Moderate activation of IKK2-NF-kB in unstressed adult mouse liver induces cytoprotective genes and lipogenesis without apparent signs of inflammation or fibrosis

Hepatic mRNA expression of genes important in NF-kB activation, inflammation, and
fibrosis in Liv-Ikk2ca mice

It is noteworthy that the data on mRNA expression is based on total RNA extracted
from whole liver and may be influenced by non-hepatocyte gene expression. The Ikk2ca
protein is Flag-tagged 9]. Hepatic mRNA expression of the Ikk2ca transgene was determined using forward and
reverse primers that specifically target the Flag tag and Ikk2 cDNA, respectively.
Ikk2ca mRNA was very low in the wild-type (Ikk2ca fl/+, Alb-cre/-) livers (Mean Ct
value ~30), whereas the Ikk2ca mRNA expression levels in Liv-Ikk2ca mice (mean Ct
value 24.7) appeared comparable to the expression levels of endogenous Ikk2 mRNA in
wild-type mice (mean Ct value 24.3), estimated by the comparable Ct values of the
Ikk2ca and mouse Ikk2 in these mice. Thus, hepatic expression of the Ikk2ca mRNA in
the current Liv-Ikk2ca mice is likely lower than the two previous Liv-Ikk2ca mouse
models in which the Ikk2ca transgene was driven by strong promoters from albumin and
liver activator protein 5], 6]. Expectedly, hepatic mRNA expression of Ikk2 and classical targets of NF-kB activation,
namely IkB?, serum amyloid a1 (Saa1), intercellular adhesion molecule 1 (Icam1), Bcl-x,
and Akt1 1], 3] were all significantly higher in 12-week-old male Liv-Ikk2ca mice than wild-type
littermates (Fig. 1a). Conversely, Liv-Ikk2ca mice had comparable Gadd45b, RelA/p65, and Nfkb1 (p50),
but moderately higher Ikk1 and much higher (6.9 fold) RelB (Fig. 1a). Interestingly, Liv-Ikk2ca mice had much higher (5.5 fold) Ikbke and 95 % higher
NF-kB inducing kinase (NIK) which activates IKK1 16] (Fig. 1a). Moreover, Liv-Ikk2ca mice had markedly higher cytokines lipocalin-2 (Lcn2, 106
fold) and A20 (9.4 fold) (Fig. 1a). Lcn2, an antimicrobial protein, protects endotoxin-induced sepsis 17] and diet-induced insulin resistance 18]. A20, an early NF-kB-responsive gene, has anti-inflammatory effects via negative
feedback regulation of the classical NF-kB pathway 19] but activation of the alternative NF-kB pathway 20].

Fig. 1. Hepatic mRNA expression of genes important in the IKK-NF-kB pathways, inflammation
and fibrosis in adult male mice with hepatocyte-specific activation of Ikk2 (Liv-Ikk2ca).
a IKK-NF-kB pathways; and b inflammation and fibrosis. N?=?6 per group, mean?±?SE. *p??0.05 versus wild-type mice

Surprisingly, markers of inflammation and fibrosis, namely Tnf?, IL-1?, IL-6, chemokine
(C-C motif) ligand 2/monocyte chemotactic protein 1 (Ccl2/Mcp-1), Ccl5, Ccl19, Ccl20,
NO synthase 2 (Nos2), transforming growth factor ? (Tgf?), ?-smooth muscle actin (?-Sma),
and collagen 1a1 (Col1a1), all remained unchanged in Liv-Ikk2ca mice (Fig. 1b), which is in a sharp contrast to marked induction of these genes as well as inflammation
and fibrosis in adult mice with neonatal hepatocyte-specific activation of IKK2 5]. In a previous study when hepatic expression of Ikk2ca was driven by the albumin
promoter, although hepatic mRNA expression of IL-1? and IL-6 were strongly increased
by 2.7 and 7.8 fold, respectively, blood levels of IL-1? remained unchanged whereas
blood levels of IL-6 only doubled in those mice 6]. Consistent with the lack of increase in hepatic mRNA expression of inflammatory
cytokines/chemokines, blood levels of IL-6 remained below the detection limit in wild-type
and Liv-Ikk2ca mice, and there was no difference in blood levels of Mcp-1 between
wild-type (96?±?12 pg/ml) and Liv-Ikk2ca mice (67?±?8 pg/ml).

