mRNA suppression by LPS in vivo is not mimicked by any one of them. The fact that 2C11 regulation by different groups of APR mediators can be additive (Chen et al., 1995) implies that these mediators cause P450 down-regulation by different mechanisms. The presence of multiple mediators and pathways suggest that P450 down-regulation during inflammation and infection may serve an as yet unknown physiological purpose. Therefore, it will be important in the future to determine how many signal transduction pathways are involved in 2C11 down-regulation, and whether they are convergent or divergent.
Acknowledgments
We are grateful to Qi Chen and Tong Li for excellent technical assistance.
Footnotes
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Send reprint requests to: Edward T. Morgan, PhD, Department of Pharmacology, Emory University, Atlanta, GA 30322.
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This work was supported by Grant GM46897 from the National Institutes of Health.
- Abbreviations used are::
- APP
- acute phase protein
- APR
- acute phase response
- 2C11
- cytochrome P450 2C11
- HGF
- hepatocyte growth factor
- IL-1
- interleukin-1
- IL-6
- interleukin-6
- IL-11
- interleukin-11
- LPS
- lipopolysaccharide
- CYP or P450
- cytochrome P450
- TGF-β
- transforming growth factor-β
- Received March 16, 1998.
- Accepted May 18, 1998.
- The American Society for Pharmacology and Experimental Therapeutics