Liver histopathology in Liv-Ikk2ca mice

Histologically, the livers of wild-type (Fig. 2a) and Liv-Ikk2ca (Fig. 2b) mice were undistinguishable. There were no signs of infiltration of inflammatory
cells or fibrosis in livers of Liv-Ikk2ca mice (Fig. 2b), which is consistent with the lack of induction of proinflammatory and fibrotic
genes in these mice (Fig. 1).

Fig. 2. Liver histopathology in adult male mice with hepatocyte-specific activation of Ikk2
(Liv-Ikk2ca). a wild-type mice; and b Liv-Ikk2ca mice. Hematoxylin and eosin (HE) staining of paraffin embedded liver
sections (5 ?m and 200 x magnification)

Hepatic mRNA expression of key antioxidative genes in Liv-Ikk2ca mice

Liv-Ikk2ca mice had 120, 85, and 80 % higher expression of antioxidative genes Nqo1,
superoxide dismutase 2 (Sod2), and glutathione peroxidase 1 (Gpx1), respectively (Fig. 3a). Conversely, Liv-Ikk2ca mice had similar Sod1, Nqo2, catalase (Cat), heme oxygenase-1
(Ho-1), epoxide hydrolase 1 (Ephx1), as well as glutamate-cysteine ligase catalytic
subunit (Gclc) and modifier subunit (Gclm), key enzymes of glutathione synthesis.

Fig. 3. Hepatic mRNA expression of key antioxidative genes and Nqo1 activities in adult male
mice with hepatocyte-specific activation of Ikk2 (Liv-Ikk2ca). a mRNA expression of antioxidative genes; and b Nqo1 activities. N?=?6 per group, mean?±?SE. *p??0.05 versus wild-type mice

To determine whether changes in mRNA expression translated into changes in protein
expression and function, we determined Nqo1 activities in liver homogenates. The 1.2
fold higher mRNA expression of Nqo1 (Fig. 3a) was associated with similarly 94 % higher activities of Nqo1 in livers of Liv-Ikk2ca
mice than wild-type mice (Fig. 3b).

Hepatic mRNA expression of genes important in metabolism of glucose, lipids, cholesterol,
and bile acids (BAs) in Liv-Ikk2ca mice

Surprisingly, Liv-Ikk2ca mice and wild-type mice had similar expression of key enzymes
for gluconeogenesis and glucose utilization, namely Pepck, G6pc, and glucokinase (Gck)
(Fig. 4a). Conversely, Liv-Ikk2ca mice had 58 % higher glycogen synthase 2 (Gys2), a key enzyme
for glycogen synthesis. Therefore, IKK2 activation in normal adult hepatocytes appears
to produce a cytoprotective gene expression profile without apparent signs of inflammation,
induction of gluconeogenic genes, or fibrosis.

Fig. 4. Hepatic expression of genes important in metabolism of glucose and lipids as well
as cholesterol and bile acids in adult male mice with hepatocyte-specific activation
of Ikk2 (Liv-Ikk2ca). a mRNA expression of genes in glucose and lipid metabolism; b mRNA expression of genes in cholesterol and bile acid metabolism. N?=?6 per group, mean?±?SE. *p??0.05 versus wild-type mice. c d Hepatic protein levels of Scd1 in adult male Liv-Ikk2ca mice. The densities of Scd1
in the gel image (top) were normalized to Gapdh (bottom). N?=?3 per group, mean?±?SE

Liv-Ikk2ca mice have 83 % higher fatty acid synthase (Fasn) and 1.2-fold higher CD36,
a key uptake transporter of fatty acids (Fig. 4a). Stearoyl-CoA desaturase 1 (Scd1) is rate-limiting for the biosynthesis of monounsaturated
fatty acids, whereas apolipoprotein E (ApoE) is essential for normal catabolism of
triglyceride-rich lipoproteins. Liv-Ikk2ca mice had 120 and 28 % higher Scd1 and ApoE,
respectively. Additionally, Liv-Ikk2ca mice had similar acyl-CoA oxidase 1 (Acox1),
a key enzyme in peroxisomal fatty acid oxidation, but 60 % lower cytochrome P450 4a14
(Cyp4a14) and a trend of lower Cyp4a12, enzymes for microsomal fatty acid oxidation
(Fig. 4a). Thus, compared to wild-type mice, livers of Liv-Ikk2ca mice had higher expression
of genes important for the uptake (CD36) and synthesis (Fasn Scd1) of fatty acids,
but lower expression of genes for microsomal fatty acid oxidation (Cyp4a14).

Cyp7a1 and Cyp8b1 are two key enzymes in the classic pathway, whereas Cyp27a1 and
Cyp7b1 are two key enzymes in the alternative pathway of BA biosynthesis from cholesterol.
Liv-Ikk2ca mice had 80 % higher Cyp7a1 and 44 % lower Cyp7b1 (Fig. 4b), but similar Cyp8b1 and Cyp27a1 (Additional file 2). Liv-Ikk2ca mice had 40 % higher Abca1, which pumps cholesterol/phospholipids into
circulation. Additionally, Liv-Ikk2ca mice had 54 % higher BA CoA: amino acid N-acyltransferase
(Baat), a key enzyme for BA conjugation, and 31 % higher Taurine transporter (TauT).
Therefore, compared to wild-type mice, livers of Liv-Ikk2ca mice had higher mRNA expression
of the key genes for BA biosynthesis (Cyp7a1) and BA conjugation (Baat and TauT).

Liv-Ikk2ca mice had 40 % lower expression of uptake transporter organic anion-transporting
polypeptide 1a1 (Oatp1a1) but 45 % higher efflux transporter Abcg2 and 57 % higher
multi-drug resistance 2 (Mdr2), the biliary efflux transporter for phospholipids (Fig. 4b). Conversely, Liv-Ikk2ca mice and wild-type mice had comparable mRNA expression of
other major BA uptake and efflux transporters (Additional file 2). In summary, Liv-Ikk2ca mice may have increased classic pathway of BA biosynthesis
(by Cyp7a1), higher capacity for BA conjugation (by Baat and TauT), and higher biliary
output of BAs (by Mdr2 and Abcg2).

To determine whether changes in mRNA expression translated into changes in protein
expression, we used Western blot to determine Scd1 protein levels in liver homogenates.
The 1.2 fold higher mRNA expression of Scd1 (Fig. 4a) was associated with similarly 78 % higher Scd1 protein in livers of Liv-Ikk2ca mice
than wild-type mice (Fig. 4c d).

Hepatic and circulating levels of triglycerides and cholesterol in Liv-Ikk2ca mice

Consistent with higher hepatic expression of lipogenic genes, hepatic triglycerides
were 61 % higher in Liv-Ikk2ca mice than in wild-type mice (Fig. 5a). Conversely, Liv-Ikk2ca mice had comparable cholesterol in liver (Fig. 5a) and similar serum levels of total cholesterol and triglycerides (data not shown).

Fig. 5. Hepatic levels of triglycerides, cholesterol, reduced GSH, endogenous reactive oxygen
species (ROS), malondialdehyde (MDA), and hydroxyproline in adult male mice with hepatocyte-specific
activation of Ikk2 (Liv-Ikk2ca). a triglycerides and cholesterol; b reduced GSH; c endogenous ROS and MDA; and d hydroxyproline. N?=?6 per group, mean?±?SE. *p??0.05 versus wild-type mice

Hepatic levels of GSH, endogenous ROS, lipid peroxidation, and hydroxyproline in Liv-Ikk2ca
mice

Activation of NF-kB has been associated with oxidative stress and steatohepatitis.
However, we found that despite moderately elevated hepatic triglycerides in Liv-Ikk2ca
mice, hepatic levels of GSH, endogenous ROS, and lipid peroxidation (MDA) remained
unchanged in Liv-Ikk2ca mice compared to wild-type mice (Fig. 5b c). Additionally, consistent with a lack of induction of fibrogenic genes, hepatic
levels of hydroxyproline, a marker of fibrosis, remained unchanged in Liv-Ikk2ca mice
(Fig. 5d).

Hepatic mRNA expression of major drug-metabolizing enzymes in Liv-Ikk2ca mice

Activation of NF-kB has been implicated in down-regulation of drug-processing genes
(DPGs) during inflammation 21]. Thus, we determined hepatic mRNA expression of major DPGs. Liv-Ikk2ca mice and wild-type
mice had similar expression of most major Cyps, (Additional file 3A). However, Liv-Ikk2ca mice had 30 and 40 % lower Cyp2c44 and Cyp2e1, respectively,
but 41 % higher Cyp3a11 (Fig. 6), the most predominant Cyp3a isoform in mouse liver 22].

Fig. 6. Hepatic mRNA expression of major cytochome P450s (Cyps) and Phase-II enzymes in adult
male mice with hepatocyte-specific activation of Ikk2 (Liv-Ikk2ca). N?=?6 per group, mean?±?SE. *p??0.05 versus wild-type mice

Liv-Ikk2ca mice and wild-type mice had similar expression of most major UDP-glucuronosyltransferases
(Ugts) and sulfotransferases (Sults) (Additional file 3B). Conversely, Liv-Ikk2ca mice had 4.2 fold, 40 %, and 21 fold higher Ugt1a9, Ugt2b34,
and Sult1e1, respectively, and 69 % higher 3?-phosphoadenosine 5?-phosphosulfate synthetase
2 (Papss2) (Fig. 6), the major enzyme for PAPS biosynthesis in liver 23].

Liv-Ikk2ca mice and wild-type mice had similar expression of most major hepatic glutathione
S-transferases (Gsts) 23], (Additional file 3C). Conversely, Liv-Ikk2ca mice had 60 % higher Gstm5 and 46 % higher Gsto1, but 30
% lower Gstp1 (Fig. 6). Thus, IKK2 activation in adult mouse hepatocytes does not down-regulate most major
DPGs.

Hepatic expression of essential transcription factors in Liv-Ikk2ca mice

Transcription factors hepatocyte nuclear factor 1? (HNF1?), HNF4?, C/EBP?, C/EBP?,
farnesoid X receptor (FXR), liver X receptor ? (LXR?), LXR?, and peroxisome proliferator-activated
receptor ? (PPAR?) as well as the co-activator PPAR? coactivator 1? (PGC1?) and the
co-inhibitor small heterodimer partner (SHP) are essential for hepatic basal expression
of genes important for nutrient and xenobiotic metabolism 24], 25]. Many of them were modestly higher in Liv-Ikk2ca mice, such as Hnf1a (45 %), Cebpa
(44 %), Cebpb (46 %), and Fxr (71 %) (Fig. 8a). Interestingly, Liv-Ikk2ca mice had 59, 78, and 32 % higher expression of 3 lipogenic
nuclear receptors, namely Ppar?, Lxr?, Lxr?, respectively, and tended to have higher
(47 %) lipogenic transcription factor Srebp-1c (Fig. 7a).

Fig. 7. Hepatic mRNA expression of key transcription factors in adult male mice with hepatocyte-specific
activation of Ikk2 (Liv-Ikk2ca). N?=?6, mean?±?SE. * p??0.05 versus wild-type mice

Xenobiotic receptors, namely aryl hydrocarbon receptor (AhR), constitutive active
receptor (CAR), Pregnane X receptor (PXR), PPAR?, and nuclear factor (erythroid-derived
2)-like 2 (NRF2) are very important in hepatic induction of DPGs by xenobiotics, and
retinoid X receptor ? (RXR?) is the obligatory heterodimer partner for FXR, CAR, PXR,
and PPARs 26]. Glucocorticoid receptor (GR), c-Jun, and c-Myc are important in inflammation and
stress responses. Liv-Ikk2ca mice had 48, 36, and 32 % higher AhR, Nrf2, and Gr, respectively,
but similar Rxra, Car, Pxr, c-jun, and c-Myc (Fig. 7b).

Because different NF-kB subunits have differential effects on gene expression, we
used Western blot to quantify hepatic nuclear and cytosolic protein levels of Ikk2,
Ikk1, and major NF-kB subunits (Fig. 8). The Ikk2ca protein was Flag-tagged. Interestingly, an additional band was detected
by Ikk2 antibody only in the cytosol of Liv-Ikk2ca mice, which might be the Ikk2ca
protein and/or post-translationally modified Ikk2 protein (Fig. 8a, upper band). Nuclear levels of Ikk2 and Ikk1 were 100 and 88 % higher in Liv-Ikk2ca
mice than wild-type mice, respectively (Fig. 8b). Nuclear levels of p65, p50, and RelB were 1.2-, 2.3-, and 8.6-fold higher in Liv-Ikk2ca
mice (Fig. 8b), whereas p65, p50 and p52 were hardly detectable in the cytosol of both Liv-Ikk2ca
mice and wild-type mice (Fig. 8a). Conversely, nuclear and cytosolic levels of precursors p100 and p105 remained unchanged
(Fig. 8), and nuclear Foxo1, a transcription factor important in the regulation of insulin
signaling and inflammation 27], 28] was very low in both strains (data not shown). Interestingly, p105, but not p100,
was clearly present in the nuclei of both strains (Fig. 8b), whereas p52 appeared to be present at much higher levels than p50 in the nuclei
in wild-type livers (Fig. 8b), which is consistent with high hepatic basal activity of Ikk1 29].

Fig. 8. Western blot quantification of cytosolic and nuclear protein levels of Ikk and NF-kB
subunits in adult male mice with hepatocyte-specific activation of Ikk2 (Liv-Ikk2ca).
a cytosol; b nuclei. The densities of Ikk and each NF-kB subunit in the gel image (top) were normalized
to Gapdh (a) or histone H3 (b). N?=?3 per group, mean?±?SE. *p??0.05 versus wild-type mice

Binding of NF-kB p50 to the promoter of genes altered in livers of Liv-Ikk2ca mice

To determine whether increases in the nuclear NF-kB subunits (Fig. 8b) translate into increases the DNA-binding of NF-kB, we used ChIP-qPCR to determine
DNA-binding of p50 in livers of Liv-Ikk2ca mice. The total amount of DNA fragments
pulled down by the p50 antibody was 12.4 fold higher in Liv-Ikk2ca mice than wild-type
mice (Fig. 9a). Additionally, results of ChIP-qPCR showed that the binding of p50 to the proximal
promoters of Ikba, Saa1, Icam1, Nqo1, Cyp2e1, and Cyp3a11 were 5–9 fold higher in
Liv-Ikk2ca mice than wild-type mice (Fig. 9b).

Fig. 9. ChIP-qPCR determination of binding of NF-kB p50 to proximal promoters of genes altered
in livers of adult male mice with hepatocyte-specific activation of Ikk2 (Liv-Ikk2ca).
a total amount of ChIPed DNA; b qPCR quantification of enrichment of DNA fragments that contain putative NF-kB binding
site. N?=?3 per group, mean?±?SE. *p??0.05 versus wild-type mice